WO2002026984A2 - Potassium channel interactors and uses therefor - Google Patents

Potassium channel interactors and uses therefor Download PDF

Info

Publication number
WO2002026984A2
WO2002026984A2 PCT/US2001/030463 US0130463W WO0226984A2 WO 2002026984 A2 WO2002026984 A2 WO 2002026984A2 US 0130463 W US0130463 W US 0130463W WO 0226984 A2 WO0226984 A2 WO 0226984A2
Authority
WO
WIPO (PCT)
Prior art keywords
seq
pcip
nucleic acid
polypeptide
amino acid
Prior art date
Application number
PCT/US2001/030463
Other languages
English (en)
French (fr)
Other versions
WO2002026984A3 (en
Inventor
Kenneth Rhodes
Maria Betty
Huai-Ping Ling
Wenqian An
Original Assignee
Millennium Pharmaceuticals Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US09/670,756 external-priority patent/US7078481B1/en
Priority to AU2001296393A priority Critical patent/AU2001296393A1/en
Priority to CA002420960A priority patent/CA2420960A1/en
Priority to EP01977260A priority patent/EP1322759A2/en
Priority to JP2002530747A priority patent/JP2004525610A/ja
Priority to IL15496201A priority patent/IL154962A0/xx
Application filed by Millennium Pharmaceuticals Inc. filed Critical Millennium Pharmaceuticals Inc.
Priority to EA200300420A priority patent/EA200300420A1/ru
Priority to MXPA03002557A priority patent/MXPA03002557A/es
Priority to KR10-2003-7004460A priority patent/KR20030074604A/ko
Priority to BR0114383-2A priority patent/BR0114383A/pt
Publication of WO2002026984A2 publication Critical patent/WO2002026984A2/en
Publication of WO2002026984A3 publication Critical patent/WO2002026984A3/en
Priority to NO20031369A priority patent/NO20031369L/no

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/08—Antiepileptics; Anticonvulsants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)

Definitions

  • Mammalian cell membranes are important to the structural integrity and activity of many cells and tissues.
  • membrane physiology is the study of trans-membrane ion channels which act to directly control a variety of pharmacological, physiological, and cellular processes.
  • Numerous ion channels have been identified including calcium, sodium, and potassium channels, each of which have been investigated to determine their roles in vertebrate and insect cells. Because of their involvement in maintaining normal cellular homeostasis, much attention has been given to potassium channels. A number of these potassium channels open in response to changes in the cell membrane potential.
  • Many voltage-gated potassium channels have been identified and characterized by their electrophysiological and pharmacological properties.
  • Potassium currents are more diverse than sodium or calcium currents and are further involved in determirring the response of a cell to external stimuli.
  • the diversity of potassium channels and their important physiological role highlights their potential as targets for developing therapeutic agents for various diseases.
  • One of the best characterized classes of potassium channels are the voltage-gated potassium channels.
  • the prototypical member of this class is the protein encoded by the Shaker gene in Drosophila melanogaster.
  • Proteins of the Shal or Kv4 family are a type of voltage-gated potassium channels that underlies many of the native A type currents that have been recorded from different primary cells.
  • Kv4 channels have a major role in the repolarization of cardiac action potentials. In neurons, Kv4 channels and the A currents they may comprise play an important role in modulation of firing rate, action potential initiation and in controlling dendritic responses to synaptic inputs.
  • the fundamental function of a neuron is to receive, conduct, and transmit signals. Despite the varied purpose of the signals carried by different classes of neurons, the form of the signal is always the same and consists of changes in the electrical potential across the plasma membrane of the neuron.
  • the plasma membrane of a neuron contains voltage-gated cation channels, which are responsible for propagating this electrical potential (also referred to as an action potential or nerve impulse) across and along the plasma membrane.
  • the Kv family of channels includes, among others: (1) the delayed-rectifier potassium channels, which repolarize the membrane after each action potential to prepare the cell to fire again; and (2) the rapidly inactivating (A-type) potassium channels, which are active predominantly at subthreshold voltages and and act to reduce the rate at which excitable cells reach firing threshold.
  • Kv channels also control the response to depolarizing, e.g., synaptic, inputs and play a role in neurotransmitter release.
  • voltage-gated potassium channels are key regulators of neuronal excitability (Hille B., Ionic Channels of Excitable Membranes, Second Edition, Sunderland, MA: Sinauer, (1992)).
  • Kv potassium channel superfamily There is tremendous structural and functional diversity within the Kv potassium channel superfamily. This diversity is generated both by the existence of multiple genes and by alternative splicing of RNA transcripts produced from the same gene. Nonetheless, the amino acid sequences of the known Kv potassium channels show high similarity. All appear to be comprised of four, pore forming ⁇ -subunits and some are known to have four cytoplasmic ( ⁇ -subunit) polypeptides (Jan L.Y. et al. (1990) Trends Neurosci 13:415-419, and Pongs, O. et al. (1995) Sem Neurosci. 7:137-146).
  • Kv channel ⁇ -subunits fall into four sub-families named for their homology to channels first isolated from Drosophila: the Kvl, or S/z ⁇ /ter-related subfamily; the Kv2, or S/z ⁇ b-related subfamily; the Kv3, or S z ⁇ w-related subfamily; and the Kv4, or Shal- related subfamily.
  • Kv4.2 and Kv4.3 are examples of Kv channel ( ⁇ -subunits of the Shal-related subfamily.
  • Kv4.3 has a unique neuroanatomical distribution in that its mRNA is highly expressed in brainstem monoaminergic and forebrain cholinergic neurons, where it is involved in the release of the neurotransmitters dopamine, norepinephrine, serotonin, and acetylcholine. This channel is also highly expressed in cortical pyramidal cells and in interneurons. (Serdio P. et ⁇ l. (1996) J Neurophys 75:2174-2179). Interestingly, the Kv4.3 polypeptide is highly expressed in neurons which express the corresponding mRNA. The Kv4.3 polypeptide is expressed in the somatodendritic membranes of these cells, where it is thought to contribute to the rapidly inactivating K+ conductance.
  • Kv4.2 mRNA is widely expressed in brain, and the corresponding polypeptide also appears to be concentrated in somatodendritic membranes where it also contributes to the rapidly inactivating K + conductance (Sheng et ⁇ l. (1992) Neuron 9:271-84).
  • These somatodendritic A-type Kv channels are likely involved in processes which underlie learning and memory, such as integration of sub-threshold synaptic responses and the conductance of back-propagating action potentials (Hoffman D.A. et ⁇ l. (1991) Nature 387:869-875).
  • proteins which interact with and modulate the activity of potassium channel proteins e.g., potassium channels having a Kv4.2 or Kv4.3 subunit, provide novel molecular targets to modulate neuronal or cardiac excitability, e.g., action potential conduction, somatodendritic excitability and neurotransmitter release, in cells expressing these channels.
  • detection of genetic lesions in the gene encoding these proteins could be used to diagnose and treat central nervous system disorders such as epilepsy, spinocerebellar ataxia, anxiety, depression, age-related memory loss, migraine, obesity, Parkinsons disease or Alzheimer's disease; or cardiovascular disorders such as heart failure, hypertension, atrial fibrillation, dilated cardiomyopathy, idiopathic cardiomyopathy, or angina.
  • central nervous system disorders such as epilepsy, spinocerebellar ataxia, anxiety, depression, age-related memory loss, migraine, obesity, Parkinsons disease or Alzheimer's disease
  • cardiovascular disorders such as heart failure, hypertension, atrial fibrillation, dilated cardiomyopathy, idiopathic cardiomyopathy, or angina.
  • the present invention is based, at least in part, on the discovery of novel nucleic acid molecules which encode gene products that interact with potassium channel proteins or possess substantial homology to the gene products of the invention that interact with potassium channel proteins (paralogs).
  • Potassium channel proteins are, for example, potassium channels having a Kv4.2 or Kv4.3 subunit.
  • the nucleic acid molecules of the invention and their gene products are referred to herein as "Potassium Channel Interacting Proteins", "PCIP”, or "KChlP" nucleic acid and protein molecules.
  • the PCIP proteins of the present invention interact with, e.g., bind to a potassium channel protein, modulate the activity of a potassium channel protein, and/or modulate a potassium channel mediated activity in a cell, e.g., a neuronal or cardiac cell.
  • the PCIP molecules of the present invention are useful as modulating agents to regulate a variety of cellular processes, e.g., neuronal or cardiac cell processes. Accordingly, in one aspect, this invention provides isolated nucleic acid molecules encoding PCIP proteins or biologically active portions- thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection of PCIP-encoding nucleic acids.
  • a PCIP nucleic acid molecule of the invention is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more identical to the nucleotide sequence (e.g., to the entire length of the nucleotide sequence) shown in SEQ ID NO:l, SEQ ID NO:3 SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:l 1, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:
  • the isolated nucleic acid molecule includes the nucleotide sequence shown SEQ ID NO:l, SEQ ID NO:3 SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:l 1, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:
  • the nucleic acid molecule includes a fragment of at least 300, 350, 400, 426, 471, or 583 nucleotides of the nucleotide sequence of SEQ ID NO:l, SEQ ID NO:3 SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:l 1, SEQ ID NO: 13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:
  • a PCIP nucleic acid molecule includes a nucleotide sequence encoding a protein having an amino acid sequence sufficiently identical to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:
  • a PCIP nucleic acid molecule includes a nucleotide sequence encoding a protein having an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more identical to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO.10, SEQ ID NO:12, SEQ ID NO-14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:70, SEQ ID NO:40
  • an isolated nucleic acid molecule encodes the amino acid sequence of lv, 9q, ⁇ l9, W28559, KChIP4a, KChIP4b, 33b07, lp, and rat 7s protein.
  • the nucleic acid molecule includes a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO-10, SEQ ID NO:12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:49, SEQ ID NO-51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:
  • nucleic acid molecule is at least 426, 471, or 583 nucleotides in length and encodes a protein having a PCIP activity (as described herein).
  • nucleic acid molecules preferably PCIP nucleic acid molecules, which specifically detect PCIP nucleic acid molecules relative to nucleic acid molecules encoding non-PCIP proteins.
  • a nucleic acid molecule is at least 426, 400-450, 471, 450-500, 500- 550, 583, 550-600, 600-650, 650-700, 700-750, 750-800 or more nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NO:l, SEQ ID NO:3 SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:ll, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:
  • nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number 98936, 98937, 98938, 98939, 98940,
  • the nucleic acid molecules are at least 15 (e.g., contiguous) nucleotides in length and hybridize under stringent conditions to nucleotides 93-126, 360-462, 732-825, 1028- 1054, or 1517-1534 of SEQ ID NO:7.
  • the nucleic acid molecules comprise nucleotides 93-126, 360-462, 732-825, 1028-1054, or 1517-1534 of SEQ ID NO.-7.
  • the nucleic acid molecules are at least 15 (e.g., contiguous) nucleotides in length and hybridize under stringent conditions to nucleotides 1-14, 49-116, 137-311, 345-410, 430-482, 503-518, 662-693, 1406-1421, 1441-1457, 1478-1494, or 1882-1959 of SEQ ID NO: 13.
  • the nucleic acid molecules comprise nucleotides 1-14, 49-116, 137-311, 345-410, 430-482, 503-518, 662-693, 1406-1421, 1441-1457, 1478-1494, or 1882-1959 of SEQ ID NO:13.
  • the nucleic acid molecules are at least 15 (e.g., contiguous) nucleotides in length and hybridize under stringent conditions to nucleotides 932-1527, 1548-1765, 1786-1871, 1908-2091, 2259-2265, or 2630-2654 of SEQ ID NO:35.
  • the nucleic acid molecules comprise nucleotides 932-1527, 1548-1765, 1786-1871, 1908-2091, 2259-2265, or 2630-2654 of SEQ ID NO.35.
  • the nucleic acid molecule encodes a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:
  • Another embodiment of the invention provides an isolated nucleic acid molecule which is antisense to a PCIP nucleic acid molecule, e.g., the coding strand of a PCIP nucleic acid molecule.
  • Another aspect of the invention provides a vector comprising a PCIP nucleic acid molecule.
  • the vector is a recombinant expression vector.
  • the invention provides a host cell containing a vector of the invention.
  • the invention also provides a method for producing a protein, preferably a PCIP protein, by culturing in a suitable medium, a host cell, e.g. , a mammalian host cell such as a non-human mammalian cell, of the invention containing a recombinant expression vector, such that the protein is produced.
  • the isolated protein, preferably a PCIP protein includes at least one calcium binding domain.
  • the protein, preferably a PCIP protein includes at least one calcium binding domain and has an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more identical to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:49, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, S
  • the protein preferably a PCIP protein, includes at least one calcium binding domain and modulates a potassium channel mediated activity.
  • the protein preferably a PCIP protein, includes at least one calcium binding domain and is encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:l, SEQ ID NO:3 SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:l l, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:
  • the invention features fragments of the proteins having the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:
  • the protein preferably a PCIP protein, has the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:
  • PCIP protein which is encoded by a nucleic acid molecule having a nucleotide sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more identical to a nucleotide sequence of SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:l 1, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56,
  • the proteins of the present invention or biologically active portions thereof can be operatively linked to a non-PCIP polypeptide (e.g., heterologous amino acid sequences) to form fusion proteins.
  • the invention further features antibodies, such as monoclonal or polyclonal antibodies, that specifically bind proteins of the invention, preferably PCIP proteins.
  • the PCIP proteins or biologically active portions thereof can be incorporated into pharmaceutical compositions, which optionally include pharmaceutically acceptable carriers.
  • the present invention provides a method for detecting the presence of a PCIP nucleic acid molecule, protein or polypeptide in a biological sample by contacting the biological sample with an agent capable of detecting a PCIP nucleic acid molecule, protein or polypeptide such that the presence of a PCIP nucleic acid molecule, protein or polypeptide is detected in the biological sample.
  • the present invention provides a method for detecting the presence of PCIP activity in a biological sample by contacting the biological sample with an agent capable of detecting an indicator of PCIP activity such that the presence of PCIP activity is detected in the biological sample.
  • the invention provides a method for modulating PCIP activity comprising contacting a cell capable of expressing PCIP with an agent that modulates PCIP activity such that PCIP activity in the cell is modulated.
  • the agent inhibits PCIP activity.
  • the agent stimulates PCIP activity.
  • the agent is an antibody that specifically binds to a PCIP protein.
  • the agent modulates expression of PCIP by modulating transcription of a PCIP gene or translation of a PCIP mRNA.
  • the agent is a nucleic acid molecule having a nucleotide sequence that is antisense to the coding strand of a PCIP mRNA or a PCIP gene.
  • the methods of the present invention are used to treat a subject having a disorder characterized by aberrant PCIP protein or nucleic acid expression or activity by administering an agent which is a PCIP modulator to the subject.
  • the PCIP modulator is a PCIP protein.
  • the PCIP modulator is a PCIP nucleic acid molecule.
  • the PCIP modulator is a peptide, peptidomimetic, or other small molecule.
  • the disorder characterized by aberrant PCIP protein or nucleic acid expression is a CNS disorder or a cardiovascular disorder.
  • the present invention also provides a diagnostic assay for identifying the presence or absence of a genetic alteration characterized by at least one of (i) aberrant modification or mutation of a gene encoding a PCIP protein; (ii) mis-regulation of the gene; and (iii) aberrant post-translational modification of a PCIP protein, wherein a wild-type form of the gene encodes a protein with a PCIP activity.
  • the invention provides a method for identifying a compound that binds to or modulates the activity of a PCIP protein, by providing an indicator composition comprising a PCIP protein having PCIP activity, contacting the indicator composition with a test compound, and determining the effect of the test compound on PCIP activity in the indicator composition to identify a compound that modulates the activity of a PCIP protein.
  • Figure 1 depicts the cDNA sequence and predicted amino acid sequence of human lv.
  • the nucleotide sequence corresponds to nucleic acids 1 to 1463 of SEQ ID NO:l .
  • the amino acid sequence corresponds to amino acids 1 to 216 of SEQ ID NO:2.
  • Figure 2 depicts the cDNA sequence and predicted amino acid sequence of rat lv.
  • the nucleotide sequence corresponds to nucleic acids 1 to 1856 of SEQ ID NO:3.
  • the amino acid sequence corresponds to amino acids 1 to 245 of SEQ ID NO:4.
  • Figure 3 depicts the cDNA sequence and predicted amino acid sequence of mouse lv.
  • the nucleotide sequence corresponds to nucleic acids 1 to 1907 of SEQ ID NO:5.
  • the amino acid sequence corresponds to amino acids 1 to 216 of SEQ ID NO:6.
  • Figure 4 depicts the cDNA sequence and predicted amino acid sequence of rat lvl.
  • the nucleotide sequence corresponds to nucleic acids 1 to 1534 of SEQ ID NO:7.
  • the amino acid sequence corresponds to amino acids 1 to 227 of SEQ ID NO:8.
  • Figure 5 depicts the cDNA sequence and predicted amino acid sequence of mouse lvl.
  • the nucleotide sequence corresponds to nucleic acids 1 to 1540 of SEQ ID NO: 9.
  • the amino acid sequence corresponds to amino acids 1 to 227 of SEQ ID NO: 10.
  • Figure 6 depicts the cDNA sequence and predicted amino acid sequence of the partial rat lvn.
  • the nucleotide sequence corresponds to nucleic acids 1 to 955 of SEQ ID NO:l 1.
  • the amino acid sequence corresponds to amino acids 1 to 203 of SEQ ID NO: 12. (The full length rat lvn sequences are set forth herein in Figure 63, see below).
  • Figure 7 depicts the cDNA sequence and predicted amino acid sequence of human 9ql.
  • the nucleotide sequence corresponds to nucleic acids 1 to 2009 of SEQ ID NO: 13.
  • the amino acid sequence corresponds to amino acids 1 to 270 of SEQ ID NO: 14.
  • Figure 8 depicts the cDNA sequence and predicted
  • the nucleotide sequence corresponds to nucleic acids 1 to 1247 of SEQ ID NO: 15.
  • the amino acid sequence corresponds to amino acids 1 to 257 of SEQ ID NO: 16.
  • Figure 9 depicts the cDNA sequence and predicted amino acid sequence of mouse 9ql.
  • the nucleotide sequence corresponds to nucleic acids 1 to 2343 of SEQ ID NO:17.
  • the amino acid sequence corresponds to amino acids 1 to 270 of SEQ ID NO:18.
  • Figure 10 depicts the cDNA sequence and predicted amino acid sequence of human 9qm.
  • the nucleotide sequence corresponds to nucleic acids 1 to 1955 of SEQ ID NO: 19.
  • the amino acid sequence corresponds to amino acids 1 to 252 of SEQ ID NO:20.
  • Figure 11 depicts the cDNA sequence and predicted amino acid sequence of rat 9qm.
  • the nucleotide sequence corresponds to nucleic acids 1 to 2300 of SEQ ID NO:21.
  • the amino acid sequence corresponds to amino acids 1 to 252 of SEQ ID NO:22.
  • Figure 12 depicts the cDNA sequence and predicted amino acid sequence of human 9qs.
  • the nucleotide sequence corresponds to nucleic acids 1 to 1859 of SEQ ID NO:23.
  • the amino acid sequence corresponds to amino acids 1 to 220 of SEQ ID NO:24.
  • Figure 13 depicts the cDNA sequence and predicted amino acid sequence of monkey 9qs.
  • the nucleotide sequence corresponds to nucleic acids 1 to 2191 of SEQ ID NO:25.
  • the amino acid sequence corresponds to amino acids 1 to 220 of SEQ ID NO:26.
  • Figure 14 depicts the cDNA sequence and predicted amino acid sequence of rat 9qc.
  • the nucleotide sequence corresponds to nucleic acids 1 to 2057 of SEQ ID NO:27.
  • the amino acid sequence corresponds to amino acids 1 to 252 of SEQ ID NO:28.
  • Figure 15 depicts the cDNA sequence and predicted amino acid sequence of rat 8t.
  • the nucleotide sequence corresponds to nucleic acids 1 to 1904 of SEQ ID NO:29.
  • the amino acid sequence corresponds to amino acids 1 to 225 of SEQ ID NO:30.
  • Figure 16 depicts the cDNA sequence and predicted amino acid sequence of human pi 9.
  • the nucleotide sequence corresponds to nucleic acids 1 to 619 of SEQ ID NO:31.
  • the amino acid sequence corresponds to amino acids 1 to 200 of SEQ ID NO:32.
  • Figure 17 depicts the cDNA sequence and predicted amino acid sequence of rat pi 9
  • the nucleotide sequence corresponds to nucleic acids 1 to 442 of SEQ ID NO:33.
  • the amino acid sequence corresponds to amino acids 1 to 109 of SEQ ID NO:34.
  • Figure 18 depicts the cDNA sequence and predicted amino acid sequence of mouse pi 9.
  • the nucleotide sequence corresponds to nucleic acids 1 to 2644 of SEQ ID NO:35.
  • the amino acid sequence corresponds to amino acids 1 to 256 of SEQ ID NO:36.
  • Figure 19 depicts the cDNA sequence and predicted amino acid sequence of human W28559.
  • the nucleotide sequence corresponds to nucleic acids 1 to 380 of SEQ ID NO:37.
  • the amino acid sequence corresponds to amino acids 1 to 126 of SEQ ID NO:38.
  • Figure 20 depicts the cDNA sequence and predicted amino acid sequence of human P193.
  • the nucleotide sequence corresponds to nucleic acids 1 to 2176 of SEQ ID NO:39.
  • the amino acid sequence corresponds to amino acids 1 to 41 of SEQ ID NO:40.
  • Figure 21 depicts a schematic representation of the rat lv, the rat 9qm, and the mouse P19 proteins, aligned to indicate the conserved domains among these proteins.
  • Figure 22 depicts the genomic DNA sequence of human 9q.
  • Figure 22 A depicts exon 1 and its flanking intron sequences (SEQ ID NO:46).
  • Figure 22B depicts exons 2- 11 and the flanking intron sequences (SEQ ID NO:47).
  • Figure 23 depicts the cDNA sequence and predicted amino acid sequence of monkey KChIP4a.
  • the nucleotide sequence corresponds to nucleic acids 1 to 2413 of SEQ ID NO:48.
  • the amino acid sequence corresponds to amino acids 1 to 233 of SEQ ID NO:49.
  • Figure 24 depicts the cDNA sequence and predicted amino acid sequence of monkey KChIP4b.
  • the nucleotide sequence corresponds to nucleic acids 1 to 1591 of SEQ ID NO:50.
  • the amino acid sequence corresponds to amino acids 1 to 233 of SEQ ID NO:51.
  • Figure 25 depicts an alignment of KChIP4a, KChIP4b, 9ql, lv, pi 9, and related human paralog (hsncspara) W28559. Amino acids identical to the consensus are shaded in black, conserved amino acids are shaded in gray.
  • Figure 26 depicts the cDNA sequence and predicted amino acid sequence of rat 33b07.
  • the nucleotide sequence corresponds to nucleic acids 1 to 2051 of SEQ ID NO:52.
  • the amino acid sequence corresponds to amino acids 1 to 407 of SEQ ID NO:53.
  • Figure 27 depicts the cDNA sequence and predicted amino acid sequence of human 33b07.
  • the nucleotide sequence corresponds to nucleic acids 1 to 4148 of SEQ ID NO: 54.
  • the amino acid sequence corresponds to amino acids 1 to 414 of SEQ ID NO:55.
  • Figure 28 depicts the cDNA sequence and predicted amino acid sequence of rat lp.
  • the nucleotide sequence corresponds to nucleic acids 1 to 2643 of SEQ ID NO:56.
  • the amino acid sequence corresponds to amino acids 1 to 267 of SEQ ID NO:57.
  • Figure 29 depicts the cDNA sequence and predicted amino acid sequence of rat 7s.
  • the nucleotide sequence corresponds to nucleic acids 1 to 2929 of SEQ ID NO:58.
  • the amino acid sequence corresponds to amino acids 1 to 270 of SEQ ID NO:59.
  • Figure 30 depicts the cDNA sequence and predicted amino acid sequence of rat 29x.
  • the nucleotide sequence corresponds to nucleic acids 1 to 1489 of SEQ ID NO:60.
  • the amino acid sequence corresponds to amino acids 1 to 351 of SEQ ID NO: 1.
  • Figure 31 depicts the cDNA sequence of rat 25r.
  • the nucleotide sequence corresponds to nucleic acids 1 to 1194 of SEQ ID NO: 62.
  • Figure 32 depicts the cDNA sequence and predicted amino acid sequence of rat 5p.
  • the nucleotide sequence corresponds to nucleic acids 1 to 600 of SEQ ID NO:63.
  • the amino acid sequence corresponds to amino acids 1 to 95 of SEQ ID NO:64.
  • Figure 33 depicts the cDNA sequence and predicted amino acid sequence of rat
  • the nucleotide sequence corresponds to nucleic acids 1 to 639 of SEQ ID NO:65.
  • the amino acid sequence corresponds to amino acids 1 to 212 of SEQ ID NO:66.
  • Figure 34 depicts the cDNA sequence and predicted amino acid sequence of rat 19r.
  • the nucleotide sequence corresponds to nucleic acids 1 to 816 of SEQ ID NO:67.
  • the amino acid sequence corresponds to amino acids 1 to 271 of SEQ ID NO:68.
  • Figure 35 depicts the cDNA sequence and predicted amino acid sequence of monkey KChIP4c.
  • the nucleotide sequence corresponds to nucleic acids 1 to 2263 of SEQ ID NO: 69.
  • the amino acid sequence corresponds to amino acids 1 to 229 of SEQ ID NO:70.
  • Figure 36 depicts the cDNA sequence and predicted amino acid sequence of monkey KChIP4d.
  • the nucleotide sequence corresponds to nucleic acids 1 to 2259 of SEQ ID NO:71.
  • the amino acid sequence corresponds to amino acids 1 to 250 of SEQ ID NO:72.
  • Figure 37 depicts an alignment of KChIP4a, KChIP4b, KChIP4c, and KChIP4d.
  • Figure 38 depicts a graph showing the current traces from CHO cells which express Kv4.2 with or without KChIP2 (9ql). Cells are voltage clamped at -80 mV and stepped from -60 mV to +50 mV for 200ms. Peak current amplitudes at the various test voltages are shown in the right panel. Figure 38 further depicts a table showing the amplitude and kinetic effects of KChIP2 (9ql) on Kv4.2. KChIP2 expression alters the peak current amplitude, inactivation and recovery from inactivation time constants, and activation V 1/2
  • Figure 39 depicts a graph showing the current traces from CHO cells which express Kv4.2 with or without KChIP3 (pl9).
  • Cells are voltage clamped at -80 mN and stepped from -60 mN to +50 mN for 200ms. Peak current amplitudes at the various test voltages are shown in the right panel.
  • Figure 39 further depicts a table showing the amplitude and kinetic effects of KChIP3 (pl9) on Kv4.2. KChIP3 causes alterations in peak current and inactivation and recovery from inactivation time constants.
  • Figure 40 depicts results from electrophysiological experiments demonstrating that coexpression of KChlPl dramatically alters the current density and kinetics of Kv4.2 channels expressed in CHO cells.
  • Figure 40A depicts current traces from a Kv4.2 transfected CHO cell. Current was evoked by depolarizing the cell sequentially from a holding potential of -80 mN to test potentials from -60 to 50 mN. Current traces are leak subtracted using a p/5 protocol. The current axis is shown at the same magnification as in (b) to emphasize the change in current amplitudes. Inset- Single current trace at 50mN at an expanded current axis to show the kinetics of current activation and inactivation.
  • Figure 40B depicts current traces as in (a), but from a cell transfected with equal amounts of D ⁇ A for Kv4.2 and KChlPl.
  • Figures 40D and 40E depict recovery from inactivation using a two pulse protocol.
  • Kv4.2 alone (D) or coexpressed with KChlPl (E) is driven into the inactivated state using a first pulse to 50 mN, then a second pulse to 50 mN is applied at varying times after the first pulse.
  • Holding potential is -80 mN before and after all pulses.
  • the time constant of recovery from inactivation is fit to a single exponential.
  • Figure 41 depicts an alignment of human KChIP family members with closely related members of the recoverin family of Ca 2+ sensing proteins.
  • HIP human hippocalcin
  • ⁇ CS1 human neuronal calcium sensor 1
  • the alignment was performed using the MegAlign program for Macintosh (version 4.00 from DNASTAR) using the Clustal method with the PAM250 residue weight table and default parameters, and shaded using BOXSHADES. Residues identical to the consensus are shaded black, conservative substitutions are shaded grey.
  • X, Y, Z and -X, -Y, -Z denote the positions of residues which are responsible for binding to the calcium ion in the EF hand.
  • Figure 42 depicts a physical map of the IOSCA region.
  • Figure 43 depicts a linkage map showing the location of h9q and known markers associating with IOSCA and epilepsy.
  • Figure 44 depicts the cDNA sequence and predicted amino acid sequence of human lvl (KChlPll).
  • the nucleotide sequence corresponds to nucleic acids 1 to 1477 of SEQ ID NO: 79.
  • the alternation of upper and lower case letters indicates the individual exons.
  • the KChlPll (KChlPl long) specific exon is the second exon in the indicated sequence.
  • the amino acid sequence corresponds to amino acids 1 to 227 of SEQ ID NO: 109.
  • Figure 45 depicts the cDNA sequence and predicted amino acid sequence of an N-terminal splice variant of human KChlPlN.
  • the nucleotide sequence corresponds to nucleic acids 1 to 1639 of SEQ ID NO:80.
  • the amino acid sequence corresponds to amino acids 1 to 232 of SEQ ID NO:81.
  • Figure 46 depicts an alignment of the N-terminal domains of the rat and human KChlPIN, indicating that this N-terminal domain is conserved between the two sequences.
  • Figure 47 depicts the genomic DNA sequence of human KCMP2 (including KChIP2 1, m, s, and N).
  • the nucleotide sequence corresponds to nucleic acids 1 to 17,803 of SEQ ID NO:74.
  • Upper case letters indicate the exons and lower case letters indicate the introns.
  • Figure 48 depicts the cDNA sequence and predicted amino acid sequence of the rat KChIP2L.
  • the nucleotide sequence corresponds to nucleic acids 1 to 1285 of SEQ ID NO:75.
  • the amino acid sequence corresponds to amino acids 1 to 270 of SEQ ID NO:76.
  • Figure 49 depicts the cDNA sequence and predicted amino acid sequence of the human 8t (KChIP2N).
  • the nucleotide sequence corresponds to nucleic acids 1 to 2076 of SEQ ID NO:77.
  • the amino acid sequence corresponds to amino acids 1 to 225 of SEQ ID NO.78.
  • Figure 50 depicts an alignment of the N-terminal domains of the rat and human KChIP2N (8t) proteins, indicating that these proteins exhibit a 96.5% identity.
  • Figure 57 depicts the cDNA sequence and predicted amino acid sequence of the full length human KChIP3.
  • the nucleotide sequence corresponds to nucleic acids 1 to 2835 of SEQ ID NO:82.
  • the amino acid sequence corresponds to amino acids 1 to 256 of SEQ ID NO: 83.
  • the alternation of upper and lower case letters indicates the individual exons.
  • Figure 52 depicts the cDNA sequence and predicted amino acid sequence of the rat KChIP3.
  • the nucleotide sequence corresponds to nucleic acids 1 to 2414 of SEQ ID NO: 84.
  • the amino acid sequence corresponds to amino acids 1 to 178 of SEQ ID NO: 84
  • Figure 53 depicts the cDNA sequence and predicted amino acid sequence of the monkey KCWP4XC (KChIP4b).
  • the nucleotide sequence corresponds to nucleic acids 1 to 1005 of SEQ ID NO:86.
  • the amino acid sequence corresponds to amino acids 1 to 127 of SEQ ID NO:87.
  • Figure 54 depicts the cDNA sequence and predicted amino acid sequence of the mouse KChIP4N2 (KChIP4c).
  • the nucleotide sequence corresponds to nucleic acids 1 to 2181 of SEQ ID NO: 88.
  • the amino acid sequence corresponds to amino acids 1 to 229 of SEQ ID NO:89.
  • Figure 55 depicts the cDNA sequence and predicted amino acid sequence of the rat KChIP4.
  • the nucleotide sequence corresponds to nucleic acids 1 to 2022 of SEQ ID NO:90.
  • the amino acid sequence corresponds to amino acids 1 to 198 of SEQ ID NO:91.
  • Figure 56 depicts the cDNA sequence and predicted amino acid sequence of the human KChIP4aS (KChIP4NlS) a shorter splice variant of KChIP4Nl.
  • the nucleotide sequence corresponds to nucleic acids 1 to 2366 of SEQ ID NO:92.
  • the amino acid sequence corresponds to amino acids 1 to 188 of SEQ ID NO: 93.
  • Figure 57 depicts the cDNA sequence and predicted amino acid sequence of the human KChIP4a (KChIP4Nl).
  • the nucleotide sequence corresponds to nucleic acids 1 to 2431 of SEQ ID NO: 94.
  • the amino acid sequence corresponds to amino acids 1 to 233 of SEQ ID NO:95.
  • Figure 58 depicts the cDNA sequence and predicted amino acid sequence of the human KChIP4c (KChIPN2).
  • the nucleotide sequence corresponds to nucleic acids 1 to 2261 of SEQ ID NO:96.
  • the amino acid sequence corresponds to amino acids 1 to 229 of SEQ ID NO:97.
  • Figure 59 depicts the cDNA sequence and predicted amino acid sequence of the human KChIP4d (KC1-IP4N3).
  • the nucleotide sequence corresponds to nucleic acids 1 to 2299 of SEQ ID NO:98.
  • the amino acid sequence corresponds to amino acids 1 to 250 of SEQ ID NO:99.
  • Figure 60 depicts the cDNA sequence and predicted amino acid sequence of the rat KChIP4Nlx, a splice variant of KChIP4Nl .
  • the nucleotide sequence corresponds to nucleic acids 1 to 2246 of SEQ ID NO: 100.
  • the amino acid sequence corresponds to amino acids 1 to 272 of SEQ ID NO: 101.
  • Figure 61 is a set of graphs depicting the competitive modulation of Kv4.3 inactivation time constant by KC1-IP4N2 and KChlPl .
  • the cRNA species injected are listed in the cRNA section with 4.3 indicating Kv4.3, 1 indicating KChlPl, and 4 indicating KCMP4.
  • the triangles above the bar graphs illustrate combination of fixed amount of KChIP4N2 or KChlPl and increasing amount of KChlPl or KChIP4N2, respectively.
  • Figure 62 depicts protein alignments indicating that the N-terminal domains of human KChlPIN and monkey KChIP4N2 are homologous and that the N-terminal domains of human/rat KChlPl and monkey KChIP4N2 are divergent.
  • Figure 63 depicts the cDNA sequence and predicted amino acid sequence of the rat KChlPIN (lvn).
  • the nucleotide sequence corresponds to nucleic acids 1 to 1856 of SEQ ID NO: 102.
  • the amino acid sequence corresponds to amino acids 1 to 232 of SEQ ID NO:103.
  • Figure 65 is a graph depicting the modulation of Kv4.3 and Kv4.3/KChIPl currents by arachidonic acid is reversible.
  • Figure 66 is a graph depicting the modulation of Kv4.3 and Kv4.3/KChIPl by fatty acids.
  • Figure 67 isa graph indicating that Arachidonic acid does not interfere with association between KChlPl and the ⁇ -terminal domain of Kv4.3.
  • A Superimposed sensograms showing that neither the association phase nor the dissociation phase of interaction between the intracellular ⁇ -terminal domain of Kv4.3 and KChlPl was qualitatively changed by 10 ⁇ M arachidonic acid in Biosensor assays.
  • B ⁇ -terminal domain of Kv4.3 and KChlPl interaction-dependent growth in selective SC-WLH medium was not altered by 10 ⁇ M of ETYA.
  • the present invention is based, at least in part, on the discovery of novel nucleic acid molecules which encode gene products that interact with potassium channel proteins or possess substantial homology to the gene products of the invention that interact with potassium channel proteins (paralogs).
  • Potassium channel proteins are, for example, potassium channels having a Kv4.2 or Kv4.3 subunit.
  • the nucleic acid molecules of the invention and their gene products are referred to herein as "Potassium Channel Interacting Proteins", "PCIP”, or "KChIP” nucleic acid and protein molecules.
  • the PCIP proteins of the present invention interact with, e.g., bind to a potassium channel protein, modulate the activity of a potassium channel protein, and/or modulate a-potassium channel mediated activity in a cell, e.g., a neuronal or cardiac cell.
  • a PCIP family when referring to the protein and nucleic acid molecules of the invention is intended to mean two or more proteins or nucleic acid molecules having a PCIP activity as defined herein.
  • Such PCIP family members can be naturally or non-naturally occurring and can be from either the same or different species.
  • a PCIP family can contain a first protein of human origin, as well as other, distinct proteins of human origin or alternatively, can contain homologues of non-human origin.
  • a PCIP activity refers to an activity exerted by a PCIP protein, polypeptide or nucleic acid molecule on a PCIP responsive cell or on a PCIP protein substrate, as determined in vivo, or in vitro, according to standard techniques.
  • a PCIP activity is a direct activity, such as an association with a PCIP- target molecule.
  • a target molecule or “binding partner” is a molecule with which a PCIP protein binds or interacts in nature, such that PCIP-mediated function is achieved.
  • a PCIP target molecule can be a non-PCIP molecule or a PCIP protein or polypeptide of the present invention.
  • a PCIP target molecule is a PCIP ligand.
  • a PCIP activity is an indirect activity, such as a cellular signaling activity mediated by interaction of the PCIP protein with a PCIP ligand. The biological activities of PCIP are described herein.
  • the PCIP proteins of the present invention can have one or more of the following activities: (1) they can interact with (e.g., bind to) a potassium channel protein or portion thereof; (2) they can regulate the phosphorylation state of a potassium channel protein or portion thereof; (3) they can associate with (e.g., bind) calcium and can, for example, act as calcium dependent kinases, e.g., phosphorylate a potassium channel or a G-protein coupled receptor in a calcium-dependent manner; (4) they can associate with (e.g., bind) calcium and can, for example, act in a calcium-dependent manner in cellular processes, e.g., act as calcium dependent transcription factors; (5) they can modulate a potassium channel mediated activity in a cell (e.g., a neuronal cell such as a sensory neuron cell or a motor neuron cell, or a cardiac cell) to, for example, beneficially affect the cell; (6) they can modulate chromatin formation in a cell, e.g.
  • a “potassium channel” includes a protein or polypeptide that is involved in receiving, conducting, and transmitting signals in an excitable cell.
  • Potassium channels are typically expressed in electrically excitable cells, e.g., neurons, cardiac, skeletal and smooth muscle, renal, endocrine, and egg cells, and can form heteromultimeric structures, e.g., composed of pore-forming and cytoplasmic subunits.
  • Examples of potassium channels include: (1) the voltage-gated potassium channels, (2) the ligand-gated potassium channels, and (3) the mechanically-gated potassium channels. For a detailed description of potassium channels, see Kandel E.R.
  • a "potassium channel mediated activity” includes an activity which involves a potassium channel, e.g. , a potassium channel in a neuronal cell or a cardiac cell, associated with receiving, conducting, and transmitting signals in, for example, the nervous system or in the heart.
  • Potassium channel mediated activities include release of neurotransmitters, e.g., dopamine or norepinephrine, from cells, e.g., neuronal or cardiac cells; modulation of resting potential of membranes, wave forms and frequencies of action potentials, and thresholds of excitation; and modulation of processes such as integration of sub-threshold synaptic responses and the conductance of back-propagating action potentials in, for example, neuronal cells or cardiac cells.
  • the PCIP proteins of the present invention modulate potassium channel mediated activities, they may be useful as novel diagnostic and therapeutic agents for potassium channel associated disorders and/or nervous system related disorders.
  • the PCIP proteins of the present invention modulate Kv4 potassium channels, e.g., potassium channels having a Kv4.2 or Kv4.3 subunit, which underlie the voltage- gated K+ current known as I t0 (transient outward current) in the mammalian heart (Kaab S. et al. (1998) Circulation 98(14):1383-93; Dixon J.E. et al. (1996) Circulation Research 79(4):659-68; Nerbonne JM (1998) Journal of Neurobiology 37(l):37-59; Barry D.M. et al. (1998) Circulation Research 83(5):560-7; Barry D.M. et al.
  • Kv4 potassium channels e.g., potassium channels having a Kv4.2 or Kv4.3 subunit
  • PCIPs of the present invention bind to and modulate potassium channels containing a Kv4.2 or Kv4.3 subunit and contain calcium binding EF-hand domains.
  • a “potassium channel associated disorder” includes a disorder, disease or condition which is characterized by a misregulation of a potassium channel mediated activity.
  • Potassium channel associated disorders can detrimentally affect conveyance of sensory impulses from the periphery to the brain and/or conductance of motor impulses from the brain to the periphery; integration of reflexes; interpretation of sensory impulses; and emotional, intellectual (e.g., learning and memory), or motor processes. Potassium channel associated disorders can further detrimentally affect electrical impulses that stimulate the cardiac muscle fibers to contract. Examples of potassium channel associated disorders include nervous system related disorders, as well as cardiovascular disorders.
  • a "nervous system related disorder” includes a disorder, disease or condition which affects the nervous system.
  • Examples of potassium channel associated disorders and nervous system related disorders include cognitive disorders, e.g., memory and learning disorders, such as amnesia, apraxia, agnosia, amnestic dysnomia, amnestic spatial disorientation, Kluver-Bucy syndrome, Alzheimer's related memory loss (Eglen R.M. (1996) Pharmacol, and Toxicol. 78(2):59-68; Perry E.K.
  • Nerv system related disorders include disorders affecting pain generation mechanisms, e.g., pain related to irritable bowel syndrome (Mitch CH. (1997) J Med. Chem. 40(4):538-46; Shannon H.E. (1997) J Pharmac. and Exp. r .erapewt c5 281(2):884-94; Bouaziz H. (1995) Anesthesia and Analgesia 80(6): 1140-4; or Guimaraes A.P. (1994) Brain Res. 647(2) :220-30) or chest pain; movement disorders (Monassi CR. (1997) Physiol. andBehav. 62(l):53-9), e.g., Parkinson's disease related movement disorders (Finn M. (1997) Pharmacol.
  • neurodegenerative disorders e.g., Alzheimer's disease, dementias related to Alzheimer's disease (such as Pick's disease), Parkinson's and other Lewy diffuse body diseases, multiple sclerosis, amyotrophic lateral sclerosis, progressive supranuclear palsy, epilepsy, spinocerebellar ataxia, epileptic syndromes, and Jakob-Creutzfieldt disease; psychiatric disorders, e.g., depression, schizophrenic disorders, Korsakoff s psychosis, ma, anxiety disorders, bipolar affective disorders, or phobic disorders; neurological disorders, e.g., migraine; spinal cord injury; stroke; and head trauma.
  • epilepsy includes a common neurological disorder caused by disturbances in the normal electrical functions of the brain. In normal brain function millions of tiny electrical charges pass from nerve cells in the brain to all parts of the body. In patients with epilepsy, this normal pattern is interrupted by sudden and unusually intense bursts of electrical energy, which may briefly affect a person's consciousness, bodily movements, or sensations. These physical changes are called epileptic seizures. There are two categories of seizures: partial seizures, which occur in one area of the brain, and generalized seizures, which affect nerve cells throughout the brain. Epilepsy may result from a brain injury before, during, or after birth; head trauma; poor nutrition; some infectious diseases; brain tumors; and some poisons. However, in many cases the cause is unknown.
  • epilepsy may be preceded by a feeling of unease or sensory discomfort called an aura, which indicates the beginning of the seizure.
  • Signs of an impending epileptic seizure which vary among patients, may include visual phenomena such as flickering lights or "sunbursts.”
  • chromosome lOq near marker D10S192: 10q22- q24 (Ottman et al. (1995) Nature Genetics 10:56-60).
  • the many forms of epilepsy include: grand mal, Jacksonian, myoclonic progressive familial, petit mal, Lennox- Gastaut syndrome, febrile seizures, psycho-motor, and temporal lobe.
  • Ataxia includes a common neurological disorder caused by disturbances in the normal electrical functions of the brain.
  • SCA1 Spinocerebellar ataxia type 1
  • HLA human major histocompatibility complex
  • SCA1 has been shown to be tightly linked to the marker D6S89 on the short arm of chromosome 6, telomeric to HLA. See, for example, L. P. W. Ranum et al., Am. J. Hum. Genet, 49, 31-41 (1991); and H. Y. Zoghbi et al., Am. J. Hum. Genet, 49, 23-30 (1991).
  • the observations described herein are particularly useful in developing treatments for infantile onset spinocerebellar ataxia (IOSCA).
  • a "cardiovascular disorder” includes a disorder affecting the cardiovascular system, e.g., the heart.
  • cardiovascular disorders include arteriosclerosis, ischemia reperfusion injury, restenosis, arterial inflammation, vascular wall remodeling, ventricular remodeling, rapid ventricular pacing, coronary microembolism, tachycardia, bradycardia, pressure overload, aortic bending, coronary artery ligation, vascular heart disease, atrial fibrilation, long-QT syndrome, congestive heart failure, sinus node disfunction, angina, heart failure, hypertension, atrial fibrillation, atrial flutter, dilated cardiomyopathy, idiopathic cardiomyopathy, myocardial infarction, coronary artery disease, coronary artery spasm, or arrhythmia.
  • the cardiovascular disorder is associated with an abnormal I t0 current.
  • PCIP family members may also have common structural characteristics, such as a common structural domain or motif or a sufficient amino acid or nucleotide sequence homology as defined herein.
  • Such PCIP family members can be naturally or non-naturally occurring and can be from either the same or different species.
  • a PCIP family can contain a first protein of human origin, as well as other, distinct proteins of human origin or alternatively, can contain homologues of non-human origin.
  • members of a PCIP family which have common structural characteristics may comprise at least one "calcium binding domain".
  • the term "calcium binding domain” includes an amino acid domain, e.g., an EF hand (Baimbridge K.G. et al. (1992) TINS 15(8): 303-308), which is involved in calcium binding.
  • a calcium binding domain has a sequence, which is substantially identical to the consensus sequence:
  • O can be I, L, N or M, and "•" indicates a position with no strongly preferred residue. Each residue listed is present in more than 25% of sequences, and those underlined are present in more than 80% of sequences.
  • Amino acid residues 116-127 and 152-163 of the monkey KChIP4a and KChIP4b proteins comprise calcium binding domains.
  • the isolated PCIP proteins of the present invention are identified based on the presence of at least one conserved carboxyl-terminal domain which includes an amino acid sequence of about 100-200 amino acid residues in length, preferably 150-200 amino acid residues in length, and more preferably 185 amino acid residues in length, and which includes three EF hands.
  • PCIP proteins of the present invention preferably contain a carboxyl-terminal domain which is at least about 70%, 71%, 74%, 15%, 16%, 80%), or more identical to the carboxyl terminal 185 amino acid residues of rat lv, rat 9q, or mouse pl9 (see Figures 21, 25, and 41).
  • the present invention provides a full length human and a partial length rat 33b07 clone and the proteins encoded by these cDNAs.
  • the present invention further provides partial length rat lp clone and the protein encoded by this cDNA.
  • the present invention provides a partial length rat 7s clone and the protein encoded by this cDNA.
  • the present invention further provides PCIP family members which represent previously identified cDNAs (29x, 25r, 5p, 7q, and 19r). These previously identified cDNAs are identified herein as PCIP family members, i.e., as molecules which have a PCIP activity, as described herein. Accordingly, the present invention provides methods for using these previously identified cD As, e.g., methods for using these cDNAs in the screening assays, the diagnostic assays, the prognostic assays, and the methods of treatment described herein.
  • PCIP molecules of the present invention were initially identified based on their ability, as determined using yeast two-hybrid assays (described in detail in
  • Example 1 to interact with the amino-terminal 180 amino acids of rat Kv4.3 subunit. Further binding studies with other potassium subunits were performed to demonstrate specificity of the PCIP for Kv4.3 and Kv4.2. In situ localization, immuno-histochemical methods, co-immunoprecipitation and patch clamping methods were then used to clearly demonstrate that the PCIPs of the present invention interact with and modulate the activity of potassium channels, particularly those comprising a 4.3 or 4.2 subunit.
  • the human, rat, and mouse cDNAs encoding the lv polypeptide are represented by SEQ ID NOs:l, 3, and 5, and shown in Figures 1, 2, and 3, respectively.
  • lv mRNA is highly expressed in neocortical and hippocampal interneurons, in the thalamic reticular nucleus and medial habenula, in basal forebrain and striatal cholinergic neurons, in the superior colliculus, and in cerebellar granule cells.
  • the lv polypeptide is highly expressed in the somata, dendrites, axons and axon terminals of cells that express lv mRNA.
  • Splice variants of the lv gene have been identified in rat and mouse and are represented by SEQ ID NOs: 7, 9, and 11 and shown in Figures 4, 5, and 6, respectively.
  • 1 v polypeptide interacts with potassium channels comprising Kv4.3 or kv4.2 subunits, but not with Kvl.l subunits.
  • the lv transcripts mRNA
  • the 8t cDNA encodes a polypeptide having a molecular weight of approximately 26 kD corresponding to SEQ ID NO:30 (see Figure 15).
  • the 8t polypeptide interacts with potassium channel comprising Kv4.3 or Kv4.2 subunits, but not with Kvl.l subunits.
  • the 8t mRNA is expressed predominantly in the heart and the brain.
  • the 8t cDNA is a splice variant of 9q.
  • Human, rat, monkey, and mouse 9q cDNA were also isolated. Splice variants include human 9ql (SEQ ID NO:13; Figure 7) rat 9ql (SEQ ID NO:15; Figure 8), mouse 9ql (SEQ ID NO:17; Figure 9), human 9qm (SEQ ID NO:19; Figure 10), rat 9qm (SEQ ID NO:21; Figure 11), human 9qs (SEQ ID NO:23; Figure 12), monkey 9qs (SEQ ID NO:25; Figure 13), and rat 9qc (SEQ ID NO:27; Figure 14).
  • the genomic DNA sequence of 9q has also be determined. Exon 1 and its flanking intron sequences (SEQ ID NO:46) are shown in Figure 22A.
  • Exons 2-11 and the flanking intron sequences are shown in Figure 22B.
  • 9q polypeptides interact with potassium channels comprising Kv4.3 or Kv4.2 subunits, but not with Kvl.l subunits.
  • the 9q proteins are expressed predominantly in the heart and the brain.
  • 9q mRNA is highly expressed in the neostriatum, hippocampal formation, neocortical pyramidal cells and interneurons, and in the thalamus, superior colliculus, and cerebellum.
  • Human, rat, and mouse PI 9 cDNA was also isolated.
  • Human P19 is shown in SEQ ID NO:31 and Figure 16; and in SEQ ID NO:39 and Figure 20 (the 3' sequence).
  • Rat P19 is shown in SEQ ID NO:33 and Figure 17, and mouse P19 is shown in SEQ ID NO:35 and Figure 18.
  • PI 9 polypeptides interact with potassium channels comprising Kv4.3 or Kv4.2 subunits, but not with Kvl .1 subunits. As determined by Northern blot analysis, the P19 transcripts (mRNA) are expressed predominantly in the brain.
  • Monkey KChIP4a and its splice variants KChIP4b, KChIP4c, and KChIP4d were also identified.
  • Monkey KChIP4a is shown in SEQ ID NO:48 and Figure 23.
  • Monkey KChIP4b is shown in SEQ ID NO:50 and Figure 24.
  • Monkey KChIP4c is shown in SEQ ID NO:69 and Figure 35.
  • Monkey KChIP4d is shown in SEQ ID NO:71 and Figure 36.
  • the nucleotide sequence of the full length rat 33b07 cDNA and the predicted amino acid sequence of the rat 33b07 polypeptide are shown in Figure 26 and in SEQ ID NOs:52 and 53, respectively.
  • the rat 33b07 cDNA encodes a protein having a molecular weight of approximately 44.7 kD and which is 407 amino acid residues in length.
  • Rat 33b07 binds rKv4.3N and rKv4.2N with slight preference for rKv4.2N in yeast 2-hybrid assays.
  • the nucleotide sequence of the full length human 33b07 cDNA and the predicted amino acid sequence of the human 33b07 polypeptide are shown in Figure 27 and in SEQ ID NOs:54 and 55, respectively.
  • the nucleotide sequence of the partial length rat lp cDNA and the predicted amino acid sequence of the rat lp polypeptide are shown in Figure 28 and in SEQ ID NOs:56 and 57, respectively.
  • the rat lp cDNA encodes a protein having a molecular weight of approximately 28.6 kD and which is 267 amino acid residues in length. Rat lp binds rKv4.3N and rKv4.2N with slight preference for rKv4.3N in yeast two-hybrid assays.
  • the nucleotide sequence of the partial length rat 7s cDNA and the predicted amino acid sequence of the rat 7s polypeptide are shown in Figure 29 and in SEQ ID NOs:58 and 59, respectively.
  • the rat 7s cDNA encodes a protein having a molecular weight of approximately 28.6 kD and which is 270 amino acid residues in length.
  • Rat 7s binds rKv4.3N and rKv4.2N with preference for rKv4.3N in yeast two-hybrid assays.
  • PCIPs were deposited with American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, NA 20110-2209, on November 17, 1998, and assigned the Accession Numbers described above. These deposits will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. These deposits were made merely as a convenience for those of skill in the art and are not an admission that a deposit is required under 35 U.S.C ⁇ 112.
  • Clones containing cDNA molecules encoding human pi 9 (clone EphP19) and human 33b07 (clone Eph33b07) were deposited with American Type Culture Collection (Manassas, NA) on July 8,1998 as Accession Number PTA-316, as part of a composite deposit representing a mixture of two strains, each carrying one recombinant plasmid harboring a particular cDNA clone.
  • the ATCC strain designation for the mixture of hP19 and h33b07 is EphP19h33b07mix).
  • an aliquot of the mixture can be streaked out to single colonies on LB plates supplemented with 100 ug/ml ampicillin, single colonies grown, and then plasmid DNA extracted using a standard minipreparation procedure.
  • a sample of the DNA minipreparation can be digested with Notl and the resultant products resolved on a 0.8% agarose gel using standard DNA electrophoresis conditions. The digest gives the following band patterns: EphP19: 7 kb 9 (single band), Eph33b07: 5.8 kb (single band).
  • nucleic acid molecules that encode PCIP proteins or biologically active portions thereof, as well as nucleic acid fragments sufficient for use as hybridization probes to identify PCIP-encoding nucleic acid molecules (e.g., PCIP mRNA) and fragments for use as PCR primers for the amplification or mutation of PCIP nucleic acid molecules.
  • nucleic acid molecule is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs.
  • the nucleic acid molecule can be single-stranded or double- stranded, but preferably is double-stranded DNA.
  • An "isolated" nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid.
  • an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
  • the isolated PCIP nucleic acid molecule can contain less than about 5 kb, 4kb, 3kb, 2kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.
  • an "isolated" nucleic acid molecule such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • a nucleic acid molecule of the present invention e.g.
  • nucleic acid molecule having the nucleotide sequence of SEQ ID NO:l, SEQ ID NO:3 SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:
  • SEQ ID NO:1 SEQ ID NO:3 SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:l 1, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:69, SEQ ID NO:71,
  • Accession Number 98936, 98937, 98938, 98939, 98940, 98941, 98942, 98943, 98944, 98945, 98946, 98947, 98948, 98949, 98950, 98951, 98991, 98993, or 98994 can be isolated by the polymerase chain reaction (PCR) using synthetic oligonucleotide primers designed based upon the sequence of SEQ ID NO:l, SEQ ID NO:3 SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:l 1, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:46,
  • a nucleic acid of the invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques.
  • the nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
  • oligonucleotides corresponding to PCIP nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
  • an isolated nucleic acid molecule of the invention comprises the nucleotide sequence shown in SEQ ID NO:l, SEQ ID NO: 3 SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:l 1, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:77, S
  • an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule which is a complement of the nucleotide sequence shown in SEQ ID NO:l, SEQ ID NO:3 SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO
  • a nucleic acid molecule which is complementary to the nucleotide sequence shown in SEQ ID NO:l, SEQ ID NO:3 SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:l l, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO.33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:77, SEQ ID
  • an isolated nucleic acid molecule of the present invention comprises a nucleotide sequence which is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more identical to the entire length of the nucleotide sequence shown in SEQ ID NO:l, SEQ ID NO:3 SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:l 1, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54,
  • nucleic acid molecule of the invention can comprise only a portion of the nucleic acid sequence of SEQ ID NO:l, SEQ ID NO:3 SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:l 1, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:77, SEQ
  • the probe/primer typically comprises substantially purified oligonucleotide.
  • the oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12 or 15, preferably about 20 or 25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75 consecutive nucleotides of a sense sequence of SEQ ID NO:l, SEQ ID NO:3 SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO-11, SEQ ID NO:13, SEQ ID NO: 15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, S
  • a nucleic acid molecule of the present invention comprises a nucleotide sequence which is 350-400, 400-450, 450-500, 500-550, 550- 600, 600-650, 650-700, 700-750, 750-800, 800-850, 850-900, 949, 950-1000, or more nucleotides in length and hybridizes under stringent hybridization conditions to a nucleic acid molecule of SEQ ID NO:l, SEQ ID NO:3 SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:l l, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:9
  • Probes based on the PCIP nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins.
  • the probe further comprises a label group attached thereto, e.g. , the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co- factor.
  • Such probes can be used as a part of a diagnostic test kit for identifying cells or tissue which misexpress a PCIP protein, such as by measuring a level of a PCIP- encoding nucleic acid in a sample of cells from a subject e.g., detecting PCIP mRNA levels or determining whether a genomic PCIP gene has been mutated or deleted.
  • a nucleic acid fragment encoding a "biologically active portion of a PCIP protein" can be prepared by isolating a portion of the nucleotide sequence of SEQ ID NO:l, SEQ ID NO:3 SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:l 1, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:69, SEQ ID NO:71, SEQ ID
  • the invention further encompasses nucleic acid molecules that differ from the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO:3 SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:l 1, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:77, SEQ ID
  • an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:
  • PCIP genes may exist among individuals within a population due to natural allelic variation.
  • gene and recombinant gene refer to nucleic acid molecules which include an open reading frame encoding a PCIP protein, preferably a mammalian PCIP protein, and can further include non-coding regulatory sequences, and introns.
  • Allelic variants of human PCIP include both functional and non-functional PCIP proteins.
  • Functional allelic variants are naturally occurring amino acid sequence variants of the human PCIP protein that maintain the ability to bind a PCIP ligand and/or modulate any of the PCIP activities described herein.
  • SEQ ID NO:2 SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89
  • Non-fonctional allelic variants are naturally occurring amino acid sequence variants of the human PCIP protein that do not have the ability to either bind a PCIP ligand and/or modulate any of the PCIP activities described herein.
  • Non-functional allelic variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO:12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO
  • Orthologues of the human PCIP protein are proteins that are isolated from non-human organisms and possess the same PCIP ligand binding and/or modulation of potassium channel mediated activities of the human PCIP protein. Orthologues of the human PCIP protein can readily be identified as comprising an amino acid sequence that is substantially identical to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:
  • nucleic acid molecules encoding other PCIP family members and, thus, which have a nucleotide sequence which differs from the PCIP sequences of SEQ ID NO:l, SEQ ID NO:3 SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO.21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:74, S
  • PCIP cDNA can be identified based on the nucleotide sequence of human PCIP.
  • nucleic acid molecules encoding PCIP proteins from different species and thus which have a nucleotide sequence which differs from the PCIP sequences of SEQ ID NO: 1 , SEQ ID NO:3 SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:l 1, SEQ ID NO: 13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:
  • Nucleic acid molecules corresponding to natural allelic variants and homologues of the PCIP cDNAs of the invention can be isolated based on their homology to the PCIP nucleic acids disclosed herein using the cDNAs disclosed herein, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.
  • an isolated nucleic acid molecule of the invention is at least 15, 20, 25, 30 or more nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:l, SEQ ID NO:3 SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:l l, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58
  • the nucleic acid is at least 30, 50, 100, 150, 200, 250, 300, 307, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 949, or 950 nucleotides in length.
  • hybridizes under stringent conditions is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60%) identical to each other typically remain hybridized to each other.
  • the conditions are such that sequences at least about 70%, more preferably at least about 80%), even more preferably at least about 85% or 90% identical to each other typically remain hybridized to each other.
  • a preferred, non-limiting example of stringent hybridization conditions includes hybridization in 4X sodium chloride/sodium citrate (SSC), at about 65-70°C (or alternatively hybridization in 4X SSC plus 50% formamide at about 42-50°C) followed by one or more washes in IX SSC, at about 65-70°C
  • a preferred, non-limiting example of highly stringent hybridization conditions includes hybridization in IX SSC, at about 65-70°C (or alternatively hybridization in IX SSC plus 50% formamide at about 42-50°C) followed by one or more washes in 0.3X SSC, at about 65-70°C
  • a preferred, non-limiting example of reduced stringency hybridization conditions includes hybridization in 4X SSC, at about 50-60°C (or alternatively hybridization in 6X SSC plus 50% formamide at about 40-45°C) followed by one or more washes in 2X SSC, at about 50-60°C Ranges intermediate to the above-recited values, e.g., at 65-70°
  • SSPE lxSSPE is 0.15M NaCl, lOmM NaH 2 PO 4 , and 1.25mM EDTA, pH 7.4
  • SSC IX SSC is 0.15M NaCl and 15mM sodium citrate
  • T m melting temperature
  • T m (°C) 81.5 + 16.6(log 10 [Na + ]) + 0.41 (%G+C) - (600/N), where N is the number of bases in the hybrid, and [Na + ] is the concentration of sodium ions in the hybridization buffer ([ a + ] for IX SSC - 0.165 M).
  • additional reagents may be added to hybridization and/or wash buffers to decrease non-specific hybridization of nucleic acid molecules to membranes, for example, nitrocellulose or nylon membranes, including but not limited to blocking agents (e.g., BSA or salmon or herring sperm carrier DNA), detergents (e.g., SDS), chelating agents (e.g., EDTA), Ficoll, PNP and the like.
  • blocking agents e.g., BSA or salmon or herring sperm carrier DNA
  • detergents e.g., SDS
  • chelating agents e.g., EDTA
  • Ficoll e.g., Ficoll, PNP and the like.
  • an additional preferred, non-limiting example of stringent hybridization conditions is hybridization in 0.25-0.5M ⁇ aH 2 PO 4 , 7% SDS at about 65°C, followed by one or more washes at 0.02M NaH 2 PO 4 , 1% SDS at 65°C (see e.g., Church and Gilbert (1984) Proc. Natl. Acad. Sci. USA 81 :1991-1995), or alternatively 0.2X SSC, 1% SDS.
  • an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID NO:l corresponds to a naturally- occurring nucleic acid molecule.
  • a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g. , encodes a natural protein).
  • nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in the sequence of SEQ ID NO:l, SEQ ID NO:3 SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:l l, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:
  • a "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of PCIP (e.g. , the sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:
  • amino acid residues that are conserved among the PCIP proteins of the present invention are predicted to be particularly unamenable to alteration.
  • additional amino acid residues that are conserved between the PCIP proteins of the present invention and other members of the PCIP family of proteins are not likely to be amenable to alteration.
  • another aspect of the invention pertains to nucleic acid molecules encoding PCIP proteins that contain changes in amino acid residues that are not essential for activity.
  • Such PCIP proteins differ in amino acid sequence from SEQ ID NO:2, SEQ ID NO.4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91
  • the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more identical to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:70, SEQ ID NO:2,
  • SEQ ID NO:l SEQ ID NO:3 SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:l 1, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO.21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:82
  • conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues.
  • a "conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • a predicted nonessential amino acid residue in a PCIP protein is preferably replaced with another amino acid residue from the same side chain family.
  • mutations can be introduced randomly along all or part of a PCIP coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for PCIP biological activity to identify mutants that retain activity.
  • SEQ ID NO:3 SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:l 1, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO:17, SEQ ID NO.T9, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:82,
  • a mutant PCIP protein can be assayed for the ability to (1) interact with (e.g., bind to) a potassium channel protein or portion thereof; (2) regulate the phosphorylation state of a potassium channel protein or portion thereof; (3) associate with (e.g., bind) calcium and, for example, act as a calcium dependent kinase, e.g., phosphorylate a potassium channel in a calcium-dependent manner; (4) associate with (e.g., bind) calcium and, for example, act as a calcium dependent transcription factor; (5) modulate a potassium channel mediated activity in a cell (e.g., a neuronal or cardiac cell) to, for example, beneficially affect the cell; (6) modulate the release of neurotransmitters; (7) modulate membrane excitability; (8) influence the resting potential of membranes; (9) modulate wave forms and frequencies of action potentials; and (10) modulate thresholds of excitation.
  • a cell e.g., a neuronal or cardiac cell
  • an antisense nucleic acid comprises a nucleotide sequence which is complementary to a "sense" nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bond to a sense nucleic acid.
  • the antisense nucleic acid can be complementary to an entire PCIP coding strand, or to only a portion thereof.
  • an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence encoding PCIP.
  • coding region refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues.
  • the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence encoding PCIP.
  • noncoding region refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids (z.e., also referred to as 5' and 3' untranslated regions).
  • antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick base pairing.
  • the antisense nucleic acid molecule can be complementary to the entire coding region of PCIP mRNA, but more preferably is an oligonucleotide which is antisense to only a portion of the coding or noncoding region of PCIP mRNA.
  • the antisense oligonucleotide can be complementary to the region surrounding the translation start site of PCIP mRNA.
  • An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.
  • an antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • an antisense nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.
  • modified nucleotides which can be used to generate the antisense nucleic acid include 5- fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4- acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2- thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D- galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5- methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5- methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'- meth
  • the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
  • the antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a PCIP protein to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation.
  • the hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix.
  • An example of a route of administration of antisense nucleic acid molecules of the invention include direct injection at a tissue site.
  • antisense nucleic acid molecules can be modified to target selected cells and then administered systemically.
  • antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens.
  • the antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.
  • the antisense nucleic acid molecule of the invention is an ⁇ -anomeric nucleic acid molecule.
  • An ⁇ -anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641).
  • the antisense nucleic acid molecule can also comprise a 2'-o- methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al.
  • an antisense nucleic acid of the invention is a ribozyme.
  • Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region.
  • ribozymes e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave PCIP mRNA transcripts to thereby inhibit translation of PCIP mRNA.
  • a ribozyme having specificity for a PCIP-encoding nucleic acid can be designed based upon the nucleotide sequence of a PCIP cDNA disclosed herein (i.e., SEQ ID NO:l, SEQ ID NO:3 SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:ll, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:69, SEQ
  • a derivative of a Tetrahymena L-19 INS R ⁇ A can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a PCIP-encoding mR ⁇ A.
  • PCIP mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J.W.
  • PCIP gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the PCIP (e.g., the PCIP promoter and/or enhancers) to form triple helical structures that prevent transcription of the PCIP gene in target cells.
  • nucleotide sequences complementary to the regulatory region of the PCIP e.g., the PCIP promoter and/or enhancers
  • the PCIP promoter and/or enhancers e.g., the PCIP promoter and/or enhancers
  • the PCIP nucleic acid molecules of the present invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule.
  • the deoxyribose phosphate backbone of the nucleic acid molecules can be modified to generate peptide nucleic acids (see Hyrup B. et al. (1996) Bioorganic & Medicinal Chemistry 4 (1): 5-23).
  • peptide nucleic acids refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained.
  • the neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength.
  • the synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup B. et al. (1996) supra; Perry-O'Keefe et al. Proc. Natl. Acad. Sci. 93: 14670-675.
  • PNAs of PCIP nucleic acid molecules can be used in therapeutic and diagnostic applications.
  • PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, for example, inducing transcription or translation arrest or inhibiting replication.
  • PNAs of PCIP nucleic acid molecules can also be used in the analysis of single base pair mutations in a gene, (e.g., by PNA- directed PCR clamping); as 'artificial restriction enzymes' when used in combination with other enzymes, (e.g., SI nucleases (Hyrup B. (1996) supra)); or as probes or primers for DNA sequencing or hybridization (Hyrup B. et al. (1996) supra; Perry- O'Keefe supra).
  • PNAs of PCIP can be modified, (e.g., to enhance their stability or cellular uptake), by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art.
  • PNA-DNA chimeras of PCIP nucleic acid molecules can be generated which may combine the advantageous properties of PNA and DNA.
  • Such chimeras allow DNA recognition enzymes, (e.g., R Ase H and DNA polymerases), to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity.
  • PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup B. (1996) supra).
  • the synthesis of PNA-DNA chimeras can be performed as described in Hyrup B. (1996) supra and Finn P.J. et al. (1996) Nucleic Acids Res. 24 (17): 3357-63.
  • a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry and modified nucleoside analogs, e.g., 5'-(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite, can be used as a between the PNA and the 5' end of DNA (Mag, M. et al. (1989) Nucleic Acid Res. 17: 5973-88). PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment (Finn P.J. et al. (1996) supra).
  • chimeric molecules can be synthesized with a 5' DNA segment and a 3' PNA segment (Peterser, K.H. et al. (1975) Bioorganic Med. Chem. Lett. 5: 1119-11124).
  • the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. US. 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCT Publication No. W088/09810) or the blood-brain barrier (see, e.g. , PCT Publication No. W089/10134).
  • peptides e.g., for targeting host cell receptors in vivo
  • agents facilitating transport across the cell membrane see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. US. 86:6553-6556; Lemaitre et al. (1987) Proc. Natl
  • oligonucleotides can be modified with hybridization- triggered cleavage agents (See, e.g., Krol et al. (1988) Bio-Techniques 6:958-976) or intercalating agents. (See, e.g., Zon (1988) Pharm. Res. 5:539-549).
  • the oligonucleotide may be conjugated to another molecule, (e.g., a peptide, hybridization triggered cross-linking agent, transport agent, or hybridization-triggered cleavage agent).
  • PCIP proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques.
  • PCIP proteins are produced by recombinant DNA techniques.
  • a PCIP protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.
  • An "isolated” or “purified” protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the PCIP protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.
  • the language “substantially free of cellular material” includes preparations of PCIP protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced.
  • the language “substantially free of cellular material” includes preparations of PCIP protein having less than about 30% (by dry weight) of non-PCIP protein (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non-PCIP protein, still more preferably less than about 10% of non-PCIP protein, and most preferably less than about 5% non-PCIP protein.
  • PCIP protein or biologically active portion thereof When the PCIP protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5%> of the volume of the protein preparation.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of PCIP protein in which the protein is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of PCIP protein having less than about 30% (by dry weight) of chemical precursors or non-PCIP chemicals, more preferably less than about 20% chemical precursors or non-PCIP chemicals, still more preferably less than about 10% chemical precursors or non-PCIP chemicals, and most preferably less than about 5% chemical precursors or non-PCIP chemicals.
  • a "biologically active portion" of a PCIP protein includes a fragment of a PCIP protein which participates in an interaction between a PCIP molecule and a non-PCIP molecule.
  • Biologically active portions of a PCIP protein include peptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of the PCIP protein, e.g., the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53
  • biologically active portions comprise a domain or motif with at least one activity of the PCIP protein, e.g., binding of a potassium channel subunit.
  • a biologically active portion of a PCIP protein can be a polypeptide which is, for example, 10, 25, 50, 100, 200, or more amino acids in length.
  • Biologically active portions of a PCIP protein can be used as targets for developing agents which modulate a potassium chamiel mediated activity.
  • a biologically active portion of a PCIP protein comprises at least one calcium binding domain.
  • a preferred biologically active portion of a PCIP protein of the present invention may contain at least one of the above-identified structural domains.
  • a more preferred biologically active portion of a PCIP protein may contain at least two of the above-identified structural domains.
  • other biologically active portions, in which other regions of the protein are deleted can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native PCIP protein.
  • the PCIP protein has an amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ
  • the PCIP protein is substantially homologous to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO
  • the PCIP protein is a protein which comprises an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more identical to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:76,
  • KChIP4a, KChIP4b, 33b07, lp, or 7s proteins have an amino acid sequence sufficiently identical to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:
  • the term "sufficiently identical" refers to a first amino acid or nucleotide sequence which contains a sufficient or minimum number of identical or equivalent (e.g., an amino acid residue which has a similar side chain) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences share common structural domains or motifs and/or a common functional activity.
  • amino acid or nucleotide sequences which share common structural domains have at least 30%, 40%), or 50% identity, preferably 60% identity, more preferably 70%-80%, and even more preferably 90-95% identity across the amino acid sequences of the domains and contain at least one and preferably two structural domains or motifs, are defined herein as sufficiently identical.
  • amino acid or nucleotide sequences which share at least 30%, 40%, or 50%, preferably 60%, more preferably 70-80%, or 90-95% identity and share a common functional activity are defined herein as sufficiently identical.
  • Preferred proteins are PCIP proteins having at least one calcium binding domain and, preferably, a PCIP activity.
  • PCIP proteins having at least one calcium binding domain
  • PCIP proteins having at least one calcium binding domain
  • sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%), and even more preferably at least 70%, 80%), or 90% of the length of the reference sequence (e.g., when aligning a second sequence to the PCIP amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:
  • amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid
  • identity is equivalent to amino acid or nucleic acid “homology”).
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J Mol. Biol.
  • the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using aNWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0 or 2.0U), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the nucleic acid and protein sequences of the present invention can further be used as a "query sequence" to perform a search against public databases to, for example, identify other family members or related sequences.
  • Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10.
  • Gapped BLAST can be utilized as described in Altschul et al, (1997) Nucleic Acids Res. 25(17):3389-3402.
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • the invention also provides PCIP chimeric or fusion proteins.
  • a PCIP "chimeric protein" or “fusion protein” comprises a PCIP polypeptide operatively linked to a non-PCIP polypeptide.
  • PCIP polypeptide refers to a polypeptide having an amino acid sequence corresponding to PCIP
  • a non-PCIP polypeptide refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to the PCIP protein, e.g., a protein which is different from the PCIP protein and which is derived from the same or a different organism.
  • the PCIP polypeptide can correspond to all or a portion of a PCIP protein.
  • a PCIP fusion protein comprises at least one biologically active portion of a PCIP protein.
  • a PCIP fusion protein comprises at least two biologically active portions of a PCIP protein.
  • the term "operatively linked" is intended to indicate that the PCIP polypeptide and the non-PCIP polypeptide are fused in-frame to each other.
  • the non-PCIP polypeptide can be fused to the N-terminus or C-terminus of the PCIP polypeptide.
  • the fusion protein is a GST-PCIP fusion protein in which the PCIP sequences are fused to the C-terminus of the GST sequences. Such fusion proteins can facilitate the purification of recombinant PCIP.
  • the fusion protein is a PCIP protein containing a heterologous signal sequence at its N-terminus.
  • expression and/or secretion of PCIP can be increased through use of a heterologous signal sequence.
  • the PCIP fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject in vivo.
  • the PCIP fusion proteins can be used to affect the bioavailability of a PCIP substrate.
  • Use of PCIP fusion proteins may be useful therapeutically for the treatment of potassium channel associated disorders such as CNS disorders, e.g., neurodegenerative disorders such as Alzheimer's disease, dementias related to Alzheimer's disease (such as Pick's disease), Parkinson's and other .Lewy diffuse body diseases, multiple sclerosis, amyotrophic lateral sclerosis, progressive supranuclear palsy, epilepsy, spinocerebellar ataxia, and Jakob-Creutzfieldt disease; psychiatric disorders, e.g., depression, schizophrenic disorders, Korsakoff s psychosis, mania, anxiety disorders, or phobic disorders; learning or memory disorders, e.g., amnesia or age-related memory loss; and neurological disorders; e
  • PCIP fusion proteins may also be useful therapeutically for the treatment of potassium channel associated disorders such as cardiovascular disorders, e.g., arteriosclerosis, ischemia reperfusion injury, restenosis, arterial inflammation, vascular wall remodeling, ventricular remodeling, rapid ventricular pacing, coronary microembolism, tachycardia, bradycardia, pressure overload, aortic bending, coronary artery ligation, vascular heart disease, atrial fibrilation or congestive heart failure.
  • cardiovascular disorders e.g., arteriosclerosis, ischemia reperfusion injury, restenosis, arterial inflammation, vascular wall remodeling, ventricular remodeling, rapid ventricular pacing, coronary microembolism, tachycardia, bradycardia, pressure overload, aortic bending, coronary artery ligation, vascular heart disease, atrial fibrilation or congestive heart failure.
  • PCIP-fusion proteins of the invention can be used as immunogens to produce anti-PCIP antibodies in a subject, to purify PCIP ligands and in screening assays to identify molecules which inhibit the interaction of PCIP with a PCIP substrate.
  • a PCIP chimeric or fusion protein of the invention is produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).
  • many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide).
  • a PCIP- encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the PCIP protein.
  • the present invention also pertains to variants of the PCIP proteins which function as either PCIP agonists (mimetics) or as PCIP antagonists.
  • Variants of the PCIP proteins can be generated by mutagenesis, e.g. , discrete point mutation or truncation of a PCIP protein.
  • An agonist of the PCIP proteins can retain substantially the same, or a subset, of the biological activities of the naturally occurring form of a PCIP protein.
  • An antagonist of a PCIP protein can inhibit one or more of the activities of the naturally occurring form of the PCIP protein by, for example, competitively modulating a potassium channel mediated activity of a PCIP protein.
  • specific biological effects can be elicited by treatment with a variant of limited function.
  • treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the protein has fewer side effects in a subject relative to treatment with the naturally occurring form of the PCIP protein.
  • variants of a PCIP protein which function as either PCIP agonists (mimetics) or as PCIP antagonists can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of a PCIP protein for PCIP protein agonist or antagonist activity.
  • a variegated library of PCIP variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library.
  • a variegated library of PCIP variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential PCIP sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of PCIP sequences therein.
  • a degenerate set of potential PCIP sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of PCIP sequences therein.
  • degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential PCIP sequences.
  • Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang, S.A. (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11 :477.
  • libraries of fragments of a PCIP protein coding sequence can be used to generate a variegated population of PCIP fragments for screening and subsequent selection of variants of a PCIP protein.
  • a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of a PCIP coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with SI nuclease, and ligating the resulting fragment library into an expression vector.
  • an expression library can be derived which encodes N-terminal, C-terminal and internal fragments of various sizes of the PCIP protein.
  • Recrusive ensemble mutagenesis (REM), a new technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify PCIP variants (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA 59:7811-7815; Delgrave et al. (1993) Protein Engineering 6(3):327-331).
  • cell based assays can be exploited to analyze a variegated
  • PCIP library For example, a library of expression vectors can be transfected into a cell line which ordinarily possesses a potassium channel mediated activity. The effect of the PCIP mutant on the potassium channel mediated activity can then be detected, e.g., by any of a number of enzymatic assays or by detecting the release of a neurotransmitter. Plasmid DNA can then be recovered from the cells which score for inhibition, or alternatively, potentiation of the potassium channel mediated activity, and the individual clones further characterized.
  • PCIP protein or a portion or fragment thereof, can be used as an immunogen to generate antibodies that bind PCIP using standard techniques for polyclonal and monoclonal antibody preparation.
  • a full-length PCIP protein can be used or, alternatively, the invention provides antigenic peptide fragments of PCIP for use as immiinogens.
  • the antigenic peptide of PCIP comprises at least 8 amino acid residues of the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID
  • the antigenic peptide comprises at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues.
  • Preferred epitopes encompassed by the antigenic peptide are regions of PCIP that are located on the surface of the protein, e.g., hydrophilic regions, as well as regions with high antigenicity.
  • a PCIP immxmogen typically is used to prepare antibodies by immunizing a suitable subject, (e.g. , rabbit, goat, mouse or other mammal) with the immunogen.
  • a suitable subject e.g. , rabbit, goat, mouse or other mammal
  • An appropriate immunogenic preparation can contain, for example, recombinantly expressed PCIP protein or a chemically synthesized PCIP polypeptide.
  • the preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent. Immunization of a suitable subject with an immunogenic PCIP preparation induces a polyclonal anti-PCIP antibody response.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds (immunoreacts with) an antigen, such as PCIP.
  • immunologically active portions of immunoglobulin molecules include F(ab) and F(ab') 2 fragments which can be generated by treating the antibody with an enzyme such as pepsin.
  • the invention provides polyclonal and monoclonal antibodies that bind PCIP.
  • monoclonal antibody or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of PCIP.
  • a monoclonal antibody composition thus typically displays a single binding affinity for a particular PCIP protein with which it immunoreacts.
  • Polyclonal anti-PCIP antibodies can be prepared as described above by immunizing a suitable subject with a PCIP immunoge ⁇ .
  • the anti-PCIP antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized PCIP.
  • ELISA enzyme linked immunosorbent assay
  • the antibody molecules directed against PCIP can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as protein A chromatography to obtain the IgG fraction.
  • antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975) Nature 256:495-497) (see also, Brown et al. (1981) J Immunol. 127:539-46; Brown et al. (1980) J. Biol. Chem .255:4980-83; Yeh et al. (1976) Proc. Natl. Acad. Sci. USA 76:2927-31; and Yeh et al. (1982) Int. J.
  • an immortal cell line typically a myeloma
  • lymphocytes typically splenocytes
  • the culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds PCIP.
  • the immortal cell line e.g., a myeloma cell line
  • the immortal cell line is derived from the same mammalian species as the lymphocytes.
  • murine hybridomas can be made by fusing lymphocytes from a mouse immunized with an immunogenic preparation of the present invention with an immortalized mouse cell line.
  • Preferred immortal cell lines are mouse myeloma cell lines that are sensitive to culture medium containing hypoxanthine, aminopterin and thymidine ("HAT medium").
  • HAT medium culture medium containing hypoxanthine, aminopterin and thymidine
  • Any of a number of myeloma cell lines can be used as a fusion partner according to standard techniques, e.g., the P3-NSl/l-Ag4-l, P3-x63-Ag8.653 or Sp2/O-Agl4 myeloma lines. These myeloma lines are available from ATCC.
  • HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol ("PEG").
  • PEG polyethylene glycol
  • Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenocytes die after several days because they are not transformed).
  • Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supernatants for antibodies that bind PCIP, e.g., using a standard ELISA assay.
  • a monoclonal anti-PCIP antibody can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with PCIP to thereby isolate immunoglobulin library members that bind PCIP.
  • Kits for generating and screening phage display libraries are commercially available (e. g. , the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01 ; and the Stratagene SurfZAPTM Phage Display Kit, Catalog No. 240612). Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, Ladner et al.
  • recombinant anti-PCIP antibodies such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention.
  • Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in Robinson et al. International Application No. PCT/US86/02269; Akira, et al. European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al. European Patent Application 173,494; Neuberger et al. PCT International Publication No. WO 86/01533; Cabilly et al.
  • An anti-PCIP antibody (e.g., monoclonal antibody) can be used to isolate PCIP by standard techniques, such as affinity chromatography or immunoprecipitation.
  • An anti-PCIP antibody can facilitate the purification of natural PCIP from cells and of recombinantly produced PCIP expressed in host cells.
  • an anti-PCIP antibody can be used to detect PCIP protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the PCIP protein.
  • Anti-PCIP antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen.
  • Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, -galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125 I, 131
  • vectors preferably expression vectors, containing a nucleic acid encoding a PCIP protein (or a portion thereof).
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • vectors e.g., non-episomal mammalian vectors
  • Other vectors are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operatively linked.
  • Such vectors are referred to herein as "expression vectors".
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and vector can be used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno- associated viruses), which serve equivalent functions.
  • the recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed.
  • "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • regulatory sequence is intended to includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like.
  • the expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fosion proteins or peptides, encoded by nucleic acids as described herein (e.g., PCIP proteins, mutant forms of PCIP proteins, fosion proteins, and the like).
  • proteins or peptides including fosion proteins or peptides, encoded by nucleic acids as described herein (e.g., PCIP proteins, mutant forms of PCIP proteins, fosion proteins, and the like).
  • the recombinant expression vectors of the invention can be designed for expression of PCIP proteins in prokaryotic or eukaryotic cells.
  • PCIP proteins can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
  • the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
  • Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein.
  • Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is introduced at the junction of the fosion moiety and the recombinant protein to enable separation of the recombinant protein from the fosion moiety subsequent to purification of the fusion protein.
  • enzymes, and their cognate recognition sequences include Factor Xa, thrombin and enterokinase.
  • Typical fosion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D.B. and Johnson, K.S.
  • GST glutathione S-transferase
  • Purified fosion proteins can be utilized in PCIP activity assays, (e.g., direct assays or competitive assays described in detail below), or to generate antibodies specific for PCIP proteins, for example.
  • a PCIP fosion protein expressed in a retroviral expression vector of the present invention can be utilized to infect bone marrow cells which are subsequently transplanted into irradiated recipients. The pathology of the subject recipient is then examined after sufficient time has passed (e.g., six (6) weeks). Examples of suitable inducible non-fosion E.
  • coli expression vectors include pTrc (Amann et al, (1988) Gene 69:301-315) and pET 1 Id (Studier et al, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60-89).
  • Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fosion promoter.
  • Target gene expression from the pET l id vector relies on transcription from a T7 gnlO-lac fosion promoter mediated by a coexpressed viral RNA polymerase (T7 gnl).
  • This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident prophage harboring a T7 gnl gene under the transcriptional control of the lacUN 5 promoter.
  • One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 119-128).
  • Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al, (1992) Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard D ⁇ A synthesis techniques.
  • the PCIP expression vector is a yeast expression vector.
  • yeast expression vectors for expression in yeast S. cerivisae include pYepSecl (Baldari, et al, (1987) Embo J. 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933- 943), pJRY88 (Schultz et al, (1987) Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego, CA), and picZ (InNitrogen Corp, San Diego, CA).
  • PCIP proteins can be expressed in insect cells using baculovirus expression vectors.
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pNL series (Lucklow and Summers (1989) Virology 170:31-39).
  • a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector.
  • mammalian expression vectors include pCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195).
  • the expression vector's control functions are often provided by viral regulatory elements.
  • commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalo virus and Simian Virus 40.
  • suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, ⁇ Y, 1989.
  • the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g. , tissue-specific regulatory elements are used to express the nucleic acid).
  • tissue-specific regulatory elements are known in the art. ⁇ on-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1 :268-277), lymphoid-specific promoters. (Calame and Eaton (1988) Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBO J.
  • promoters are also encompassed, for example the murine hox promoters (Kessel and Grass (1990) Science 249:374-379) and the ⁇ -fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).
  • the invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner which allows for expression (by transcription of the DNA molecule) of an RNA molecule which is antisense to PCIP mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue specific or cell type specific expression of antisense RNA.
  • the antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced.
  • a high efficiency regulatory region the activity of which can be determined by the cell type into which the vector is introduced.
  • host cell and "recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • a host cell can be any prokaryotic or eukaryotic cell.
  • a PCIP protein can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and transfection are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), and other laboratory manuals.
  • a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest.
  • selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexate.
  • Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding a PCIP protein or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
  • a host cell of the invention such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) a PCIP protein.
  • the invention forther provides methods for producing a PCIP protein using the host cells of the invention.
  • the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding a PCIP protein has been introduced) in a suitable medium such that a PCIP protein is produced.
  • the method further comprises isolating a PCIP protein from the medium or the host cell.
  • the host cells of the invention can also be used to produce non-human transgenic animals.
  • a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which PCIP-coding sequences have been introduced.
  • Such host cells can then be used to create non-human transgenic animals in which exogenous PCIP sequences have been introduced into their genome or homologous recombinant animals in which endogenous PCIP sequences have been altered.
  • Such animals are useful for studying the function and or activity of a PCIP and for identifying and/or evaluating modulators of PCIP activity.
  • a "transgenic animal” is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene.
  • Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, and the like.
  • a transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal.
  • a "homologous recombinant animal” is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous PCIP gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.
  • a transgenic animal of the invention can be created by introducing a PCIP- encoding nucleic acid into the male pronuclei of a fertilized oocyte, e.g. , by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal.
  • a nonhuman homologue of a human PCIP gene such as a mouse or rat PCIP gene
  • a PCIP gene homologue such as another PCIP family member, can be isolated based on hybridization to the PCIP cDNA sequences of SEQ ID NO:l, SEQ ID NO:3 SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO.21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54,
  • Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene.
  • a tissue-specific regulatory sequence(s) can be operably linked to a PCIP transgene to direct expression of a PCIP protein to particular cells.
  • transgenic founder animal can be identified based upon the presence of a PCIP transgene in its genome and/or expression of PCIP mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene encoding a PCIP protein can further be bred to other transgenic animals carrying other transgenes.
  • a vector which contains at least a portion of a PCIP gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the PCIP gene.
  • the PCIP gene can be a human gene (e.g. , the cDNA of SEQ ID NO: 1), but more preferably, is a non- human homologue of a human PCIP gene (e.g. , the cDNA of SEQ ID NO:3 or 5).
  • a mouse PCIP gene can be used to construct a homologous recombination vector suitable for altering an endogenous PCIP gene in the mouse genome.
  • the vector is designed such that, upon homologous recombination, the endogenous PCIP gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a "knock out" vector).
  • the vector can be designed such that, upon homologous recombination, the endogenous PCIP gene is mutated or otherwise altered but still encodes functional protein (e.g. , the upstream regulatory region can be altered to thereby alter the expression of the endogenous PCIP protein).
  • the altered portion of the PCIP gene is flanked at its 5' and 3' ends by additional nucleic acid sequence of the PCIP gene to allow for homologous recombination to occur between the exogenous PCIP gene carried by the vector and an endogenous PCIP gene in an embryonic stem cell.
  • the additional flanking PCIP nucleic acid sequence is of sufficient length for successful homologous recombination with the endogenous gene.
  • flanking DNA both at the 5' and 3' ends
  • are included in the vector see e.g., Thomas, K.R. and Capecchi, M. R. (1987) Cell 51:503 for a description of homologous recombination vectors).
  • the vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced PCIP gene has homologously recombined with the endogenous PCIP gene are selected (see e.g., Li, E. et al. (1992) Cell 69:915).
  • the selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras (see e.g., Bradley, A. in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E.J. Robertson, ed. (IRL, Oxford, 1987) pp. 113-152).
  • a chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term.
  • Progeny harboring the homologously recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously recombined DNA by germline transmission of the transgene.
  • Methods for constructing homologous recombination vectors and homologous recombinant animals are described further in Bradley, A.
  • transgenic non-humans animals can be produced which contain selected systems which allow for regulated expression of the transgene.
  • One example of such a system is the cre/loxP recombinase system of bacteriophage PI .
  • a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al (1991) Science 251:1351-1355. If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required.
  • Such animals can be provided through the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.
  • Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, I. et al. (1997) Nature 385:810- 813 and PCT International Publication Nos. WO 97/07668 and WO 97/07669.
  • a cell e.g., a somatic cell, from the transgenic animal can be isolated and induced to exit the growth cycle and enter G 0 phase.
  • the quiescent cell can then be fused, e.g. , through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated.
  • the recontructed oocyte is then cultured such that it develops to morula or blastocyte and then transferred to pseudopregnant female foster animal.
  • the offspring borne of this female foster animal will be a clone of the animal from which the cell, e.g., the somatic cell, is isolated.
  • compositions suitable for administration typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a fragment of a PCIP protein or an anti-PCIP antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets.
  • the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules.
  • Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed.
  • Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fosidic acid derivatives.
  • Transmucosal admimstration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • suppositories e.g., with conventional suppository bases such as cocoa butter and other glycerides
  • retention enemas for rectal delivery.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio
  • LD50/ED50 Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.
  • a therapeutically effective amount of protein or polypeptide ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight.
  • an effective dosage ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight.
  • an effective dosage ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/
  • treatment of a subject with a therapeutically effective amount of a protein, polypeptide, or antibody can include a single treatment or, preferably, can include a series of treatments.
  • a subject is treated with antibody, protein, or polypeptide in the range of between about 0.1 to 20 mg/kg body weight, one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks.
  • the effective dosage of antibody, protein, or polypeptide used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result and become apparent from the results of diagnostic assays as described herein.
  • the present invention encompasses agents which modulate expression or activity.
  • An agent may, for example, be a small molecule.
  • small molecules include, but are not limited to, peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e,.
  • heteroorganic and organometallic compounds having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds. It is understood that appropriate doses of small molecule agents depends upon a number of factors within the ken of the ordinarily skilled physician, veterinarian, or researcher.
  • the dose(s) of the small molecule will vary, for example, depending upon the identity, size, and condition of the subject or sample being treated, further depending upon the route by which the composition is to be administered, if applicable, and the effect which the practitioner desires the small molecule to have upon the nucleic acid or polypeptide of the invention.
  • Exemplary doses include milligram or microgram amounts of the small molecule per kilogram of subject or sample weight (e.g. , about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram. It is furthermore understood that appropriate doses of a small molecule depend upon the potency of the small molecule with respect to the expression or activity to be modulated. Such appropriate doses may be determined using the assays described herein.
  • a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained.
  • the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.
  • an antibody may be conjugated to a therapeutic moiety such as a cytotoxin, a therapeutic agent or a radioactive metal ion.
  • a cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.
  • Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g.
  • the drug moiety is not to be construed as limited to classical chemical therapeutic agents.
  • the drug moiety may be a protein or polypeptide possessing a desired biological activity.
  • proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, alpha.
  • -interferon beta.-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or, biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophase colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.
  • IL-1 interleukin-1
  • IL-2 interleukin-2
  • IL-6 interleukin-6
  • GM-CSF granulocyte macrophase colony stimulating factor
  • G-CSF granulocyte colony stimulating factor
  • an antibody can be conjugated to a second antibody to Form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980.
  • the nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors.
  • Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Patent 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054- 3057).
  • the pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.
  • the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
  • compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • nucleic acid molecules, proteins, protein homologues, and antibodies described herein can be used in one or more of the following methods: a) screening assays; b) predictive medicine (e.g., diagnostic assays, prognostic assays, monitoring clinical trials, and pharmacogenetics); and c) methods of treatment (e.g., therapeutic and prophylactic).
  • a PCIP protein of the invention has one or more of the following activities: (1) it interacts with (e.g., binds to) a potassium channel protein or portion thereof; (2) it regulates the phosphorylation state of a potassium channel protein or portion thereof; (3) it associates with (e.g., binds to) calcium and can, for example, act as a calcium dependent kinase, e.g., phosphorylate a potassium channel or a G-protein coupled receptor in a calcium-dependent manner; (4) it associates with (e.g., binds to) calcium and can, for example, act as a calcium dependent transcription factor; (5) it modulates a potassium channel mediated activity in a cell (e.g., a neuronal or cardiac cell) to, for example, beneficially affect the cell; (6) it modulates chromatin formation in a cell, e.g., a neuronal or cardiac cell; (7) it modulates vesicular traffic and protein transport in a cell,
  • the isolated nucleic acid molecules of the invention can be used, for example, to express PCIP protein (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect PCIP mRNA (e.g., in a biological sample) or a genetic alteration in a PCIP gene, and to modulate PCIP activity, as described further below.
  • PCIP proteins can be used to treat disorders characterized by insufficient or excessive production of a PCIP substrate or production of PCIP inhibitors.
  • PCIP proteins can be used to screen for naturally occurring PCIP substrates, to screen for drags or compounds which modulate PCIP activity, as well as to treat disorders characterized by insufficient or excessive production of PCIP protein or production of PCIP protein forms which have decreased or aberrant activity compared to PCIP wild type protein (e.g., CNS disorders such as neurodegenerative disorders, e.g., Alzheimer's disease, dementias related to Alzheimer's disease (such as Pick's disease), Parkinson's and other Lewy diffuse body diseases, multiple sclerosis, amyotrophic lateral sclerosis, progressive supranuclear palsy, epilepsy, spinocerebellar ataxia, and Jakob-Creutzfieldt disease; psychiatric disorders, e.g., depression, schizophrenic disorders, Korsakoff s psychosis, mania, anxiety disorders, bipolar affective disorders, or phobic disorders; learning or memory disorders, e.g., amnesia or age-related memory loss;
  • the invention provides a method (also referred to herein as a "screening assay") for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) which bind to PCIP proteins, have a stimulatory or inhibitory effect on, for example, PCIP expression or PCIP activity, or have a stimulatory or inhibitory effect on, for example, the expression or activity of PCIP substrate.
  • modulators i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) which bind to PCIP proteins, have a stimulatory or inhibitory effect on, for example, PCIP expression or PCIP activity, or have a stimulatory or inhibitory effect on, for example, the expression or activity of PCIP substrate.
  • the invention provides assays for screening candidate or test compounds which are substrates of a PCIP protein or polypeptide or biologically active portion thereof. In another embodiment, the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of a PCIP protein or polypeptide or biologically active portion thereof.
  • the test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the 'one-bead one-compound' library method; and synthetic library methods using affinity chromatography selection.
  • the biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, K.S. (1997) . Anti cancer Drug Des. 12:145).
  • an assay is a cell-based assay in which a cell which expresses a PCIP protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to modulate PCIP activity, e.g., binding to a potassium channel or a portion thereof, is determined. Determining the ability of the test compound to modulate PCIP activity can be accomplished by monitoring, for example, the release of a neurotransmitter, e.g., dopamine, form a cell which expresses PCIP such as a neuronal cell, e.g., a substantia nigra neuronal cell, or a cardiac cell.
  • a neurotransmitter e.g., dopamine
  • determining the ability of the test compound to modulate PCIP activity can be accomplished by monitoring, for example, the I t0 current or the release of a neurotransmitter from a cell which expresses PCIP such as a cardiac cell.
  • Currents in cells e.g., the I t0 current
  • the cell can be of mammalian origin.
  • Determining the ability of the test compound to modulate the ability of PCIP to bind to a substrate can be accomplished, for example, by coupling the PCIP substrate with a radioisotope or enzymatic label such that binding of the PCIP substrate to PCIP can be determined by detecting the labeled PCIP substrate in a complex.
  • compounds e.g., PCIP substrates
  • compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.
  • a compound e.g., PCIP substrate
  • a microphysiometer can be used to detect the interaction of a compound with PCIP without the labeling of either the compound or the PCIP. McConnell, H. M. et al. (1992) Science 257:1906-1912.
  • a compound e.g., PCIP substrate
  • a microphysiometer can be used to detect the interaction of a compound with PCIP without the labeling of either the compound or the PCIP. McConnell, H. M. et al. (1992) Science 257:1906-1912.
  • microphysiometer e.g., Cytosensor
  • LAPS light-addressable potentiometric sensor
  • an assay is a cell-based assay comprising contacting a cell expressing a PCIP target molecule (e.g., a potassium channel or a fragment thereof) with a test compound and determining the ability of the test compound to modulate (e.g. stimulate or inhibit) the activity of the PCIP target molecule. Determining the ability of the test compound to modulate the activity of a PCIP target molecule can be accomplished, for example, by determining the ability of the PCIP protein to bind to or interact with the PCIP target molecule, e.g. , a potassium channel or a fragment thereof.
  • a PCIP target molecule e.g., a potassium channel or a fragment thereof
  • Determining the ability of the PCIP protein or a biologically active fragment thereof, to bind to or interact with a PCIP target molecule can be accomplished by one of the methods described above for determining direct binding. In a preferred embodiment, determining the ability of the PCIP protein to bind to or interact with a PCIP target molecule can be accomplished by determining the activity of the target molecule.
  • the activity of the target molecule can be determined by detecting induction of a cellular second messenger of the target (i.e., intracellular Ca ⁇ "1" , diacylglycerol, IP3, and the like), detecting catalytic/enzymatic activity of the target an appropriate substrate, detecting the induction of a reporter gene (comprising a target-responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g., luciferase), or detecting a target-regulated cellular response such as the release of a neurotransmitter.
  • a cellular second messenger of the target i.e., intracellular Ca ⁇ "1" , diacylglycerol, IP3, and the like
  • detecting catalytic/enzymatic activity of the target an appropriate substrate detecting the induction of a reporter gene (comprising a target-responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g.,
  • an assay of the present invention is a cell-free assay in which a PCIP protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to bind to the PCIP protein or biologically active portion thereof is determined.
  • Preferred biologically active portions of the PCIP proteins to be used in assays of the present invention include fragments which participate in interactions with non-PCIP molecules, e.g., potassium channels or fragments thereof, or fragments with high surface probability scores. Binding of the test compound to the PCIP protein can be determined either directly or indirectly as described above.
  • the assay includes contacting the PCIP protein or biologically active portion thereof with a known compound which binds PCIP to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a PCIP protein, wherein determining the ability of the test compound to interact with a PCIP protein comprises determining the ability of the test compound to preferentially bind to PCIP or biologically active portion thereof as compared to the known compound.
  • the assay is a cell-free assay in which a PCIP protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the PCIP protein or biologically active portion thereof is determined.
  • Determining the ability of the test compound to modulate the activity of a PCIP protein can be accomplished, for example, by determining the ability of the PCIP protein to bind to a PCIP target molecule by one of the methods described above for determining direct binding. Determining the ability of the PCIP protein to bind to a PCIP target molecule can also be accomplished using a technology such as real-time Biomolecular Interaction Analysis (BIA). Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705.
  • BIOA Biomolecular Interaction Analysis
  • BIOA is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the optical phenomenon of surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.
  • SPR surface plasmon resonance
  • determining the ability of the test compound to modulate the activity of a PCIP protein can be accomplished by determining the ability of the PCIP protein to further modulate the activity of a downstream effector of a PCIP target molecule.
  • the activity of the effector molecule on an appropriate target can be determined or the binding of the effector to an appropriate target can be determined as previously described.
  • the cell-free assay involves contacting a PCIP protein or biologically active portion thereof with a known compound which binds the PCIP protein to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the PCIP protein, wherein determimng the ability of the test compound to interact with the PCIP protein comprises determining the ability of the PCIP protein to preferentially bind to or modulate the activity of a PCIP target molecule.
  • the cell-free assays of the present invention are amenable to use of both soluble and/or membrane-bound forms of isolated proteins.
  • a membrane-bound form of an isolated protein e.g., a potassium channel
  • a solubilizing agent such that the membrane-bound form of the isolated protein is maintained in solution.
  • non-ionic detergents such as n
  • binding of a test compound to a PCIP protein, or interaction of a PCIP protein with a target molecule in the presence and absence of a candidate compound can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and micro-centrifoge tubes.
  • a fosion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix.
  • glutathione-S-transferase/ PCIP fosion proteins or glutathione-S- transferase/target fosion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtitre plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or PCIP protein, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtitre plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above.
  • glutathione sepharose beads Sigma Chemical, St. Louis, MO
  • glutathione derivatized microtitre plates which are then combined with the test compound or the test compound and either the non-adsorbed target protein or PCIP protein, and the mixture incubated under conditions conducive
  • the complexes can be dissociated from the matrix, and the level of PCIP binding or activity determined using standard techniques.
  • Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention.
  • a PCIP protein or a PCIP target molecule can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated PCIP protein or target molecules can be prepared from biotin-NHS (N- hydroxy-succinimide) using techniques known in the art (e.g. , biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
  • antibodies reactive with PCIP protein or target molecules but which do not interfere with binding of the PCIP protein to its target molecule can be derivatized to the wells of the plate, and unbound target or PCIP protein trapped in the wells by antibody conjugation.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies reactive with the PCIP protein or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the PCIP protein or target molecule.
  • candidate or test compounds or agents are tested for their ability to inhibit or stimulate a PCIP molecule's ability to modulate vesicular traffic and protein transport in a cell, e.g., a neuronal or cardiac cell, using the assays described in, for example, Komada M. et al (1999) Genes E>ev.l3(l l):1475-85, and Roth M.G. et al. (1999) Chem. Phys. Lipids. 98(l-2):141-52, the contents of which are incorporated herein by reference.
  • candidate or test compounds or agents are tested for their ability to inhibit or stimulate a PCIP molecule's ability to regulate the phosphorylation state of a potassium channel protein or portion thereof, using for example, an in vitro kinase assay.
  • a PCIP target molecule e.g., an immunoprecipitated potassium channel from a cell line expressing such a molecule
  • radioactive ATP e.g., [ ⁇ - ⁇ P] ATP
  • the immunoprecipitated PCIP target molecule e.g., the potassium channel
  • the immunoprecipitated PCIP target molecule can be separated by SDS-polyacrylamide gel electrophoresis under reducing conditions, transferred to a membrane, e.g., a PNDF membrane, and autoradiographed.
  • a membrane e.g., a PNDF membrane
  • Phosphoaminoacid analysis of the phosphorylated substrate can also be performed in order to determine which residues on the PCIP substrate are phosphorylated.
  • the radiophosphorylated protein band can be excised from the SDS gel and subjected to partial acid hydrolysis.
  • the products can then be separated by one-dimensional electrophoresis and analyzed on, for example, a phosphoimager and compared to ninhydrin-stained phosphoaminoacid standards.
  • candidate or test compounds or agents are tested for their ability to inhibit or stimulate a PCIP molecule's ability to associate with (e.g., bind) calcium, using for example, the assays described in Liu L. (1999) Cell Signal 11(5):317-24 and Kawai T. et al. (1999) Oncogene 18(23):3471-80, the contents of which are incorporated herein by reference.
  • candidate or test compounds or agents are tested for their ability to inhibit or stimulate a PCIP molecule's ability to modulate chromatin formation in a cell, using for example, the assays described in Okuwaki M. et al. (1998) J Biol. Chem. 273(51):34511-8 and Miyaji-Yamaguchi M. (1999) J. Mol. Biol. 290(2): 547-557, the contents of which are incorporated herein by reference.
  • candidate or test compounds or agents are tested for their ability to inhibit or stimulate a PCIP molecule's ability to modulate cellular proliferation, using for example, the assays described in Baker F.L. et al. (1995) CellProlif 28(1):1-15, Cheviron N. et al. (1996) Cell Prolif. 29(8):437-46, Hu Z.W. et al. (1999) J Pharmacol. Exp. Ther. 290(l):28-37 and Elliott K. et al. (1999) Oncogene 18(24) :3564-73, the contents of which are incorporated herein by reference.
  • candidate or test compounds or agents are tested for their ability to inhibit or stimulate a PCIP molecule's ability to regulate the association of a potassium channel protein or portion thereof with the cellular cytoskeleton, using for example, the assays described in Gonzalez C. et al. (1998) Cell Mol. Biol. 44(7): 1117-27and Chia C.P. et al. (1998) Exp. Cell Res.244(l):340-8, the contents of which are incorporated herein by reference.
  • candidate or test compounds or agents are tested for their ability to inhibit or stimulate a PCIP molecule's ability to modulate membrane excitability, using for example, the assays described in Bar-Sagi D. et al (1985) J Biol. Chem. 260(8):4740-4 and Barker J.L. et al. (1984) Neurosci. Lett. 47(3):313-8, the contents of which are incorporated herein by reference.
  • candidate or test compounds or agents are tested for their ability to inhibit or stimulate a PCIP molecule's ability to modulate cytokine signaling in a cell, e.g., a neuronal or cardiac cell, the assays described in Nakashima Y. et al. (1999) J Bone Joint Surg. Am. 81(5):603-15, the contents of which are incorporated herein by reference.
  • modulators of PCIP expression are identified in a method wherein a cell is contacted with a candidate compound and the expression of PCIP mRNA or protein in the cell is determined.
  • the level of expression of PCIP mRNA or protein in the presence of the candidate compound is compared to the level of expression of PCIP mRNA or protein in the absence of the candidate compound.
  • the candidate compound can then be identified as a modulator of PCIP expression based on this comparison. For example, when expression of PCIP mRNA or protein is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of PCIP mRNA or protein expression. Alternatively, when expression of PCIP mRNA or protein is less
  • the candidate compound is identified as an inhibitor of PCIP mRNA or protein expression.
  • the level of PCIP mRNA or protein expression in the cells can be determined by methods described herein for detecting PCIP mRNA or protein.
  • the PCIP proteins can be used as "bait proteins" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Patent No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J Biol. Chem.
  • PCIP-binding proteins proteins which bind to or interact with PCIP
  • PCIP-binding proteins proteins which bind to or interact with PCIP
  • PCIP-binding proteins are also likely to be involved in the propagation of signals by the PCIP proteins or PCIP targets as, for example, downstream elements of a PCIP- mediated signaling pathway.
  • PCIP-binding proteins are likely to be PCIP inhibitors.
  • the two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains.
  • the assay utilizes two different DNA constructs.
  • the gene that codes for a PCIP protein is fosed to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4).
  • a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein (“prey" or "sample”) is fosed to a gene that codes for the activation domain of the known transcription factor.
  • the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the PCIP protein.
  • a reporter gene e.g., LacZ
  • This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model.
  • an agent identified as described herein e.g., a PCIP modulating agent, an antisense PCIP nucleic acid molecule, a PCIP-specific antibody, or a PCIP-binding partner
  • an agent identified as described herein can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent.
  • an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent.
  • this invention pertains to uses of novel agents identified by the above-described screening assays for treatments, e.g., treatments of a CNS disorder or a cardiovascular disorder, as described herein.
  • cDNA sequences identified herein can be used in numerous ways as polynucleotide reagents. For example, these sequences can be used to: (i) map their respective genes on a chromosome; and, thus, locate gene regions associated with genetic disease; (ii) identify an individual from a minute biological sample (tissue typing); and (iii) aid in forensic identification of a biological sample. These applications are described in the subsections below.
  • this sequence can be used to map the location of the gene on a chromosome. This process is called chromosome mapping. Accordingly, portions or fragments of the PCIP nucleotide sequences, described herein, can be used to map the location of the PCIP genes on a chromosome. The mapping of the PCIP sequences to chromosomes is an important first step in correlating these sequences with genes associated with disease.
  • PCIP genes can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp in length) from the PCIP nucleotide sequences. Computer analysis of the PCIP sequences can be used to predict primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the PCIP sequences will yield an amplified fragment.
  • Somatic cell hybrids are prepared by fusing somatic cells from different mammals (e.g. , human and mouse cells). As hybrids of human and mouse cells grow and divide, they gradually lose human chromosomes in random order, but retain the mouse chromosomes. By using media in which mouse cells cannot grow, because they lack a particular enzyme, but human cells can, the one human chromosome that contains the gene encoding the needed enzyme, will be retained. By using various media, panels of hybrid cell lines can be established. Each cell line in a panel contains either a single human chromosome or a small number of human chromosomes, and a foil set of mouse chromosomes, allowing easy mapping of individual genes to specific human chromosomes. (D'Eustachio P. et al. (1983) Science 220:919-924). Somatic cell hybrids containing only fragments of human chromosomes can also be produced by using human chromosomes with translocations and deletions.
  • mammals e.g.
  • PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular sequence to a particular chromosome. Three or more sequences can be assigned per day using a single thermal cycler. Using the PCIP nucleotide sequences to design oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes. Other mapping strategies which can similarly be used to map a PCIP sequence to its chromosome include in situ hybridization (described in Fan, Y. et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6223-27), pre-screening with labeled flow-sorted chromosomes, and pre-selection by hybridization to chromosome specific cDNA libraries.
  • Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step.
  • Chromosome spreads can be made using cells whose division has been blocked in metaphase by a chemical such as colcemid that disrupts the mitotic spindle.
  • the chromosomes can be treated briefly with trypsin, and then stained with Giemsa. A pattern of light and dark bands develops on each chromosome, so that the chromosomes can be identified individually.
  • the FISH technique can be used with a DNA sequence as short as 500 or 600 bases.
  • clones larger than 1,000 bases have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection.
  • 1,000 bases, and more preferably 2,000 bases will suffice to get good results at a reasonable amount of time.
  • Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents corresponding to noncoding regions of the genes actually are preferred for mapping purposes. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping. Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. (Such data are found, for example, in N.
  • differences in the D ⁇ A sequences between individuals affected and unaffected with a disease associated with the PCIP gene can be determined. If a mutation is observed in some or all of the affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent of the particular disease. Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that D ⁇ A sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymorphisms.
  • the PCIP sequences of the present invention can also be used to identify individuals from minute biological samples.
  • the United States military, for example, is considering the use of restriction fragment length polymorphism (RFLP) for identification of its personnel.
  • RFLP restriction fragment length polymorphism
  • an individual's genomic D ⁇ A is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identification.
  • This method does not suffer from the current limitations of "Dog Tags" which can be lost, switched, or stolen, making positive identification difficult.
  • the sequences of the present invention are useful as additional D ⁇ A markers for RFLP (described in U.S. Patent 5,272,057).
  • sequences of the present invention can be used to provide an alternative technique which determines the actual base-by-base D ⁇ A sequence of selected portions of an individual's genome.
  • the PCIP nucleotide sequences described herein can be used to prepare two PCR primers from the 5' and 3' ends of the sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it.
  • Panels of corresponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences.
  • the sequences of the present invention can be used to obtain such identification sequences from individuals and from tissue.
  • the PCIP nucleotide sequences of the invention uniquely represent portions of the human genome. Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. It is estimated that allelic variation between individual humans occurs with a frequency of about once per each 500 bases.
  • Each of the sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes.
  • Non- coding sequences can comfortably provide positive individual identification with a panel of perhaps 10 to 1,000 primers which each yield a noncoding amplified sequence of 100 bases. If predicted coding sequences are used, a more appropriate number of primers for positive individual identification would be 500-2,000.
  • a panel of reagents from PCIP nucleotide sequences described herein is used to generate a unique identification database for an individual, those same reagents can later be used to identify tissue from that individual.
  • Using the unique identification database positive identification of the individual, living or dead, can be made from extremely small tissue samples.
  • DNA-based identification techniques can also be used in forensic biology. Forensic biology is a scientific field employing genetic typing of biological evidence found at a crime scene as a means for positively identifying, for example, a perpetrator of a crime.
  • PCR technology can be used to amplify DNA sequences taken from very small biological samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, or semen found at a crime scene. The amplified sequence can then be compared to a standard, thereby allowing identification of the origin of the biological sample.
  • sequences of the present invention can be used to provide polynucleotide reagents, e.g., PCR primers, targeted to specific loci in the human genome, which can enhance the reliability of DNA-based forensic identifications by, for example, providing another "identification marker" (i.e. another DNA sequence that is unique to a particular individual).
  • an "identification marker” i.e. another DNA sequence that is unique to a particular individual.
  • actual base sequence information can be used for identification as an accurate alternative to patterns formed by restriction enzyme generated fragments.
  • Sequences targeted to noncoding regions are particularly appropriate for this use as greater numbers of polymorphisms occur in the noncoding regions, making it easier to differentiate individuals using this technique.
  • Examples of polynucleotide reagents include the PCIP nucleotide sequences or portions thereof, having a length of at least 20 bases, preferably at least 30 bases.
  • the PCIP nucleotide sequences described herein can forther be used to provide polynucleotide reagents, e.g. , labeled or labelable probes which can be used in, for example, an in situ hybridization technique, to identify a specific tissue, e.g., brain tissue. This can be very useful in cases where a forensic pathologist is presented with a tissue of unknown origin. Panels of such PCIP probes can be used to identify tissue by species and/or by organ type. In a similar fashion, these reagents, e.g. , PCIP primers or probes can be used to screen tissue culture for contamination (i.e. screen for the presence of a mixture of different types of cells in a culture).
  • the present invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, and monitoring clinical trials are used for prognostic (predictive) purposes to thereby treat an individual prophylactically. Accordingly, one aspect of the present invention relates to diagnostic assays for determining PCIP protein and/or nucleic acid expression as well as PCIP activity, in the context of a biological sample (e.g., blood, serum, cells, tissue) to thereby determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with aberrant PCIP expression or activity.
  • a biological sample e.g., blood, serum, cells, tissue
  • the invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with PCIP protein, nucleic acid expression or activity. For example, mutations in a PCIP gene can be assayed in a biological sample. Such assays can be used for prognostic or predictive purpose to thereby phophylactically treat an individual prior to the onset of a disorder characterized by or associated with PCIP protein, nucleic acid expression or activity.
  • Another aspect of the invention pertains to monitoring the influence of agents (e.g., drags, compounds) on the expression or activity of PCIP in clinical trials.
  • agents e.g., drags, compounds
  • An exemplary method for detecting the presence or absence of PCIP protein or nucleic acid in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting PCIP protein or nucleic acid (e.g. , mRNA, genomic DNA) that encodes PCIP protein such that the presence of PCIP protein or nucleic acid is detected in the biological sample.
  • a compound or an agent capable of detecting PCIP protein or nucleic acid e.g. , mRNA, genomic DNA
  • a preferred agent for detecting PCIP mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to PCIP mRNA or genomic DNA.
  • the nucleic acid probe can be, for example, a full-length PCIP nucleic acid, such as the nucleic acid of SEQ ID NO:l, SEQ ID NO:3 SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:l l, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:75, S
  • a preferred agent for detecting PCIP protein is an antibody capable of binding to PCIP protein, preferably an antibody with a detectable label.
  • Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab')2) can be used.
  • the term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled.
  • Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin.
  • biological sample is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. That is, the detection method of the invention can be used to detect PCIP mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo.
  • in vitro techniques for detection of PCIP mRNA include Northern hybridizations and in situ hybridizations.
  • In vitro techniques for detection of PCIP protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence.
  • In vitro techniques for detection of PCIP genomic DNA include Southern hybridizations.
  • in vivo techniques for detection of PCIP protein include introducing into a subject a labeled anti-PCIP antibody.
  • the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
  • the biological sample contains protein molecules from the test subject.
  • the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subject.
  • a preferred biological sample is a serum sample or cerebrospinal fluid isolated by conventional means from a subject.
  • the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting PCIP protein, mRNA, or genomic DNA, such that the presence of PCIP protein, mRNA or genomic DNA is detected in the biological sample, and comparing the presence of PCIP protein, mRNA or genomic DNA in the control sample with the presence of PCIP protein, mRNA or genomic DNA in the test sample.
  • kits for detecting the presence of PCIP in a biological sample can comprise a labeled compound or agent capable of detecting PCIP protein or mRNA in a biological sample; means for determining the amount of PCIP in the sample; and means for comparing the amount of PCIP in the sample with a standard.
  • the compound or agent can be packaged in a suitable container.
  • the kit can further comprise instructions for using the kit to detect PCIP protein or nucleic acid.
  • the diagnostic methods described herein can furthermore be utilized to identify subjects having or at risk of developing a disease or disorder associated with aberrant PCIP expression or activity.
  • the assays described herein can be utilized to identify a subject having or at risk of developing a disorder associated with a misregulation in PCIP protein activity or nucleic acid expression, such as a neurodegenerative disorder, e.g., Alzheimer's disease, dementias related to Alzheimer's disease (such as Pick's disease), Parkinson's and other Lewy diffuse body diseases, multiple sclerosis, amyotrophic lateral sclerosis, progressive supranuclear palsy, epilepsy, spinocerebellar ataxia, and Jakob-Creutzfieldt disease; a psychiatric disorder, e.g., depression, schizophrenic disorders, Korsakoff s psychosis, mania, anxiety disorders, bipolar affective disorders, or
  • the prognostic assays can be utilized to identify a subject having or at risk for developing a disorder associated with a misregulation in PCIP protein activity or nucleic acid expression, such as a potassium channel associated disorder.
  • the present invention provides a method for identifying a disease or disorder associated with aberrant PCIP expression or activity in which a test sample is obtained from a subject and PCIP protein or nucleic acid (e.g., mRNA or genomic DNA) is detected, wherein the presence of PCIP protein or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with aberrant PCIP expression or activity.
  • a test sample refers to a biological sample obtained from a subject of interest.
  • a test sample can be a biological fluid (e.g. , serum), cell sample, or tissue.
  • the prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with aberrant PCIP expression or activity.
  • an agent e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate
  • agents e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate
  • agents e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate
  • such methods can be used to determine whether a subject can be effectively treated with an agent for a CNS disorder or a cardiovascular disorder.
  • the present invention provides methods for determining whether a subject can be effectively treated with an agent for a disorder associated with aberrant PCIP expression or activity in which a test sample is obtained and PCIP protein or nucleic acid expression or activity is detected (e.g., wherein the abundance of PCIP protein or nucleic acid expression or activity is diagnostic for a subject that can be administered the agent to treat a disorder associated with aberrant PCIP expression or activity).
  • the methods of the invention can also be used to detect genetic alterations in a PCIP gene, thereby determining if a subject with the altered gene is at risk for a disorder characterized by misregulation in PCIP protein activity or nucleic acid expression, such as a CNS disorder or a cardiovascular disorder.
  • the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic alteration characterized by at least one of an alteration affecting the integrity of a gene encoding a PCIP-protein, or the mis-expression of the PCIP gene.
  • such genetic alterations can be detected by ascertaining the existence of at least one of 1) a deletion of one or more nucleotides from a PCIP gene; 2) an addition of one or more nucleotides to a PCIP gene; 3) a substitution of one or more nucleotides of a PCIP gene, 4) a chromosomal rearrangement of a PCIP gene; 5) an alteration in the level of a messenger RNA transcript of a PCIP gene, 6) aberrant modification of a PCIP gene, such as of the methylation pattern of the genomic DNA, 7) the presence of a non- wild type splicing pattern of a messenger RNA transcript of a PCIP gene, 8) a non- wild type level of a PCIP-protein, 9) allelic loss of a PCIP gene, and 10) inappropriate post- translational modification of a PCIP-protein.
  • assays known in the art which can be used for detecting alterations in a PCIP
  • detection of the alteration involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) Proc. Natl. Acad. Sci.
  • PCR polymerase chain reaction
  • LCR ligation chain reaction
  • This method can include the steps of collecting a sample of cells from a subject, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a PCIP gene under conditions such that hybridization and amplification of the PCIP-gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample.
  • nucleic acid e.g., genomic, mRNA or both
  • PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein.
  • Alternative amplification methods include: self sustained sequence replication (Guatelli, J.C et al, (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh, D.Y. et al, (1989) Proc. Natl. Acad. Sci. USA 86:1173- 1177), Q-Beta Replicase (Lizardi, P.M.
  • mutations in a PCIP gene from a sample cell can be identified by alterations in restriction enzyme cleavage patterns.
  • sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA.
  • sequence specific ribozymes see, for example, U.S. Patent No. 5,498,531 can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.
  • genetic mutations in PCIP can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, to high density arrays containing hundreds or thousands of oligonucleotides probes (Cronin, M.T. et al. (1996) Human Mutation 7: 244-255; Kozal, M.J. et al. (1996) Nature Medicine 2: 753- 759).
  • a sample and control nucleic acids e.g., DNA or RNA
  • high density arrays containing hundreds or thousands of oligonucleotides probes e.g., DNA or RNA
  • genetic mutations in PCIP can be identified in two dimensional arrays containing light-generated DNA probes as described in Cronin, M.T. et al supra.
  • a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes. This step allows the identification of point mutations. This step is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected.
  • Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.
  • any of a variety of sequencing reactions known in the art can be used to directly sequence the PCIP gene and detect mutations by comparing the sequence of the sample PCIP with the corresponding wild-type (control) sequence.
  • Examples of sequencing reactions include those based on techniques developed by Maxam and Gilbert ((1977) Proc. Natl. Acad. Sci. USA 74:560) or Sanger ((1977) Proc. Natl. Acad. Sci. USA 74:5463). It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays ((1995) Biotechniques 19:448), including sequencing by mass spectrometry (see, e.g., PCT International Publication No.
  • WO 94/16101 Cohen et al. (1996) Adv. Chromatogr. 36:127-162; and Griffin et al (1993) Appl. Biochem. Biotechnol. 38:147-159).
  • Other methods for detecting mutations in the PCIP gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science 230:1242).
  • Myers et al. (1985) Science 230:1242 Myers et al. (1985) Science 230:1242).
  • the art technique of "mismatch cleavage" starts by providing heteroduplexes of formed by hybridizing (labeled) RNA or DNA containing the wild-type PCIP sequence with potentially mutant RNA or DNA obtained from a tissue sample.
  • RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with SI nuclease to enzymatically digesting the mismatched regions.
  • either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, for example, Cotton et al. (1988) Proc. Natl Acad Sci USA 85:4397; Saleeba et al. (1992) Methods Enzymol. 217:286-295.
  • the control DNA or RNA can be labeled for detection.
  • the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called "DNA mismatch repair" enzymes) in defined systems for detecting and mapping point mutations in PCIP cDNAs obtained from samples of cells.
  • DNA mismatch repair enzymes
  • the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662).
  • a probe based on a PCIP sequence e.g., a wild-type PCIP sequence
  • a cDNA or other DNA product from a test cell(s).
  • the duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, for example, U.S. Patent No. 5,459,039.
  • alterations in electrophoretic mobility will be used to identify mutations in PCIP genes.
  • SSCP single strand conformation polymorphism
  • the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al. (1985) Nature 313:495).
  • DGGE denaturing gradient gel electrophoresis
  • DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR.
  • a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem 265:12753).
  • oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki et al. (1986) Nature 324:163); Saiki etal (1989) Proc. Natl Acad. Sci USA 86:6230).
  • Such allele specific oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.
  • Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al. (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3' end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner (1993) Tibtech 11 :238).
  • amplification may also be performed using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad. Sci USA 88:189). In such cases, ligation will occur only if there is a perfect match at the 3' end of the 5' sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.
  • the methods described herein may be performed, for example, by utilizing prepackaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving a PCIP gene.
  • any cell type or tissue in which PCIP is expressed may be utilized in the prognostic assays described herein.
  • Monitoring the influence of agents (e.g., drugs) on the expression or activity of a PCIP protein can be applied not only in basic drag screening, but also in clinical trials.
  • agents e.g., drugs
  • the effectiveness of an agent determined by a screening assay as described herein to increase PCIP gene expression, protein levels, or upregulate PCIP activity can be monitored in clinical trials of subjects exhibiting decreased PCIP gene expression, protein levels, or downregulated PCIP activity.
  • the effectiveness of an agent determined by a screening assay to decrease PCIP gene expression, protein levels, or downregulate PCIP activity can be monitored in clinical trials of subjects exhibiting increased PCIP gene expression, protein levels, or upregulated PCIP activity.
  • a PCIP gene and preferably, other genes that have been implicated in, for example, a potassium channel associated disorder can be used as a "read out" or markers of the phenotype of a particular cell.
  • genes, including PCIP that are modulated in cells by treatment with an agent (e.g., compound, drug or small molecule) which modulates PCIP activity (e.g., identified in a screening assay as described herein) can be identified.
  • an agent e.g., compound, drug or small molecule
  • cells can be isolated and RNA prepared and analyzed for the levels of expression of PCIP and other genes implicated in the potassium channel associated disorder, respectively.
  • the levels of gene expression can be quantified by northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of protein produced, by one of the methods as described herein, or by measuring the levels of activity of PCIP or other genes.
  • the gene expression pattern can serve as a marker, indicative of the physiological response of the cells to the agent. Accordingly, this response state may be determined before, and at various points during treatment of the individual with the agent.
  • the present invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g. , an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) including the steps of (i) obtaining a pre-administration sample from a subject prior to administration of the agent; (ii) detecting the level of expression of a PCIP protein, mRNA, or genomic DNA in the preadmimstration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression or activity of the PCIP protein, mRNA, or genomic DNA in the post-administration samples; (v) comparing the level of expression or activity of the PCIP protein, mRNA, or genomic DNA in the pre- administration sample with the PCIP protein, mRNA, or genomic DNA in the post administration sample or samples; and (vi) altering the administration of the agent to the subject accordingly.
  • an agent e.
  • increased administration of the agent may be desirable to increase the expression or activity of PCIP to higher levels than detected, i.e., to increase the effectiveness of the agent.
  • decreased admimstration of the agent may be desirable to decrease expression or activity of PCIP to lower levels than detected, i.e. to decrease the effectiveness of the agent.
  • PCIP expression or activity may be used as an indicator of the effectiveness of an agent, even in the absence of an observable phenotypic response.
  • the present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant PCIP expression or activity.
  • treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics.
  • “Pharmacogenomics”, as used herein, refers to the application of genomics technologies such as gene sequencing, statistical genetics, and gene expression analysis to drugs in clinical development and on the market.
  • the term refers the study of how a patient's genes determine his or her response to a drug (e.g., a patient's "drag response phenotype", or "drag response genotype”.)
  • another aspect of the invention provides methods for tailoring an individual's prophylactic or therapeutic treatment with either the PCIP molecules of the present invention or PCIP modulators according to that individual's drag response genotype.
  • Pharmacogenomics allows a clinician or physician to target prophylactic or therapeutic treatments to patients who will most benefit from the treatment and to avoid treatment of patients who will experience toxic drug-related side effects.
  • the invention provides a method for preventing in a subject, a disease or condition associated with an aberrant PCIP expression or activity, by administering to the subject a PCIP or an agent which modulates PCIP expression or at least one PCIP activity.
  • Subjects at risk for a disease which is caused or contributed to by aberrant PCIP expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein.
  • Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the PCIP aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression.
  • a PCIP, PCIP agonist or PCIP antagonist agent can be used for treating the subject. The appropriate agent can be determined based on screening assays described herein.
  • the modulatory method of the invention involves contacting a cell with a PCIP or agent that modulates one or more of the activities of PCIP protein activity associated with the cell.
  • An agent that modulates PCIP protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring target molecule of a PCIP protein (e.g., a PCIP substrate), a PCIP antibody, a PCIP agonist or antagonist, a peptidomimetic of a PCIP agonist or antagonist, or other small molecule.
  • the agent stimulates one or more PCIP activities.
  • Such stimulatory agents include active PCIP protein and a nucleic acid molecule encoding PCIP that has been introduced into the cell.
  • the agent inhibits one or more PCIP activities.
  • inhibitory agents include antisense PCIP nucleic acid molecules, anti-PCIP antibodies, and PCIP inhibitors.
  • CNS disorders such as neurodegenerative disorders, e.g., Alzheimer's disease, dementias related to Alzheimer's disease (such as Pick's disease), Parkinson's and other Lewy diffuse body diseases, multiple sclerosis, amyotrophic lateral sclerosis, progressive supranuclear palsy, epilepsy, and Jakob- Creutzfieldt disease; psychiatric disorders, e.g., depression, schizophrenic disorders, Korsakoff s psychosis, mania, anxiety disorders, bipolar affective disorders, or phobic disorders; learning or memory disorders, e.g., amnesia or age-related memory loss; neurological disorders, e.g., migraine; pain disorders, e.g., hyperalgesia or pain associated with muscoloskeletal disorders; spinal cord injury; stroke; and head trauma; or cardiovascular disorders, e.g., arteriosclerosis, ischemia reperfusion injury, restenosis, arterial inflammation, vascular wall remodeling, ventricular remodeling, rapid
  • the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g. , upregulates or downregulates) PCIP expression or activity.
  • an agent e.g., an agent identified by a screening assay described herein
  • the method involves administering a PCIP protein or nucleic acid molecule as therapy to compensate for reduced or aberrant PCIP expression or activity.
  • a preferred embodiment of the present invention involves a method for treatment of a PCIP associated disease or disorder which includes the step of administering a therapeutically effective amount of a PCIP antibody to a subject.
  • a therapeutically effective amount of antibody i.e., an effective dosage
  • treatment of a subject with a therapeutically effective amount of an antibody can include a single treatment or, preferably, can include a series of treatments.
  • a subject is treated with antibody in the range of between about 0.1 to 20 mg/kg body weight, one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks.
  • the effective dosage of antibody used for treatment may increase or decrease over the course of a particular treatment.
  • Changes in dosage may result from the results of diagnostic assays as described herein.
  • Stimulation of PCIP activity is desirable in situations in which PCIP is abnormally downregulated and/or in which increased PCIP activity is likely to have a beneficial effect.
  • stimulation of PCIP activity is desirable in situations in which a PCIP is downregulated and/or in which increased PCIP activity is likely to have a beneficial effect.
  • inhibition of PCIP activity is desirable in situations in which PCIP is abnormally upregulated and/or in which decreased PCIP activity is likely to have a beneficial effect.
  • PCIP molecules of the present invention as well as agents, or modulators which have a stimulatory or inhibitory effect on PCIP activity (e.g., PCIP gene expression) as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) potassium channel associated disorders associated with aberrant PCIP activity (e.g, CNS disorders such as neurodegenerative disorders, e.g., Alzheimer's disease, dementias related to Alzheimer's disease (such as Pick's disease), Parkinson's and other Lewy diffuse body diseases, multiple sclerosis, amyotrophic lateral sclerosis, progressive supranuclear palsy, epilepsy, spinocerebellar ataxia, and Jakob-Creutzfieldt disease; psychiatric disorders, e.g., depression, schizophrenic disorders, Korsakoff s psychosis, mania, anxiety disorders, bipolar affective disorders, or phobic disorders; learning or memory disorders, e.g., am
  • pharmacogenomics i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug
  • Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drag.
  • a physician or clinician may consider applying knowledge obtained in relevant pharmacogenomics studies in determining whether to administer a PCIP molecule or PCIP modulator as well as tailoring the dosage and/or therapeutic regimen of treatment with a PCIP molecule or PCIP modulator.
  • Pharmacogenomics deals with clinically significant hereditary variations in the response to drags due to altered drag disposition and abnormal action in affected persons. See, for example, Eichelbaum, M. et al. (1996) Clin. Exp. Pharmacol. Physiol. 23(10-11) :983-985 and Linder, M.W. et al. (1997) Clin. Chem. 43(2):254-266.
  • two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drags act on the body (altered drug action) or genetic conditions transmitted as single factors altering the way the body acts on drugs (altered drug metabolism). These pharmacogenetic conditions can occur either as rare genetic defects or as naturally-occurring polymorphisms.
  • G6PD glucose-6-phosphate dehydrogenase deficiency
  • oxidant drugs anti-malarials, sulfonamides, analgesics, nitroforans
  • a genome-wide association relies primarily on a high-resolution map of the human genome consisting of already known gene-related markers (e.g., a "bi- allelic” gene marker map which consists of 60,000-100,000 polymorphic or variable sites on the human genome, each of which has two variants.)
  • gene-related markers e.g., a "bi- allelic” gene marker map which consists of 60,000-100,000 polymorphic or variable sites on the human genome, each of which has two variants.
  • Such a high-resolution genetic map can be compared to a map of the genome of each of a statistically significant number of patients taking part in a Phase II/III drag trial to identify markers associated with a particular observed drag response or side effect.
  • such a high resolution map can be generated from a combination of some ten-million known single nucleotide polymorphisms (SNPs) in the human genome.
  • SNPs single nucleotide polymorphisms
  • a "SNP" is a common alteration that occurs in a single nucleotide base in a stretch of
  • a SNP may occur once per every 1000 bases of DNA.
  • a SNP may be involved in a disease process, however, the vast majority may not be disease- associated.
  • individuals Given a genetic map based on the occurrence of such SNPs, individuals can be grouped into genetic categories depending on a particular pattern of SNPs in their individual genome. In such a manner, treatment regimens can be tailored to groups of genetically similar individuals, taking into account traits that may be common among such genetically similar individuals.
  • a method termed the "candidate gene approach” can be utilized to identify genes that predict drag response.
  • a gene that encodes a drags target e.g., a PCIP protein of the present invention
  • all common variants of that gene can be fairly easily identified in the population and it can be determined if having one version of the gene versus another is associated with a particular drag response.
  • the activity of drag metabolizing enzymes is a major determinant of both the intensity and duration of drag action.
  • drug metabolizing enzymes e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymes C YP2D6 and C YP2C 19
  • NAT 2 N-acetyltransferase 2
  • C YP2D6 and C YP2C 19 cytochrome P450 enzymes
  • These polymorphisms are expressed in two phenotypes in the population, the extensive metabolizer (EM) and poor metabolizer (PM). The prevalence of PM is different among different populations.
  • the gene coding for CYP2D6 is highly polymorphic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quite frequently experience exaggerated drug response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, PM show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-formed metabolite morphine. The other extreme are the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification.
  • a method termed the "gene expression profiling" can be utilized to identify genes that predict drug response.
  • a drag e.g., a PCIP molecule or PCIP modulator of the present invention
  • a drag can give an indication whether gene pathways related to toxicity have been turned on.
  • Information generated from more than one of the above pharmacogenomics approaches can be used to determine appropriate dosage and treatment regimens for prophylactic or therapeutic treatment an individual. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with a PCIP molecule or PCIP modulator, such as a modulator identified by one of the exemplary screening assays described herein.
  • the PCIP molecules of the invention are also useful as markers of disorders or disease states, as markers for precursors of disease states, as markers for predisposition of disease states, as markers of drag activity, or as markers of the pharmacogenomic profile of a subject.
  • the presence, absence and/or quantity of the PCIP molecules of the invention may be detected, and may be correlated with one or more biological states in vivo.
  • the PCIP molecules of the invention may serve as surrogate markers for one or more disorders or disease states or for conditions leading up to disease states.
  • a "surrogate marker” is an objective biochemical marker which correlates with the absence or presence of a disease or disorder, or with the progression of a disease or disorder (e.g., with the presence or absence of a tumor). The presence or quantity of such markers is independent of the causation of the disease. Therefore, these markers may serve to indicate whether a particular course of treatment is effective in lessening a disease state or disorder.
  • Surrogate markers are of particular use when the presence or extent of a disease state or disorder is difficult to assess through standard methodologies (e.g., early stage tumors), or when an assessment of disease progression is desired before a potentially dangerous clinical endpoint is reached (e.g., an assessment of cardiovascular disease may be made using cholesterol levels as a surrogate marker, and an analysis of HIN infection may be made using HIV R ⁇ A levels as a surrogate marker, well in advance of the undesirable clinical outcomes of myocardial infarction or folly-developed AIDS).
  • Examples of the use of surrogate markers in the art include: Koomen et al (2000) J Mass. Spectrom. 35:258-264; and James (1994) AIDS Treatment News Archive 209.
  • a "pharmacodynamic marker” is an objective biochemical marker which correlates specifically with drug effects.
  • the presence or quantity of a pharmacodynamic marker is not related to the disease state or disorder for which the drug is being administered; therefore, the presence or quantity of the marker is indicative of the presence or activity of the drag in a subject.
  • a pharmacodynamic marker may be indicative of the concentration of the drug in a biological tissue, in that the marker is either expressed or transcribed or not expressed or transcribed in that tissue in relationship to the level of the drug. In this fashion, the distribution or uptake of the drag may be monitored by the pharmacodynamic marker.
  • the presence or quantity of the pharmacodynamic marker may be related to the presence or quantity of the metabolic product of a drug, such that the presence or quantity of the marker is indicative of the relative breakdown rate of the drag in vivo.
  • Pharmacodynamic markers are of particular use in increasing the sensitivity of detection of drug effects, particularly when the drug is administered in low doses. Since even a small amount of a drag may be sufficient to activate multiple rounds of marker (e.g., a PCIP marker) transcription or expression, the amplified marker may be in a quantity which is more readily detectable than the drag itself.
  • the marker may be more easily detected due to the nature of the marker itself; for example, using the methods described herein, anti-PCIP antibodies may be employed in an immune-based detection system for a PCIP protein marker, or PCIP-specific radiolabeled probes may be used to detect a PCIP mRNA marker.
  • a pharmacodynamic marker may offer mechanism-based prediction of risk due to drug treatment beyond the range of possible direct observations. Examples of the use of pharmacodynamic markers in the art include: Matsuda et al. US 6,033,862; Hattis et al. (1991) Env. Health Perspect. 90:229-238; Schentag (1999) Am. J. Health-Syst. Pharm. 56 Suppl. 3:S21-S24; and icolau (1999) Am. J. Health-Syst. Pharm. 56 Suppl. 3:S16-S20.
  • the PCIP molecules of the invention are also useful as pharmacogenomic markers.
  • a "pharmacogenomic marker” is an objective biochemical marker which correlates with a specific clinical drug response or susceptibility in a subject (see, e.g., McLeod et al. (1999) Eur. J. Cancer 35(12):1650-1652).
  • the presence or quantity of the pharmacogenomic marker is related to the predicted response of the subject to a specific drug or class of drugs prior to administration of the drag.
  • a drug therapy which is most appropriate for the subject, or which is predicted to have a greater degree of success, may be selected.
  • RNA, or protein e.g., PCIP protein or RNA
  • a drug or course of treatment may be selected that is optimized for the treatment of the specific tumor likely to be present in the subject.
  • the presence or absence of a specific sequence mutation in PCIP DNA may correlate PCIP drug response.
  • the use of pharmacogenomic markers therefore permits the application of the most appropriate treatment for each subject without having to administer the therapy.
  • the first 180 amino acids of rKv4.3 (described in Serdio P. et al. (1996) J. Neurophys 75:2174-2179) were amplified by PCR and cloned in frame into pGBT9 resulting in plasmid pFWA2, (hereinafter "bait").
  • This bait was transformed into the two-hybrid screening strain HF7c and tested for expression and self-activation. The bait was validated for expression by Western blotting.
  • the rKv4.3 bait did not self-activate in the presence of 10 mM 3-amino-l,2,3-Triazole (3-AT). Library constraction
  • Rat mid brain tissue was provided by Wyeth-Ayerst Research (Monmouth Junction, NJ). Total cellular RNA was extracted from the tissues using standard techniques (Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1989)). mRNA was prepared using a Poly- A Spin mRNA Isolation Kit from New England Biolabs (Beverly, MA).
  • cDNA from the mRNA sample was synthesized using a cDNA Synthesis Kit from Stratagene (La Jolla, CA) and ligated into pACT2's EcoRI and Xhol sites, giving rise to a two-hybrid library.
  • Two-hybrid screens were carried out essentially as described in Bartel, P. et al. (1993) "Using the Two-Hybrid System to Detect Polypeptide-Polypeptide Interactions" in Cellular Interactions in Development: A Practical Approach, Hartley, D.A. ed. Oxford University Press, Oxford, pp. 153-179, with a bait-library pair of rkv4.3 bait-rat mid brain library.
  • a filter disk beta-galactosidase (beta-gal) assay was performed essentially as previously described (Brill et al. (1994) Mol. Biol. Cell. 5:297-312).
  • Plasmids that were positive for both reporter gene activity were scored and fish, plasmids were isolated from yeast, transformed into E. coli strain KC8, DNA plasmids were purified and the resulting plasmids were sequenced by conventional methods (Sanger F. et al. (1977) PNAS, 74: 5463-67).
  • Positive interactor clones were subjected to a binding specificity test where they were exposed to a panel of related and unrelated baits by a mating scheme previously described (Finley R.L. Jr. et al. (1994) PNAS, 91(26):12980-12984). Briefly, positive fish plasmids were transformed into Y187 and the panel of baits were transformed into HF7c. Transformed fish and bait cells were streaked out as stripes on selective medium plates, mated on YPAD plates, and tested for reporter gene activity. Analysis
  • PCIP nuleotides were analyzed for nucleic acid hits by the BLASTN 1.4.8MP program (Altschul et al. (1990) Basic Local Alignment Search Tool. J. Mol. Biol. 215: 403-410).
  • PCIP proteins were analyzed for polypeptide hits by the BLASTP 1.4.9MP program.
  • HEK 293 and CHO cells were used for recordings 1-3 days after a transient transfection.
  • Whole-cell currents were recorded from cells expressing GFP, identified by their green fluorescence.
  • Electrodes pulled from filamented borosilicate glass (Sutter Instrument Co, Novato, CA) had an initial resistance of 3-5 MOhms. After Gigaseal and raptured whole-cell configuration access, resistance was less than 10 MOhms.
  • Xenopus oocyte studies Frogs underwent no more than two surgeries and surgeries were performed by well established techniques. Frogs were anesthetized with ice. Total cRNA (1— 10 ng) was injected into stage IN Xenopus oocytes that were harvested the previous day. The Xenopus oocytes were incubated in ND96 containing (in mM) 96 NaCl, 2 KCl, 1.8 CaCl 2 , 1 MgCl 2 , 5 HEPES, pH 7.6, plus (Gentamycin 50 ⁇ g/ml) at 18°C. Xenopus oocytes were studied 3-7 days after injection.
  • the Kv4.3 gene coding sequence (coding for the first 180 amino acids) was amplified by PCR and cloned into pGBT9 creating a GAL4 DNA-binding domain- Kv4.3(l-180) gene fosion (plasmid pFWA2). HF7c was transformed with this construct. The resulting strain grew on synthetic complete medium lacking L- tryptophan but not on synthetic complete medium lacking L-tryptophan and L-histidine in the presence of lOmM 3 -AT demonstrating that the ⁇ GAL4 DNA-binding domain ⁇ - ⁇ vKv4.3(l-180) ⁇ gene fosion does not have intrinsic transcriptional activation activity higher than the threshhold allowed by lOmM 3 -AT .
  • a yeast two-hybrid assay was performed in which a plasmid containing a ⁇ GAL4 DNA-binding domain ⁇ - ⁇ rKv4.3(l-180) ⁇ gene fosion was introduced into the yeast two-hybrid screening strain HF7c described above. HF7c was then transformed with the rat mid brain two-hybrid library. Approximately six million transformants were obtained and plated in selection medium. Colonies that grew in the selection medium and expressed the beta-galactosidase reporter gene were forther characterized and subjected to retransformation and specificity assays. The retransfo ⁇ nation and specificity tests yielded three PCIP clones (rat 1 v, 8t, and 9qm) that were able to bind to the Kv4.3 polypeptide.
  • the foil length sequences for the rat lv gene, and partial sequences for 8t and 9q genes were derived as follows.
  • the partial rat PCIP sequences were used to prepare probes, which were then used to screen, for example, rat mid brain cDNA libraries. Positive clones were identified, amplified and sequenced using standard techniques, to obtain the foil length sequence. Additionally, a rapid amplification of the existing rat PCIP cDNA ends (using for example, 5' RACE, by Gibco, BRL) was used to complete the 5' end of the transcript.
  • a cDNA library made from a human hippocampus (Clontech, Palo Alto, CA) was screened under low stringency conditions as follows: Prehybridization for 4 hours at 42°C in Clontech Express Hyb solution, followed by overnight hybridization at 42°C
  • the probe used was a PCR- generated fragment including nucletides 49-711 of the rat sequence labeled with 32p dCTP.
  • the filters were washed 6 times in 2XSSC/0.1% SDS at 55°C. The same conditions were used for secondary screening of the positive isolates.
  • Clones thus obtained were sequenced using an ABI automated DNA Sequencing system, and compared to the rat sequences shown in SEQ ID NO: 3 as well as to known sequences from the GenBank database.
  • the largest clone from the library screen was subsequently subcloned into pBS-KS+ (Stratagene, La Jolla, CA) for sequence verification.
  • the 515 base pair clone was determined to represent the human homolog of the lv gene, encompasing 211 base pairs of 5' UTR and a 304 base pair coding region.
  • 3' RACE was used according to the manufacturers instructions (Clontech Advantage PCR kit).
  • the mouse lv shown in SEQ ID NO: 5 and the rat lvl splice variant shown in SEQ ID NO: 7 was isolated using a two-hybrid assay as described in Example 1.
  • the mouse lvl splice variant shown in SEQ ID NO: 7 was isolated by screening a mouse brain cDNA library, and the rat lvn splice variant shown in SEQ ID NO:l 1 was isolated by BLAST searching.
  • Rat 9ql (SEQ ID NO: 15) was isolated by database mining, rat 9qm (SEQ ID NO: 21) was isolated by a two-hybrid assay, and rat 9qc (SEQ ID NO:27) was identified by database mining.
  • Human 9ql (SEQ ID NO: 13), and human 9qs (SEQ ID NO: 23) were identified as described in Example 2.
  • Mouse 9ql (SEQ ID NO: 17), monkey 9qs (SEQ ID NO:25), human pl93 (SEQ ID NO:39), rat pl9 (SEQ ID NO:33), and mouse pi 9 (SEQ ID NO: 35) were identified by database mining.
  • Rat 8t (SEQ ID NO:29) was identified using a two-hybrid assay.
  • the sequence of W28559 (SEQ ID NO:37) was identified by database mining and sequencing of the identified EST with Genbank Accession Number AI352454. The protein sequence was found to contain a
  • the human genomic 9q sequence (SEQ ID NOs:46 and 47) was isolated by screening a B AC genomic DNA library (Reasearch Genetics) using primers which were designed based on the sequence of the human 9qm cDNA. Two positive clones were identified (44802 and 721117) and sequenced.
  • EXAMPLE 5 EXPRESSION OF IV, 8T, AND 9Q mRNA IN RAT
  • Northern blots hybridized with the rat lv probe revealed a single band at 2.3kb only in the lane containing brain RNA, suggesting that 1 v expression is brain specific.
  • Northern blots probed with the rat 8t probe revealed a major band at 2.4kb. The rat 8t band was most intense in the lane containing heart RNA and there was also a weaker band in the lane containing brain RNA.
  • Northern blots hybridized with the 9q cDNA probe revealed a major band at 2.5kb and a minor band at over 4kb with predominant expression in brain and heart. The minor band may represent incompletely spliced or processed 9q mRNA. The results from the northern blots further indicated that pi 9 is expressed predominantly in the heart.
  • oligonucleotide primers were designed to amplify a fragment of 3'- or 5 '-untranslated region of the target cDNA and in addition, add the promoter recognition sequences for T7 and T3 polymerase.
  • 5-ATTAACCCTCACTAAAGGGACACTACTGTTTAAGCTCAAG-3 (T3, reverse, antisense; SEQ ID NO:43).
  • the underlined, bases correspond to the T7 and T3 promoter sequences.
  • 1 v mRNA is highly expressed in cortical, hippocampal, and striatal interneurons, the reticlar nucleus of the thalamus, the medial habenula, and in cerebellar granule cells, lv mRNA is expressed at moderate levels in midbrain nuclei including the substantia nigra and superior colliculus, in several other thalamic nuclei, and in the medial septal and diagonal band nuclei of the basal forebrain.
  • the probe used to analyze the expression of 8t and 9q hybridizes to a region of the 3 -untranslated region that is identical in the 8t and 9q mRNAs, this probe generates a composite image that reveals that 8t/9q mRNA is expressed widely in brain in a pattern that partly overlaps with that for 1 v as described above.
  • 8t/9q mRNA is highly expressed in the striatum, hippocampal formation, cerebellar granule cells, and neocortex.
  • 8t/9q mRNA is expressed at moderate levels in the midbrain, thalamus, and brainstem.
  • 8t./9q mRNA appears to be concentrated in interneurons in addition to principal cells, and in all regions 8t/9q expression appears to be concentrated in neurons as apposed to glial cells.
  • Single- and double-label immunohistochemistry revealed that the PCIP and Kv4 polypeptides are precisely colocalized in many of the cell types and brain regions where PCIP and Kv4 mRNAs are coexpressed.
  • COS1 and CHO cells were transiently transfected with individual PCIPs (KChlPl, KChIP2, KChIP3) alone or together with Kv4.2 or Kv4.3 using the lipofectamine plus procedure essentially as described by the manufacturer (Boehringer Mannheim). Forty-eight hours after the transfection, cells were washed, fixed, and processed for immunofluorescent visualization as described previously (Bekele-Arcuri et al. (1996) Neuropharmacology, 35:851-865). Affinity-purified rabbit polyclonal or mouse monoclonal antibodies to the Kv4 channel or the PCIP protein were used for immunofluorescent detection of the target proteins.
  • the PCIPs When expressed alone, the PCIPs were diffusely distributed throughout the cytoplasm of COS- 1 and CHO cells, as would be expected for cytoplasmic proteins. In contrast, when expressed alone, the Kv4.2 and Kv4.3 polypeptides were concentrated within the perinuclear ER and Golgi compartments, with some immunoreactivity concentrated in the outer margins of the cell. When the PCIPs were coexpressed with Kv4 ⁇ -subunits, the characteristic diffose PCIP distribution changed dramatically, such that the PCIPs precisely colocalized with the Kv4 ⁇ -subunits.
  • Kv4.3 ⁇ C Kv4.3 ⁇ C
  • KChlPl and KCMP2 precisely colocalized with Kv4.3 ⁇ C in cotransfected cells, and moreover, Kv4.3 ⁇ C was efficiently coimmunoprecipitated by PCIP antibodies, indicating that the interaction of these PCIPs with Kv4 ⁇ -subunits does not require the cytoplasmic C-terminus of the channel.
  • Kv4- and PCIP-specific antibodies were used for single and double-label immunohistochemical analyses and for reciprocal coimmunoprecipitation analyses of rat brain membranes. Immunohistochemical staining of rat brain sections indicated that KChlPl and KChIP2 colocalize with Kv4.2 and Kv4.3 in a region and cell type-specific manner.
  • KChlPl colocalized with Kv4.3 in hippocampal interneurons, cerebellar granule cells, and cerebellar glomeruli, a specialized synaptic arrangement between the dendrites of cerebellar basket and golgi cells and mossy fiber terminals.
  • KChIP2 colocalized with Kv4.3 and Kv4.2 in the dendrites of granule cells in the dentate gyrus, in the apical and basal dendrites of hippocampal and neocortical pyramidal cells, and in several subcortical structures including the striatum and superior colliculus.
  • PCIPs e.g., KChlPl (lv), KChIP2 (9ql), and KChIP3 (pl9)
  • Kv4.2 and Kv4.3 were expressed alone and in combination with individual PCIPs.
  • CHO cells were transiently-transfected with cDNA using the DOTAP lipofection method as described by the manufacturer (Boehringer Mannheim, Inc.). Transfected cells were identified by cotransfecting enhanced GFP along with the genes of interest and subsequently determining if the cells contained green GFP fluorescence. Currents in CHO cells were measured using the patch-clamp technique (Hamill et al. 1981. Pfluegers Arch. 391: 85-100).
  • KChlPs 1, 2 and 3 have distinct N-termini but share considerable amino acid identity within the C-terminal "core" domain. Despite their distinct N-termini, the effects of KChIP2 and KChIP3 on Kv4.2 current density and kinetics were strikingly similar to those produced by KChlPl (Tablel). Thus to confirm that the conserved C- terminal core domain, which contains all three EF-hands, is sufficient to modulate Kv4 current density and kinetics, N-terminal truncation mutants of KChlPl and KChIP2 were prepared.
  • KChlPl ⁇ N2-31 and KChIP2 ⁇ N2-67 mutants truncated KChlPl and KChIP2, respectively, to the C-terminal 185 amino acid core sequence.
  • Coexpression of KChlPl ⁇ N2-31 or KChIP2 ⁇ N2-67 with Kv4.2 in CHO cells produced changes in Kv4.2 current density and kinetics that were indistinguishable from the effects produced by full-length KChlPl or KChIP2 (Tablel).
  • KChlPl was coexpressed with Kvl.4 and Kv2.1 in Xenopus oocytes.
  • Xenopus oocytes were injected with 1-3 ng/oocyte of cRNA which was prepared using standard in vitro transcription techniques (Sambrook et al. 1989. Molecular Cloning: a laboratory manual, Cold Spring Harbor Press). Currents in Xenopus oocytes were measured with a two-electrode voltage clamp.
  • KChlPl did not appear to have any effect on Kvl.4 or Kv2.1 currents (Table2), indicating that these functional effects may be specific for Kv4 channels.
  • KChlPl Since these KChlPs bind Ca2+, another important question is to determine whether the effects of KChlPl on Kv4.2 currents are Ca2+-dependent. This question was addressed indirectly by introducing point mutations within each of KChlPl 's EF- hand domains: one mutant has point mutations in the first two EF hands (D 199 to A, G 104 to A, D 135 to A, and G 140 to A) and the other one has point muations in all three EF hands (D 199 to A, G 104 to A, D 135 to A, G M0 to A, D lg3 to A, and G 188 to A).
  • Kv4.3ch a chimeric Kv4.3 subunit
  • the C- terminal 10 amino acids from rKvl.4 were used because they associate with PSD-95 and confer the ability to associate with PSD-95 to the Kv4.3 protein when fosed to the Kv4.3 C-terminus.
  • Expression of Kv4.3ch in COS-1 cells revealed that the Kv4.3ch polypeptide was trapped in the perinuclear cytoplasm, with minimal detectable Kv4.3ch immunoreactivity at the outer margins of the cell.
  • Kv4.3ch was co-expressed with PSD-95, PSD-95 became trapped in the perinuclear cytoplasm and co-localized with Kv4.3ch.
  • EXAMPLE 12 CHARACTERIZATION OF THE PCIP PROTEINS
  • amino acid sequences of the PCIP proteins were compared to amino acid sequences of known proteins and various motifs were identified.
  • the lv polypeptide is a novel polypeptide which includes 216 amino acid residues. Domains that are putatively involved in calcium binding (Linse, S. and Forsen, S. (1995) Advances in Second Messenger and Phosphoprotein Research 30, Chapter 3, p89-151, edited by
  • NO:30 is a novel polypeptide which includes 225 amino acid residues. Calcium binding domains that are putatively involved in calcium binding (Linse, S. and Forsen, S.
  • the 9q polypeptide is a novel polypeptide which includes calcium binding domains that are putatively involved in calcium binding (Linse, S. and Forsen, S. (1995) Advances in Second Messenger and Phosphoprotein Research 30, Chapter 3, p89-151, edited by Means, AR., Raven Press, Ltd., New York (see Figure 21).
  • the pi 9 polypeptide is a novel polypeptide which includes calcium binding domains that are putatively involved in calcium binding (Linse, S. and Forsen, S. (1995,) Advances in Second Messenger and Phosphoprotein Research 30, Chapter 3, p89-151, edited by Means, AR., Raven Press, Ltd., New York (see Figure 21).
  • a BLASTN 2.0.7 search (Altschul et al. (1990) J Mol. Biol. 215:403) of the nucleotide sequence of rat lvl revealed that the rat lvl is similar to the rat cDNA clone RMUAH89 (Accession Number AA849706).
  • the rat 1 vl nucleic acid molecule is 98% identical to the rat cDNA clone RMUAH89 (Accession Number AA849706) over nucleotides 1063 tol488.
  • a BLASTN 2.0.7 search (Altschul et al. (1990) J Mol. Biol. 215:403) of the nucleotide sequence of human 9ql revealed that the human 9ql is similar to the human cDNA clone 1309405 (Accession Number AA757119).
  • the human 9 ql nucleic acid molecule is 98% identical to the human cDNA clone 1309405 (Accession Number AA757119) over nucleotides 937 tol405.
  • a BLASTN 2.0.7 search (Altschul et al. (1990) J Mol. Biol. 215:403) of the nucleotide sequence of mouse PI 9 revealed that the mouse PI 9 is similar to the Mus musculus cDNA clone MNCb-7005 (Accession Number AU035979).
  • the mouse P19 nucleic acid molecule is 98% identical to the Mus musculus cDNA clone MNCb-7005 (Accession Number AU035979) over nucleotides 1 to 583.
  • PCIP is expressed as a recombinant glutathione-S-transferase (GST) fosion polypeptide in E. coli and the fosion polypeptide is isolated and characterized.
  • GST glutathione-S-transferase
  • PCIP is fosed to GST and this fosion polypeptide is expressed in E. coli, e.g., strain BI21.
  • Expression of the GST-PCIP fusion protein in BI21 is induced with IPTG.
  • the recombinant fosion polypeptide is purified from crude bacterial lysates of the induced BI21 strain by affinity chromatography on glutathione beads. Using polyacrylamide gel electrophoretic analysis of the polypeptide purified from the bacterial lysates, the molecular weight of the resultant fosion polypeptide is determined.
  • Rat lv and 9ql were cloned into pGEX-6p-2 (Pharmacia).
  • the resulting recombinant fosion proteins were expressed in E. coli cells and purified following art known methods (described in, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).
  • the identities of the purified proteins were verified by western blot analysis using antibodies raised against peptide epitopes of rat lv and 9ql.
  • This vector contains an SV40 origin of replication, an ampicillin resistance gene, an E. coli replication origin, a CMV promoter followed by a polylinker region, and an SV40 intron and polyadenylation site.
  • a DNA fragment encoding the entire PCIP protein and an HA tag (Wilson et al. (1984) Cell 37:767) or a FLAG tag fosed in-frame to its 3' end of the fragment is cloned into the polylinker region of the vector, thereby placing the expression of the recombinant protein under the control of the CMV promoter.
  • the PCIP DNA sequence is amplified by PCR using two primers.
  • the 5' primer contains the restriction site of interest followed by approximately twenty nucleotides of the PCIP coding sequence starting from the initiation codon; the 3' end sequence contains complementary sequences to the other restriction site of interest, a translation stop codon, the HA tag or FLAG tag and the last 20 nucleotides of the PCIP coding sequence.
  • the PCR amplified fragment and the pCDNA/Amp vector are digested with the appropriate restriction enzymes and the vector is dephosphorylated using the CIAP enzyme (New England Biolabs, Beverly, MA).
  • the two restriction sites chosen are different so that the PCIP gene is inserted in the correct orientation.
  • the ligation mixture is transformed into E. coli cells (strains HB101, DH5a, SURE, available from Stratagene Cloning Systems, La Jolla, CA, can be used), the transformed culture is plated on ampicillin media plates, and resistant colonies are selected. Plasmid DNA is isolated from transformants and examined by restriction analysis for the presence of the correct fragment. COS cells are subsequently transfected with the PCIP-pcDNA/Amp plasmid
  • DNA using the calcium phosphate or calcium chloride co-precipitation methods, DEAE- dextran-mediated transfection, lipofection, or electroporation.
  • Other suitable methods for transfecting host cells can be found in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.
  • the expression of the PCIP polypeptide is detected by radiolabelling ( 35 S-methionine or 35 S-cysteine available from NEN, Boston, MA, can be used) and immunoprecipitation (Harlow, E. and Lane, D.
  • HA specific monoclonal antibody A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988
  • the cells are labelled for 8 hours with 35 S-methionine (or 35 S-cysteine).
  • the culture media are then collected and the cells are lysed using detergents (RIP A buffer, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC, 50 mM Tris, pH 7.5). Both the cell lysate and the culture media are precipitated with an HA specific monoclonal antibody. Precipitated polypeptides are then analyzed by SDS-PAGE.
  • DNA containing the PCIP coding sequence is cloned directly into the polylinker of the pCDN A/Amp vector using the appropriate restriction sites.
  • the resulting plasmid is transfected into COS cells in the manner described above, and the expression of the PCIP polypeptide is detected by radiolabelling and immunoprecipitation using a PCIP specific monoclonal antibody.
  • Rat lv was cloned into the mammalian expresssion vector pRBG4.
  • the human foil length pi 9 sequence was identified using RACE PCR.
  • the sequence of pl9 (also referred to as KChIP3) is shown in Figure 16.
  • the amino acid sequence of human pl9 is 92% identical to the mouse pl9 gene (SEQ ID NO:35).
  • TBLASTN searches using the protein sequence of human pi 9 revealed that human pi 9 is homologous to two sequences, Calsenilin (described in (1998) Nature Medicine 4: 1177- 1181) and DREAM, a Ca2+-dependent regulator of prodynorphin and c-fos transcription (described in Carrion et al. (1999) Nature 398: 80-84).
  • Human pl9 is 100%> identical at the nucleotide level to Calsenilin (but extends 3' to the published sequence) and 99% identical at the nucleotide level to DREAM.
  • pi 9 (as well as other PCIP family members) to co-localize with presenilin and act as transcription factors is determined using art known techniques such as northern blots, in situ hybridization, ⁇ -gal assays, DNA mobility assays (described in, for example, Carrion et al. (1999) Nature 398:80) and DNA mobility supershift assays, using antibodies specific for KChlPs.
  • MONKEY KChIP4 In this example, the identification and characterization of the genes encoding monkey KChIP4a (jlkbd352e01tl) and alternatively spliced monkey KChIP4b Glkbb231c04tl), KChIP4c (jlkxa053c02), and KChIP4d (jlkx015bl0) is described. TBLASTN searches in proprietary databases with the sequence of the known PCIP family members, lead to the identification of four clones Jlkbb231c04tl,jlkbd352e01tl, jlkxa053c02, and jlkx015bl0. The four monkey clones were obtained and sequenced.
  • sequences of proprietary monkey clones jlkbb231c04tl and jlkbd352e01tl were found to correspond to alternately spliced variants of an additional PCIP family member, referred to herein as KChIP4.
  • Clone jlkbb23 lc04tl contains a 822bp deletion relative to jlkbd352e01tl (presumably due to splicing out of an exon), resulting in the loss of the final EF hand domain.
  • the final EF hand domain is preserved, and the C-terminus is highly homologous to that of PCIP family members lv, 9ql, and pi 9.
  • Overall identity in the homologous C-termini among KChIP4, lv, 9ql, and pi 9 ranged from 71%-80% at the amino acid level (alignments were performed using the CLUSTALW).
  • Monkey KChIP4c and KChIP4d were discovered by BLASTN search using monkey KChIP4a as a query for searching a proprietary database.
  • the nucleotide sequence of the monkey KChIP4a cDNA and the predicted amino acid sequence of the KChIP4a polypeptide are shown in Figure 23 and in SEQ ID NOs:48 and 49, respectively.
  • the nucleotide sequence of the monkey KChIP4b cDNA and the predicted amino acid sequence of the KChIP4b polypeptide are shown in Figure 24 and in SEQ ID NOs:50 and 51, respectively.
  • the nucleotide sequence of the monkey KChIP4c cDNA and the predicted amino acid sequence of the KChIP4c polypeptide are shown in Figure 35 and in SEQ ID NOs:69 and 70, respectively.
  • Rat KChIP4 is predominantly expressed in the brain, and weakly in the kidney, but not in the heart, brain, spleen, lung, liver, skeletal muscle or testes, as indicated by northern blot experiments in which a northern blot purchased from Clontech was probed with a DNA fragment from the 3 '-untranslated region of rat KCUP4.
  • Partial rat 33b07 (clone name 9o) was isolated as a positive clone from the yeast two-hybrid screen described above, using rKv4.3N as bait.
  • the f ll length rat 33b07 clone was identified by mining of proprietary databases.
  • the nucleotide sequence of the full length rat 33b07 cDNA and the predicted amino acid sequence of the rat 33b07 polypeptide are shown in Figure 26 and in SEQ ID NOs:52 and 53, respectively.
  • the rat 33b07 cDNA encodes a protein having a molecular weight of approximately 44.7 kD and which is 407 amino acid residues in length.
  • Rat 33b07 binds rKv4.3N and rKv4.2N with slight preference for rKv4.2N in yeast 2-hybrid assays. In contrast, rat 33b07 does not bind rKvl.lN, indicating that the rat 33b07-Kv4N interaction is specific.
  • Rat 33b07 is expressed predominantly in the brain as determined by northern blot analysis.
  • the human 33b07 ortholog (clone 106d5) was also identified by mining of proprietary databases.
  • the nucleotide sequence of the foil length human 33b07 cDNA and the predicted amino acid sequence of the human 33b07 polypeptide are shown in Figure 27 and in SEQ ID NOs:54 and 55, respectively.
  • the human 33b07 cDNA encodes a protein having a molecular weight of approximately 45.1 kD and which is 414 amino acid residues in length.
  • TSP(Y)s Testes-specific proteins
  • NAPs Nucleosome Assembly Proteins
  • Human SET is also called HLA-DR associated protein II (PHAPII) (Hoppe- Seyler (1994) Biol. Chem. 375:113-126) and in some cases is associated with acute undifferentiated leukemia (AUL) as a result of a translocation event resulting in the formation of a SET-CAN fosion gene (Von Lindern M. et al. (1992) Mol. Cell. Biol. 12:3346-3355).
  • An alternative spliced form of SET is also called Template Activating Factor-I alpha (TAF). TAF is found to be associated with myeloid leukemogenesis (Nagata K. et al. (1995) Proc. Natl. Acad. Sci.
  • Human SET is also a potent protein inhibitor of phosphatase 2A (Adachi Y. et al. (1994) J. Biol. Chem. 269:2258-2262). NAPs may be involved in modulating chromatin formation and contribute to regulation of cell proliferation (Simon H.U. et al. (1994) Biochem. J. 297, 389-397).
  • 33b07 may function as a protein inhibitor of phosphatase, an oncogene, and/or a chromatin modulator.
  • the homology of 33b07 to SET, a protein phosphatase inhibitor, is of particular interest.
  • Many channels, in particular the Kv4 channels (with which 33b07 is associated), are known to be regulated by phosphorylation by PKC and PKA ((1998) J Neuroscience 18(10): 3521-3528; Am J Physiol 273: H1775-86 (1997)).
  • 33b07 may modulate Kv4 activity by regulating the phosphorylation status of the potassium channel.
  • RAT lp In this example, the identification and characterization of the gene encoding rat lp is described. Partial rat lp was isolated as a positive clone from the yeast two-hybrid screen described above, using rKv4.3N as a bait.
  • the nucleotide sequence of the partial length rat lp cDNA and the predicted amino acid sequence of the rat lp polypeptide are shown in Figure 28 and in SEQ ID NOs:56 and 57, respectively.
  • the rat lp cDNA encodes a protein having a molecular weight of approximately 28.6 kD and which is 267 amino acid residues in length.
  • Rat lp binds rKv4.3N and rKv4.2N with slight preference for rKv4.3N in yeast two-hybrid assays. In contrast, lp does not bind rKvl.lN, indicating that the lp-Kv4N interaction is specific.
  • Rat lp is predominantly expressed in the brain as determined by northern blot analysis.
  • the rat lp protein is 58% identical to the human Restin over amino acid residues 105 tol82, 55% identical to the human Restin over amino acid residues 115 to 186, 22% identical to the human Restin over amino acid residues 173 to 246, 22% identical to the human Restin over amino acid residues 169 to 218, and 58% identical to the human Restin over amino acid residues 217 to 228.
  • Restin is also named Reed-Sternberg intermediate filament associated protein.
  • Reed-Sternberg cells are the tumoral cells diagnostic for Hodgkin's disease. It is suggested that Restin overexpression may be a contributing factor in the progression of Hodgkin's disease (Bilbe G. et al. (1992) EMBO J. 11 : 2103-13) and Restin appears to be an intermediate filament associated protein that links endocytic vesicles to microtubules (Pierre P, et al. (1992) Cell 70 (6), 887-900).
  • the cytoskeleton regulates the activity of potassium channels (see, for example, Honore E, et al. (1992) EMBO J. 11 :2465-2471 and Levin G, et al. (1996) J Biol.
  • the rat lp protein may be associated with the cytoskeleton and may modulate the activity of potassium channels, e.g., Kv4, via its association to the cytoskeleton.
  • potassium channels e.g., Kv4
  • Rat 7s Partial rat 7s was isolated as a positive clone from the yeast two-hybrid screen described above, using rKv4.3N as a bait.
  • Rat 7s is the rat ortiiolog of the human vacuolar H(+)-ATPase catalytic subunit A (Accession Number P38606 and B46091) described in, for example, van Hille B. et al. (1993) J. Biol. Chem. 268 (10), 7075-7080.
  • the nucleotide sequence of the partial length rat 7s cDNA and the predicted amino acid sequence of the rat 7s polypeptide are shown in Figure 29 and in SEQ ID NOs:58 and 59, respectively.
  • the rat 7s cDNA encodes a protein having a molecular weight of approximately 28.6 kD and which is 270 amino acid residues in length.
  • Rat 7s binds rKv4.3N and rKv4.2N with preference for rKv4.3N in yeast two- hybrid assays. In contrast, 7s does not bind rKvl .IN, indicating that the 7s-Kv4N interaction is specific. Rat 7s is expressed at significantly higher levels in the brain and the kidney than in the lung, liver, heart, testes, and skeletal muscle, as determined by northern blot analysis.
  • Rat 29x was isolated as a positive clone from the yeast two-hybrid screen described above, using rKv4.3N as a bait.
  • Rat 25r is a splice variant of 29x. They differ in the 5' untranslated region, but are identical in the coding region and at the amino acid level.
  • the nucleotide sequence of the rat 29x cDNA and the predicted amino acid sequence of the rat 29x polypeptide are shown in Figure 30 and in SEQ ID NOs:60 and 61, respectively.
  • the rat 29x cDNA encodes a protein having a molecular weight of approximately 40.4 kD and which is 351 amino acid residues in length.
  • the nucleotide sequence of the rat 25r cDNA is shown in Figure 31 and in SEQ ID NO:62.
  • the rat 25r cDNA encodes a protein having a molecular weight of approximately 40.4 kD and which is 351 amino acid residues in length.
  • Rat 29x is expressed in the spleen, lung, kidney, heart, brain, testes, skeletal muscle and liver, with the highest level of expression being in the spleen and the lowest being in the liver.
  • Rat 29x binds rKv4.3N and rKv4.2N with slight preference for rKv4.3N in yeast two-hybrid assays. In contrast, 29x does not bind rKvl .IN, indicating that the 29x- Kv4N interaction is specific. Rat 29x is identical at the amino acid level to rat SOCS-1 (Suppressor Of
  • SH2 domain also referred to a Src Homology 2 domain, includes a protein domain of about 100 amino acids in length which is involved in binding of phosphotyrosine residues, e.g., phosphotyrosine residues in other proteins.
  • the target site is called an SH2-binding site.
  • the SH2 domain has a conserved 3D structure consisting of two alpha helices and six to seven beta-strands.
  • the core of the SH2 domain is formed by a continuous beta-meander composed of two connected beta- sheets (Kuriyan J. et al. (1997) Curr. Opin. Struct. Biol. 3:828-837).
  • SH2 domains function as regulatory modules of intracellular signaling cascades by interacting with high affinity to phosphotyrosine-containing target peptides in a sequence-specific and strictly phosphorylation-dependent manner (Pawson T. (1995) Nature 373:573-580). Some proteins contain multiple SH2 domains, which increases their affinity for binding to phosphoproteins or confers the ability to bind to different phosphoproteins.
  • Rat 29x contains an SH2 domain at amino acid residues 219-308 of SEQ ID NO:61.
  • Tyrosine phosphorylation regulates potassium channel activity (Prevarskaya N.B. et al. (1995) J. Biol. Chem. 270:24292-24299). JAK kinase phoshorylates proteins at tyrosines and is implicated in the regulation of channel activity (Prevarskaya N.B. et al. supra). Accordingly, based on its homology to SOCS-1, JAB, and SSI-1, rat 29x may modulate the activity of potassium channels, e.g., Kv4, by modulating JAK kinase activity.
  • Rat 5p was isolated as a positive clone from the yeast two-hybrid screen described above, using rKv4.3N as a bait.
  • the nucleotide sequence of the rat 5pc DNA and the predicted amino acid sequence of the rat 5p polypeptide are shown in Figure 32 and in SEQ ID NOs:63 and 64, respectively.
  • the rat 5p cDNA encodes a protein having a molecular weight of approximately 11.1 kD and which is 95 amino acid residues in length.
  • Rat 5p binds rKv4.3N and rKv4.2N with similar strength in yeast two-hybrid assays. In contrast, 5p does not bind rKvl.lN, indicating that the 5p-Kv4N interaction is specific.
  • Rat 5p is expressed in the spleen, lung, skeletal muscle, heart, kidney, brain, liver, and testes, as determined by northern blot analysis.
  • the rat 5p is identical to rat Calpactin I light chain or P10 (Accession Number P05943).
  • P10 binds and induces the dimerization of annexin II (p36).
  • P10 may function as a regulator of protein phosphorylation in that the p36 monomer is the preferred target of a tyrosine-specific kinase (Masiakowski P. et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 85 (4): 1277-1281).
  • rat 5p may modulate the activity of potassium channels, e.g., Kv4, by modulating the activity of a tyrosine- specific kinase.
  • Rat 7q was described. Rat 7q was isolated as a positive clone from the yeast two-hybrid screen described above, using rKv4.3N as a bait. Full length rat 7q was obtained by RACE PCR.
  • the nucleotide sequence of the rat 7q cDNA and the predicted amino acid sequence of the rat 7q polypeptide are shown in Figure 33 and in SEQ ID NOs:65 and 66, respectively.
  • the rat 7q cDNA encodes a protein having a molecular weight of approximately 23.5 kD and which is 212 amino acid residues in length.
  • Rat 7q binds rKv4.3N and rKv4.2N with same strength in yeast two-hybrid assays. In contrast, 7q does not bind rKvl . IN, indicating that the 7q-Kv4N interaction is specific.
  • Rat 7q is expressed in the heart, brain, spleen, lung, liver, skeletal muscle, kidney, and testes, as determined by northern blot analysis.
  • Rat 7q is identical to RAB2 (rat RAS-related protein, Accession Number P05712) at the amino acid level. RAB2 appears to be involved in vesicular traffic and protein transport (Touchot N. et al. (1987) Proc. Natl. Acad. Sci. U.S.A. 84 (23): 8210- 8214). Accordingly, based on its homology to RAB2, rat 7q may be involved in potassium channel, e.g., Kv4, trafficking.
  • EXAMPLE 23 IDENTIFICATION AND CHARACTERIZATION OF
  • Partial rat 19r was isolated as a positive clone from the yeast two- hybrid screen described above, using rKv4.3N as a bait. Full length rat 19r was obtained by RACE PCR.
  • the nucleotide sequence of the rat 19r cDNA and the predicted amino acid sequence of the rat 19r polypeptide are shown in Figure 34 and in SEQ ID NOs:67 and 68, respectively.
  • the rat 19r cDNA encodes a protein having a molecular weight of approximately 31.9 kD and which is 271 amino acid residues in length.
  • Rat 19r is expressed in the heart, brain, spleen, lung, liver, skeletal muscle, kidney, and testes, as determined by northern blot analysis.
  • Rat 19r binds rKv4.3N and rKv4.2N with slight preference for rKv4.3N in yeast two-hybrid assays. In contrast, 19r does not bind rKvl . IN, indicating that the 19r-Kv4N interaction is specific.
  • Rat 19r is identical to Rat phosphatidylinositol (PTDINS) transfer protein alpha (PTDINSTP, Accession Number M25758 or P16446) described in Dickeson S.K. et al. (1989) J Biol. Chem. 264:16557-16564.
  • PTDINSTP is believed to be involved in phospholipase C-beta (PLC-beta) signaling, phosphatidylinositol transfer protein (Ptdlns-TP) synthesis, secrettory vesicle formation, and enhancement of phosphatidylinositol 3-kinase (Ptdlns 3-kinase) activity (Cunningham E. et al.
  • rat 19r may modulate potassium chamiel, e.g., Kv4, activity via the PLC-beta signaling pathway and/or the Ptdlns 3-kinase signaling pathway.
  • Rat pl9r may also be involved in potassium channel, e.g., Kv4, trafficking.
  • the human PCIP 9q was chromosomally mapped using a radiation hybrid panel (Panel GB4).
  • h9q mapped to a region of chromosome lOq that had been previously shown to contain a linkage with partial epilepsy, namely D10S192: 10q22-q24 (Ottman et al. (1995) Nature Genetics 10:56-60) (see Figure 43).
  • D10S192: 10q22-q24 Ottman et al. (1995) Nature Genetics 10:56-60
  • the present invention clearly demonstrates that the 9q family of proteins can serve as targets for developing anti-epilepsy drugs and as targets for medical intervention of epilepsy.
  • h9q mapped to a region of chromosome lOq that had been previously shown to contain a linkage with IOSCA, namely D10S192 and D10S1265: 10q24- Nikali (Genomics 39:185-191 (1997)) (see Figures 42 and 43).
  • the present invention clearly demonstrates that the 9q family of proteins can serve as targets for developing anti-spinocerebellar ataxia drags and as targets for medical intervention of spinocerebellar ataxia.
  • EXAMPLE 25 ARACHIDONIC ACID MODULATION OF KV4/KCMP
  • KChlP-dependent Arachidonic Acid was shown to inhibit recombinant Kv4 current expressed inXenopus oocytes (Villarroel, A. and Schwarz, T. L. (1996) J Neuroscience 16:2522- 32). However, the modulation was only observed with peak current amplitude whereas the current kinetic parameters were not affected by the presence of AA. In contrast, recordings of membrane patches from hippocampal neurons showed that in additional to suppression of peak amplitude, AA changed the kinetic parameters of the A-current by Kv4 channels (Keros, S. and McBain, C. J. (1997) J Neuroscience 17: 3476-87).
  • inactivation time constant was considerably reduced (note: inactivation time constant is in inversely correlated to rate of inactivation. Therefore, inactivation was sped up (Keros (1997) supra).
  • KChlPs were the missing auxiliary subunits that accounted for the above kinetic discrepancy was investigated by expressing Kv4 alone or together with KChlPs in both CHO cells and Xenopus oocytes, and measuring their inactivation time constants (using art known techniques as described in, for example, An et al. (2000) Nature 403:553-6; Keros, S. and McBain, C. J. (1997) J Neuroscience 17: 3476-87; and Nillarroel, A.
  • AA also suppressed peak amplitude ofKv4/KChIP current in both CHO cells and Xenopus oocytes (Tables 3 and 4). This indicates that modulation of peak amplitude of Kv4currents is independent of KChlPs.
  • the arachidonic acid effects on the A-current were also investigated in a neuronal system (cultured primary cerebellar granule neurons) where both Kv4 and KChlPs are present.
  • TEA (10 mM) was applied to block a small sustained outward component.
  • Inactivation time constants of the A current in the absence and presence of 10 ⁇ M arachidonic acid were 44 ⁇ 5 ms and 21 ⁇ 3 ms (mean + SEM), respectively.
  • the corresponding peak amplitude was reduced from 2.0 ⁇ 0.6 nA to 1.2 ⁇ 0.4 nA.
  • Arachidonic acid modulation of Kv4/KChIP current is concentration-dependent and reversible The effects of different concentrations of arachidonic acid on Kv4/KChIP current was studied in Xenopus oocytes. Because the physiological concentrations of arachidonic acid are often under 10 ⁇ M (Needleman, et al, 1986 Annu Rev Biochem 55:69-102; Anderson and Welsh, 1990, Proc Natl Acad Sci U S A 87:7334-8; Meves, 1994, Prog Neurobiol 43 : 175-86), arachidonic acid was tested in the 1 - 10 ⁇ M range.
  • the concentration-dependent block of peak amplitude of the Kv4.3 current was independent of the presence of KChlPl (see Figure 64 A). Further, the slope of amplitude reduction as a function of increasing concentrations was very similar with or without the presence of KChlPs. Peak current block did not appear to saturate up to 10 ⁇ M. Nillarroel and Schwarz, (1996) J Neurosci 16:2522-32 reported that the IC 50 of arachidonic acid on Kv4 ⁇ subunits was approximately 8 ⁇ M in oocytes. The inactivation time constant in the absence of KChlPl was unchanged at all arachidonic acid concentrations tested.
  • arachidonic acid analogs with distinct stractural features were studied: ⁇ - linolenic acid (18:3 c9) has three cis double bonds instead of four double bonds, linolelaidic acid (18:219) has two trans double bonds instead of four cis double bonds, 5,8,11,14-eicosatetraynoic acid (ETYA, 20:4 n5) has four triple bonds instead of double bonds found in arachidonic acid (n indicates position of the first triple bond), and 5,8,11- eicosatriynoic acid (ETI, 20:3 n5) has three triple bonds.
  • Figure 66A shows that the peak amplitude of Kv4.3 current was inhibited significantly compared with no-fatty acid control by 10 ⁇ M of ⁇ -linolenic acid, ETI, ETYA, and arachidonic acid, independent of the presence of KChlP 1. The percent inhibition of amplitude of Kv4 alone and
  • Kv4/KChIP was not significantly different for these fatty acids.
  • a small, statistically significant block of Kv4 current amplitude by 10 ⁇ M linolelaidic acid was observed in the presence of KChlPl but not absence of KChlPl when the values were compared to their respective controls.
  • Kv4.3 and Kv4.3/KChIP KChlP 1 there was no significant difference when comparing Kv4.3 and Kv4.3/KChIP KChlP 1.
  • the experiments were performed in phosphate buffered saline (PBS), pH 7.4, with 1 mM CaCl 2 and 0.05% polysorbate P-20.
  • Anti-GST antibody (Biacore AB) was coupled to 3 flowcells of a CM-5 chip (Biacore AB) at a level of 2000 resonance units (RUs) using amine coupling. The final flowcell was activated and blocked with ethanolamine to use as a reference control surface.
  • the GST-Kv4.3N terminal domain was captured on two of the anti- GST flowcells and GST alone was bound to the third anti-GST flowcell at levels of 150 RUs.
  • Diploid strains containing bait (the N-terminal domain of Kv4.3 or the empty vector pGBT9) and fish (KChlPl) plasmids were obtained as described in (An, et al, 2000).
  • strains were grown to saturation before they were inoculated at equal OD600 value into 5 ml of synthetic complete-TrpLeuHis drop-out (SC-WLH) medium that selects for interaction-dependent growth or 5 ml of SC-WL medium that is nonselective in the presence or absence of 10 ⁇ M ETYA.
  • SC-WLH synthetic complete-TrpLeuHis drop-out
  • 5 mM 3 -AT (3-amino-l,2,4-triazole) was included in the media to suppress weak self-activating activity from the Kv4.3 N-terminal domain bait. Cultures were grown for 17 hours at 30°C and OD600 values were read by a spectrophotometer.
  • the surface plasmon resonance measurement (Biosensor) was first used to monitor the association and dissociation phases of Kv4-KChIP interaction in the presence and absence of arachidonic acid.
  • the intracellular N-terminal domain of Kv4.3 was expressed as a GST fusion protein (GST-Kv4.3N) and immobilized to the surface of a Biosensor chip. Recombinant KChlPl protein was passed over the chip surface in the presence and absence of 10 ⁇ M arachidonic acid.
  • Kv4/KChIP is more sensitive to AA modulation than is Kyl.l/Kv ⁇ l
  • the pore-forming alpha subunits of ion channels including those of potassium channels, often do not work alone. They associate with auxiliary subunits and these auxiliary subunits can change channel activities dramatically. Therefore, it is more useful to study alpha subunits in combination with their auxiliary subunits as the physiologically relevant channels are complexes of alpha-auxiliary subunits.
  • Kv4 alpha subunits were shown to be by far more sensitive to AA inhibition than alpha subunits of several other voltage-gated potassium channels (e.g. , Kvl .1) (Villarroel (1996) supra).
  • this paper examined AA modulation of only the alpha subunits of the channels. It was not known whether Kv4 current would still be more sensitive to AA modulation than would be other channel currents if all channels were to be tested in the presence of their cognate auxiliary subunits.
  • Kv4.3/KChIPl Kvl.l/Kv ⁇ l
  • Kv ⁇ l which is one of the classic potassium channel beta subunits, dramatically changes Kvl.l kinetics.
  • Kv4.3/KChIPl and Kvl.l/Kv ⁇ l were expressed respectively in Xenopus oocytes and their resulting currents were recorded in the presence or absence of 10 ⁇ M AA.
  • EXAMPLE 26 K-CHANNEL INTERACTING PROTEIN-2 (KCMP2)
  • KChIP2 genes are highly conserved at the amino acid level among human, rat, and mouse. Multiple human splice variants were identified by database mining and cDNA library screening. Alternative splicing gives rise to N-terminal domains that are variable in length, but the core C-terminal domain is sufficient for associating with and modulating Kv4.
  • the human KChIP2 gene spans approximately 18 kb in the q23 region of human chromosome 10 between WI-8488 and WI-6750. This region is syntenic to mouse chromosome 19 between D19Mit40 and D 19Mitl 1.
  • a rat variant discovered by database mining changed the last five amino acids and maintained its ability to associate with and modulate Kv4. Therefore, these multiple variants of KChIP2 appear to function similarly in Kv4 modulation.
  • EXAMPLE 27 KCMPIL FUNCTION AND EXPRESSION RT-PCR was performed to examine tissue expression of the rat KChlP 11
  • RNA from heart, brain, lung, spleen, liver, skeletal muscle, kidney, and testes were purchased from Clontech.
  • RT-PCR was performed using the One-step RT-PCR kit from Clontech with amplifying 5' primer GGTACCTTCTCGTCCCTGCAGACCAAACAAAG (SEQ ID NO:104) and 3' primer CGGTAAAGGACTTGCAGTTCTCTC (SEQ ID NO: 105) with the modifications on PCR condition: 50°C forl hour; 94°C for 3 minutes; 50 cycles of 94°C for 30 seconds, 65°C for 30 seconds, and 68°C for 2 minutes.
  • the 5' primer is KChlPl -specific.
  • KChlPl and KChlPll can be amplified by the same primer set, giving two different sized PCR products that separate into two bands by electrophoresis.
  • a KChlPl 1-specific band was only observed in brain, indicating it is specifically expressed in the brain.
  • the same reaction also showed a strong KChlPl -specific signal in the brain and a barely visible band in skeletal muscle. No KChlPl or KChlPll signals were observed in any other tissues examined.
  • KChlPll expression is brain-specific whereas KChlPl expression is brain predominant with a very low level of expression in skeletal muscle.
  • KChlPll The function of KChlPll in Xenopus oocytes was also examined.
  • Kv4.3 cRNA was injected into Xenopus oocytes either with or without KChlPll cRNA. Similar to KChlPl, KChlPll increased peak amplitude of Kv4.3 from 15 ⁇ 4 ⁇ to 55 ⁇ 7 ⁇ A and increased inactivation time constant from 56 ⁇ 4 to 100 ⁇ 8 ms (Table 5).
  • KChlPll co-associates with Kv4 in • the brain.
  • the insertion of extra amino acids in the KChlPll protein may be important for unknown functions, and the DNA sequence encoding these amino acids may be used as a specific gene marker for detecting cell tissue and/or cell type specific expression of this particular splice variant.
  • the DNA and protein sequences specific to the Kchlll splice variant are identical between rat and human. So, functional data obtained with KChlPll molecules from one species also apply to that from the other species.
  • EXAMPLE 28 KChlPIN FUNCTION AND EXPRESSION
  • the expression of rat KChlPIN was examined using the Taqman technique with the probe GGCAAAGAAGCGCGATTTT (SEQ ID NO: 106), forward primer TCCCGGGTAGGCAAGCA (SEQ ID NO: 107), and reverse primer CCTGCTCAAGCCCAGCACTGCA (SEQ ID NO: 108).
  • the probe is specific to KChlPIN.
  • KChlPIN is predominantly expressed in dorsal root ganglion (DRG), and at low levels in spinal cord and brain.
  • DRG dorsal root ganglion
  • KChlPIN The function of KChlPIN in Xenopus oocytes was also examined.
  • Kv4.3 cRNA was injected into Xenopus oocytes either with or without KChlPIN cRNA.
  • KChlPIN did not affect peak amplitude of Kv4.3 (15 ⁇ 4 vs. 18 ⁇ 3 without or with KChlPIN, Table 5).
  • KChlPIN caused a much greater increase of inactivation time constant of Kv4.3 than KChlPl or KChlPll (32- fold increase by KChlPIN vs. ⁇ 2 fold increase by KChlPl or KChlPll; Table 5).
  • KChlPIN modulates Kv4 current in vitro in a manner distinct from KChlPl or KChlPll.
  • the increase of the inactivation time constant by KChlPIN was considerably bigger as opposed to the increase mediated by KChlPl or KChlPll.
  • KChlPIN was able to change the fast inactivating Kv4.3 current (nearly completely inactivated within 200 ms) to nearly non-inactivating for a 500 ms second +40 volts pulse.
  • KChlPIN at the particular concentration tested, did not affect peak amplitude of Kv4. Because all KChlPl splice variants share the C-terminal 196 amino acids, these data point to an important and distinct function of the unique 36-amino acid N-terminal domain of KChlPIN.
  • KChIP2 splice variants modulate Kv4 current similar to KChIP2m (Table 6). Since there is an extremely high homology at the amino acid level between the rat and human KChIP2s (>95%), it is believed that the results obtained using KChIP2 molecules from one species will be similar to the results for KChIP2 molecules from other species.
  • KChIP4 The ability of KChIP4 to associate with Kv4 was also examined using a yeast 2- hybrid assay.
  • the H domain of KChIP4 (the C-terminal 185 amino acids) which is common to all N-terminal splice variants of KChIP4 and homologous to other KChlPs, was expressed as "fish” and the N-terminal domains of Kv4.3, Kv4.2 were expressed as "baits" (Kv4.3N, Kv4.2N, respectively) using standard techniques.
  • KChIP4H associated with Kv4.3N and Kv4.2N, but not with Kvl .IN or other control baits both in a growth assay and in a ⁇ -galactosidase assay.
  • EXAMPLE 31 FUNCTIONAL ANALYSIS OF KCMP4N2 KChIP4N2, unlike KChlPl, kCHIP2, and KCMP3, showed a dose-dependent effect on the peak amplitude of Kv4.3 when these were co-injected into Xenopus oocytes (Table 7).
  • KChIP4N2 suppressed Kv4.3 current amplitude, whereas more diluted concentrations of KChIP4N2 either enhanced or had no effect ontKv4 current amplitude (Table 7)
  • KCHP4N2 unlike KChlPl, kCHIP2, and KChIP3, also showed a dose- dependent effect on the inactivation kinetics of Kv4.3 when these were co-injected into Xenopus oocytes (Table 7).
  • KChIP4 converted the fast- inactivating Kv4.3 current into an almost non-inactivating current (e.g., at 5x dilution of stock the current curve was too slow to decrease over time to fit and obtain an inactivation time constant).
  • the inactivation time constants gradually decreased toward the value obtained in the absence of KChIP4N2.
  • the N-terminal domain of KChIP4N2 is necessary for the observed action of KChIP4N2. Deletion of the N-terminal domain essentially abolished the effects of the wild type KChIP4N2 on the peak amplitude and the inactivation time constant of Kv4.3 (Table 8).
  • KChIP4N2 The action of the N-terminal domain of KChIP4N2 seems to be dominant over other KChlP molecules.
  • KChlPl H which is homologous to other KChlPs.
  • KChlPl H When co-expressed with Kv4, KChlPl H modulated Kv4 current almost identically to KChlPl, and produced a modulation profile that is quite different from that produced by KChIP4N2 (previous filing, [An F. et al. (2000) Nature 403:553-556).
  • KChIP4 and other KChlPs associate with the Kv4 N-terminal domain (Kv4N)
  • Kv4N Kv4 N-terminal domain
  • KChIP4N2 and other KChlPs will give rise to currents with hybrid profiles that are quantitatively and qualitatively similar or different from the parental profiles. It is conceivable that KCMP4N2 and other KChlPs are co-expressed in certain cell types in vivo (e.g., in the brain). Therefore, depending on the in vivo concentrations in a particular cell type, KChIP4N2 and other KChlPs may produce quite different currents even though the pore-forming alpha subunits are the same Kv4 molecules.
  • N-terminal domain carries a dominant modulatory function that can be separated from the functions of the H domain (binding to Kv4 and modulating Kv4 current amplitute and kinetics as described in An et al, supra, but in a manner that is different from those of the KChIP4N2's N-terminal domain). Consequently, it is conceivable that the N-terminal domain of KChIP4N2 interacts with parts of the potassium channel other than the N-te minal domain of Kv4.
  • Kv4 is likely important for controlling the movement of potassium ions through the channel, given KChIP4N2's dramatic effect on inactivation kinetics. It is then possible to use the N-terminal domain of KChIP4N2 as a tool for designing and conducting protein/peptide/compound screens using this distinct activity as a read out. Using these screening assays it is possible to obtain proteins/peptides/compounds that modulate Kv4 activity in a KChlP dependent or independent manner.
  • KChlPIN and KChIP4N2 share similar Kv4 current modulating characteristics. Both can convert fast-inactivating Kv4 currents into almost non-inactivating currents. Both can have no effect on peak amplitude of Kv4. These are characteristically different from the actions of KChlPl, KChIP2, and KChIP3.
  • the N-terminal domains of human KChlPIN and monkey KChIP4N2 were aligned (using Megalign, DNA Star), they showed a considerable homology (Figure 62) suggesting the existence of a protein motif that underlies the distinct modulation by KChlPIN and KChIP4N2.
  • the N-terminal domains of human/rat KChlPl and monkey KChIP4N2 were quite divergent (Figure 62).
  • EXAMPLE 32 FUNCTIONAL ANALYSIS OF KCMP4N1 AND KCMP4N3
  • KCMP4N1 and KChIP4N3 were co-injected with Kv4.3 cRNA into Xenopus oocytes.
  • the modulation effects of these proteins on Kv4.3 are summarized in table 9. Both increased the inactivation time constant of Kv4.3. While KChIP4N3 increased the peak amplitude of Kv4.3, KChIP4Nl statistically had no significant (ns) effect on Kv4.3 amplitude.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Zoology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Neurosurgery (AREA)
  • Biophysics (AREA)
  • Neurology (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Pain & Pain Management (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Plant Pathology (AREA)
  • Toxicology (AREA)
  • Cardiology (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
PCT/US2001/030463 2000-09-27 2001-09-27 Potassium channel interactors and uses therefor WO2002026984A2 (en)

Priority Applications (10)

Application Number Priority Date Filing Date Title
BR0114383-2A BR0114383A (pt) 2000-09-27 2001-09-27 Interatuadores de canais de potássio e aplicações dos mesmos
CA002420960A CA2420960A1 (en) 2000-09-27 2001-09-27 Potassium channel interactors and uses therefor
EP01977260A EP1322759A2 (en) 2000-09-27 2001-09-27 Potassium channel interactors and uses therefor
JP2002530747A JP2004525610A (ja) 2000-09-27 2001-09-27 カリウムチャネル相互作用因子及びその利用方法
IL15496201A IL154962A0 (en) 2000-09-27 2001-09-27 Potassium channel interactors and uses therefor
AU2001296393A AU2001296393A1 (en) 2000-09-27 2001-09-27 Potassium channel interactors and uses therefor
EA200300420A EA200300420A1 (ru) 2000-09-27 2001-09-27 Выделенная молекула нуклеиновой кислоты, выделенный полипептид и способ его получения, способ обнаружения их присутствия в образце (варианты), клетка-хозяин (варианты), антитело, набор (варианты), способ модулирования активности полипептида, способ идентификации (варианты) и применение соединения, способ выявления субъекта (варианты) и способ лечения субъекта с нарушением, связанным с калиевыми каналами (варианты)
MXPA03002557A MXPA03002557A (es) 2000-09-27 2001-09-27 Interactores de canales del potasio y sus usos.
KR10-2003-7004460A KR20030074604A (ko) 2000-09-27 2001-09-27 칼륨 채널 상호작용물질 및 이의 용도
NO20031369A NO20031369L (no) 2000-09-27 2003-03-26 Kalium-kanal interagerende forbindelser, og anvendelser derav

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US09/670,756 US7078481B1 (en) 1998-11-20 2000-09-27 Potassium channel interactors and uses therefor
US09/670,756 2000-09-27
US09/703,094 2000-10-31
US09/703,094 US7556938B1 (en) 1998-11-20 2000-10-31 Nucleic acids encoding potassium channel interactors

Publications (2)

Publication Number Publication Date
WO2002026984A2 true WO2002026984A2 (en) 2002-04-04
WO2002026984A3 WO2002026984A3 (en) 2003-03-13

Family

ID=27100393

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2001/030463 WO2002026984A2 (en) 2000-09-27 2001-09-27 Potassium channel interactors and uses therefor

Country Status (13)

Country Link
EP (1) EP1322759A2 (xx)
JP (1) JP2004525610A (xx)
KR (1) KR20030074604A (xx)
CN (1) CN1498271A (xx)
AU (1) AU2001296393A1 (xx)
BR (1) BR0114383A (xx)
CA (1) CA2420960A1 (xx)
CZ (1) CZ20031154A3 (xx)
EA (1) EA200300420A1 (xx)
IL (1) IL154962A0 (xx)
MX (1) MXPA03002557A (xx)
NO (1) NO20031369L (xx)
WO (1) WO2002026984A2 (xx)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1572102A2 (en) * 2002-11-01 2005-09-14 Decode Genetics EHF. Human type ii diabetes gene-kv channel-interacting protein (kchip1) located on chromosome 5
US7067312B1 (en) 2001-09-19 2006-06-27 Myriad Genetics, Inc. PN7718 nucleic acids and use thereof
CN102272603A (zh) * 2008-11-06 2011-12-07 巴斯夫欧洲公司 杀虫剂的筛选测定法

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105483276B (zh) * 2016-02-01 2019-06-11 成都望路医药技术有限公司 Kcnip4基因及其表达产物在直肠腺癌诊疗中的应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998016185A2 (en) * 1996-10-17 1998-04-23 Nps Pharmaceuticals, Inc. Potassium channel blocking compounds and their use
WO1999049038A2 (en) * 1998-03-26 1999-09-30 Incyte Pharmaceuticals, Inc. Human calcium-binding proteins
WO2000031133A2 (en) * 1998-11-20 2000-06-02 Millennium Pharmaceuticals, Inc. Potassium channel interactors and uses therefor
WO2000070049A2 (en) * 1999-05-19 2000-11-23 Incyte Genomics, Inc. Extracellular signaling molecules
WO2001053312A1 (en) * 1999-12-23 2001-07-26 Hyseq, Inc. Novel nucleic acids and polypeptides

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998016185A2 (en) * 1996-10-17 1998-04-23 Nps Pharmaceuticals, Inc. Potassium channel blocking compounds and their use
WO1999049038A2 (en) * 1998-03-26 1999-09-30 Incyte Pharmaceuticals, Inc. Human calcium-binding proteins
WO2000031133A2 (en) * 1998-11-20 2000-06-02 Millennium Pharmaceuticals, Inc. Potassium channel interactors and uses therefor
WO2000070049A2 (en) * 1999-05-19 2000-11-23 Incyte Genomics, Inc. Extracellular signaling molecules
WO2001053312A1 (en) * 1999-12-23 2001-07-26 Hyseq, Inc. Novel nucleic acids and polypeptides

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
AN W FRANK ET AL: "Modulation of A-type potassium channels by a family of calcium sensors." NATURE (LONDON), vol. 403, no. 6769, 3 February 2000 (2000-02-03), pages 553-556, XP002209980 ISSN: 0028-0836 *
BUXBAUM ET AL.: "Calsenilin: a calcium-binding protein that interacts with the presenilins and regulates the levels of a presenilin fragment" NATURE MEDICINE, vol. 10, October 1998 (1998-10), pages 1177-1181, XP002132975 cited in the application *
CARRION ET AL.: "DREAM is a Ca2+-regulated transcriptional repressor" NATURE, vol. 398, 4 March 1999 (1999-03-04), pages 80-84, XP000700251 cited in the application *
CASTAGNA ET AL.: "Molecular characteristics of mammalian and insect amino acid transporters: implications for amino acid homeostasis" THE JOURNAL OF EXPERIMENTAL BIOLOGY, vol. 200, 1997, pages 269-286, XP002132978 *
LOMBARDI ET AL.: "Structure-activity relationships of the Kvbeta1 inactivation domain and its putative receptor probed using peptide analogs of voltage-gated potassium channel alpha- and beta-subunits" THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 273, no. 46, 13 November 1998 (1998-11-13), pages 30092-30096, XP002132979 *
VAN HILLE ET AL.: "Identification of two subunit A isoforms of the vacuolar H+-ATPase in human osteoclastoma" THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 268, no. 10, 1993, pages 7075-7080, XP002132977 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7067312B1 (en) 2001-09-19 2006-06-27 Myriad Genetics, Inc. PN7718 nucleic acids and use thereof
EP1572102A2 (en) * 2002-11-01 2005-09-14 Decode Genetics EHF. Human type ii diabetes gene-kv channel-interacting protein (kchip1) located on chromosome 5
EP1572102A4 (en) * 2002-11-01 2006-06-14 Decode Genetics Ehf HUMAN DIABETES TYPE II: PATHWAY OF THE KV-PROTEIN INTERACTION GENE (KCHIP1) LOCATED ON CHROMOSOME 5
CN102272603A (zh) * 2008-11-06 2011-12-07 巴斯夫欧洲公司 杀虫剂的筛选测定法

Also Published As

Publication number Publication date
IL154962A0 (en) 2003-10-31
EP1322759A2 (en) 2003-07-02
BR0114383A (pt) 2005-04-12
CA2420960A1 (en) 2002-04-04
JP2004525610A (ja) 2004-08-26
NO20031369D0 (no) 2003-03-26
MXPA03002557A (es) 2004-09-10
NO20031369L (no) 2003-05-22
KR20030074604A (ko) 2003-09-19
WO2002026984A3 (en) 2003-03-13
CZ20031154A3 (cs) 2003-09-17
EA200300420A1 (ru) 2004-04-29
CN1498271A (zh) 2004-05-19
AU2001296393A1 (en) 2002-04-08

Similar Documents

Publication Publication Date Title
US8133689B2 (en) Members of the capsaicin/vanilloid receptor family of proteins and uses thereof
US6518398B1 (en) ERG potassium channel
US6780987B1 (en) β-cap73 control of normal and abnormal cell migration
US6225085B1 (en) LRSG protein and nucleic acid molecules and uses therefor
WO2000052164A2 (en) Potassium channel molecules and uses therefor
AU775713B2 (en) Potassium channel interactors and uses therefor
US6361971B1 (en) Nucleic acid molecules encoding potassium channel interactors and uses therefor
EP1322759A2 (en) Potassium channel interactors and uses therefor
US7517659B2 (en) Potassium channel molecules and uses therefor
AU4101599A (en) Novel secreted and membrane-associated proteins and uses therefor
US6689581B1 (en) Potassium channel interactors and uses therefor
US7556938B1 (en) Nucleic acids encoding potassium channel interactors
US20020081658A1 (en) 18610, a novel human transient receptor and uses thereof
US7439029B2 (en) Human 9q polypeptides method
US6756212B1 (en) Isolated proteins and nucleic acid molecules having homology to the NIP2 protein and uses thereof
ZA200103995B (en) Potassium channel interactors and uses therefor.
WO2002026983A2 (en) 56115, a novel human twik potassium channel and uses therefor
US20030049727A1 (en) 25658, a novel human calcium channel subunit and uses thereof
US20020177148A1 (en) FBH58295FL, a novel human amino acid transporter and uses thereof
EP1792913A1 (en) Novel secreted and membrane-associated proteins and uses therefor
WO2000017236A2 (en) Apoptosis related protein and uses therefor
WO2001081416A2 (en) Human ion channel and uses thereof

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PH PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 154962

Country of ref document: IL

WWE Wipo information: entry into national phase

Ref document number: 2420960

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: PA/a/2003/002557

Country of ref document: MX

WWE Wipo information: entry into national phase

Ref document number: 1020037004460

Country of ref document: KR

Ref document number: 2002530747

Country of ref document: JP

WWE Wipo information: entry into national phase

Ref document number: 525282

Country of ref document: NZ

WWE Wipo information: entry into national phase

Ref document number: 2001296393

Country of ref document: AU

WWE Wipo information: entry into national phase

Ref document number: 2001977260

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: PV2003-1154

Country of ref document: CZ

WWE Wipo information: entry into national phase

Ref document number: 200300420

Country of ref document: EA

WWE Wipo information: entry into national phase

Ref document number: 018191681

Country of ref document: CN

WWP Wipo information: published in national office

Ref document number: 2001977260

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWP Wipo information: published in national office

Ref document number: PV2003-1154

Country of ref document: CZ

WWP Wipo information: published in national office

Ref document number: 1020037004460

Country of ref document: KR

WWR Wipo information: refused in national office

Ref document number: PV2003-1154

Country of ref document: CZ

WWW Wipo information: withdrawn in national office

Ref document number: 2001977260

Country of ref document: EP