WO2001066743A2 - Nouvelle molecule de canal sodique et son utilisation - Google Patents

Nouvelle molecule de canal sodique et son utilisation Download PDF

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WO2001066743A2
WO2001066743A2 PCT/US2001/006933 US0106933W WO0166743A2 WO 2001066743 A2 WO2001066743 A2 WO 2001066743A2 US 0106933 W US0106933 W US 0106933W WO 0166743 A2 WO0166743 A2 WO 0166743A2
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twik
ofthe
nucleic acid
polypeptide
protein
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PCT/US2001/006933
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WO2001066743A3 (fr
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Rory A. J. Curtis
Maria Alexandra Glucksmann
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Millennium Pharmaceuticals, Inc.
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Priority to AU2001241978A priority Critical patent/AU2001241978A1/en
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Publication of WO2001066743A3 publication Critical patent/WO2001066743A3/fr

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    • 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

Definitions

  • K + channels are ubiquitous proteins which are involved in the setting ofthe resting membrane potential as well as in the modulation ofthe electrical activity of cells.
  • K + channels influence action potential waveforms, firing frequency, and neurotransmitter secretion (Rudy, B. (1988) Neuroscience, 25, 729-749; Hille, B. (1992) Ionic Channels of Excitable Membranes, 2nd Ed.).
  • non- excitable cells they are involved in hormone secretion, cell volume regulation and potentially in cell proliferation and differentiation (Lewis et al. (1995) Annu. Rev. Immunol, 13, 623-653).
  • K + channels arises mainly from the existence of a great number of genes coding for pore-forming subunits, as well as for other associated regulatory subunits.
  • Two main structural families of pore-forming subunits have been identified. The first one consists of subunits with a conserved hydrophobic core containing six transmembrane domains (TMDs). These K + channel ⁇ subunits participate in the formation of outward rectifier voltage-gated (Kv) and Ca + -dependent
  • the fourth TMD contains repeated positive charges involved in the voltage gating of these channels and hence in their outward rectification (Logothetis et al. (1992) Neuron, 8, 531-540; Bezanilla et al. (1994) Biophys. J. 66, 1011-1021).
  • the second family of pore-forming subunits have only two TMDs. They are essential subunits of inward-rectifying (IRK), G-protein-coupled (GIRK) and ATP- sensitive (K ⁇ TP) K + channels.
  • IRK inward-rectifying
  • GIRK G-protein-coupled
  • K ⁇ TP ATP- sensitive
  • P domain A conserved domain, called the P domain, is present in all members of both families (Pongs, O. (1993) J Membr. Biol, 136, 1-8; Heginbotham et al. (1994) Biophys. J. 66,1061-1067; Mackinnon, R. (1995) Neuron, 14, 889-892; Pascual et al, (1995) Neuron., and 14, 1055-1063).
  • This domain is an essential element ofthe aqueous K + -selective pore. In both groups, the assembly of four subunits is necessary to form a functional K + channel (Mackinnon, R. (1991) Nature, 350, 232- 235; Yang et al, (1995) Neuron, 15, 1441-1447.
  • the domain responsible for this selective association is localized in the N-terminal region and is conserved between members ofthe same subgroup. This domain is necessary for hetero- but not homomultimeric assembly within a subfamily and prevents co-assembly between subfamilies. Recently, pore-forming subunits with two TMDs were also shown to co-assemble to form heteropolymers (Duprat et al. (1995) Biochem. Biophys. Res. Commun., 212, 657-663. This heteropolymerization seems necessary to give functional GIRKs. IRKs are active as homopolymers but also form heteropolymers.
  • TWIK-1 New structural types of K + channels were identified recently in both humans and yeast. These channels have two P domains in their functional subunit instead of only one (Ketchum et al. (1995) Nature, 376, 690-695; Lesage et al. (1996) J Biol. Chem, 271, 4183-4187; Lesage et ⁇ . (1996) EMBO J., 15, 1004-1011; Reid et al. (1996) Receptors Channels 4, 51-62).
  • the human channel called TWIK-1 has four TMDs. TWIK-1 is expressed widely in human tissues and is particularly abundant in the heart and the brain. TWIK-1 currents are time independent and inwardly rectifying. These properties suggest that TWIK-1 channels are involved in the control ofthe background K + membrane conductance (Lesage et ⁇ . (1996) EMBO J., 15, 1004-1011).
  • TWIK-6 nucleic acid and protein molecules novel members ofthe TWIK (for Tandem of P domains in a Weak Inward rectifying K + channel) family of potassium channels, referred to herein as TWIK-6 nucleic acid and protein molecules.
  • the TWIK-6 molecules ofthe present invention are useful as targets for developing modulating agents to regulate a variety of cellular processes. Accordingly, in one aspect, this invention provides isolated nucleic acid molecules encoding TWIK proteins or biologically active portions thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection of TWIK- encoding nucleic acids.
  • a TWIK-6 nucleic acid molecule ofthe invention is at least 50%, 55%, 60%, 65%, 70%, 73%, 74%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the nucleotide sequence (e.g., to the entire length ofthe nucleotide sequence) shown in SEQ ID NO:l or 3 or SEQ ID NO:4 or 6 or the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC as Accession Number , or a complement thereof.
  • the isolated nucleic acid molecule includes the nucleotide sequence shown SEQ ID NO:l or 3, or SEQ ID NO:4 or 6, or a complement thereof.
  • the nucleic acid molecule includes SEQ ID NO: 3 or 6 and nucleotides 1-102 of SEQ ID NO:l or 4.
  • the nucleic acid molecule includes SEQ ID NO:3 or 6 and nucleotides 1306-1528 of SEQ ID NO:l or 4.
  • the nucleic acid molecule consists ofthe nucleotide sequence shown in SEQ ID NO:l or 3 or SEQ ID NO:4 or 6.
  • the nucleic acid molecule includes a fragment of at least 1051 nucleotides (e.g., 1051 contiguous nucleotides) ofthe nucleotide sequence of SEQ ID NO: 1 or 3 or SEQ ID NO:4 or 6, or a complement thereof.
  • a TWIK-6 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 or 5 or an amino acid sequence encoded by the DNA insert ofthe plasmid deposited with ATCC as Accession Number .
  • a TWIK-6 nucleic acid molecule includes a nucleotide sequence encoding a protein having an amino acid sequence at least 50%, 55%, 60%, 65%, 67%, 68%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the entire length ofthe amino acid sequence of SEQ ID NO: 2 or 5 or the amino acid sequence encoded by the DNA insert ofthe plasmid deposited with ATCC as Accession
  • an isolated nucleic acid molecule encodes the amino acid sequence of human TWIK-6.
  • the nucleic acid molecule includes a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID NO:2 or 5 or the amino acid sequence encoded by the
  • the nucleic acid molecule is at least 1051 nucleotides in length. In a further preferred embodiment, the nucleic acid molecule is at least 1051 nucleotides in length and encodes a protein having a TWIK-6 activity (as described herein).
  • nucleic acid molecules preferably TWIK-6 nucleic acid molecules, which specifically detect TWIK-6 nucleic acid molecules relative to nucleic acid molecules encoding non-TWIK-6 proteins.
  • a nucleic acid molecule is at least 1050, 1051 - 1100, 1100-1150, 1150-1200, 1200-1250, 1250-1300, 1300-1350, 1350-1400, 1400-1450, 1450-1500, 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 or 4, the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC as Accession Number , or a complement thereof.
  • the nucleic acid molecules are at least 15 (e.g., 15 contiguous) nucleotides in length and hybridize under stringent conditions to nucleotides 1-156 of SEQ ID NO:l or 4. In other preferred embodiments, the nucleic acid molecules comprise nucleotides 1-156 of SEQ ID NO:l or 4.
  • the nucleic acid molecule encodes a naturally occurring allelic variant (e.g., an allelic variant as described in Example 1, where a single base change of A to G at nucleotide position 596 was identified) of a polypeptide comprising the amino acid sequence of SEQ ID NO:2 or an amino acid sequence encoded by the DNA insert ofthe plasmid deposited with ATCC as Accession Number , wherein the nucleic acid molecule hybridizes to a nucleic acid molecule comprising SEQ ID NO:l or 3 or SEQ ID NO:4 or 6 under stringent conditions.
  • Another embodiment ofthe invention provides an isolated nucleic acid molecule which is antisense to a TWIK-6 nucleic acid molecule, e.g., the coding strand of a TWIK-6 nucleic acid molecule.
  • Another aspect ofthe invention provides a vector comprising a TWIK-6 nucleic acid molecule.
  • the vector is a recombinant expression vector.
  • the invention provides a host cell containing a vector ofthe invention.
  • the invention provides a host cell containing a nucleic acid molecule ofthe invention.
  • the mvention also provides a method for producing a protein, preferably a TWIK-6 protein, by culturing in a suitable medium, a host cell, e.g., a mammalian host cell such as a non-human mammalian cell, ofthe invention containing a recombinant expression vector, such that the protein is produced.
  • an isolated TWIK-6 protein includes at least one transmembrane domain.
  • an isolated TWIK-6 protein includes a TWIK-related ion channel domain.
  • an isolated TWIK-6 protein includes a potassium channel protein domain.
  • an isolated TWIK-6 protein includes a P-loop domain.
  • an isolated TWIK-6 protein includes at least one transmembrane domain and one or more ofthe following domains: a TWIK-related ion channel domain, a P- loop domain, and a potassium channel protein domain.
  • an isolated TWIK-6 protein includes at least one transmembrane domain, a TWIK-related ion channel domain, a P-loop domain, and a potassium channel protein domain.
  • a TWIK-6 protein includes at least one transmembrane domain and has an amino acid sequence at least about 50%, 55%, 60%, 65%, 67%, 68%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:2 or 5, or the amino acid sequence encoded by the DNA insert ofthe plasmid deposited with ATCC as Accession Number .
  • a TWIK-6 protein includes a TWIK-related ion channel domain and has an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:2 or 5, or the amino acid sequence encoded by the
  • a TWIK-6 protein includes a potassium channel protein domain and has an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:2 or 5, or the amino acid sequence encoded by the DNA insert ofthe plasmid deposited with ATCC as Accession Number .
  • a TWIK-6 protein includes at least one P-loop domain and has an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:2 or 5, or the amino acid sequence encoded by the DNA insert ofthe plasmid deposited with ATCC as Accession Number .
  • a TWIK-6 protein includes at least one transmembrane domain and one or more of a TWIK-related ion channel domain,, a P-loop domain, and/or a potassium channel protein domain, and has an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:2 or 5, or the amino acid sequence encoded by the DNA insert ofthe plasmid deposited with ATCC as Accession Number .
  • a TWIK-6 protein includes at least one transmembrane domain and has a TWIK-6 activity (as described herein).
  • a TWIK-6 protein includes at least one transmembrane 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.T or 3 or SEQ ID NO:4 or 6.
  • a TWIK-6 protein includes a TWIK-related ion channel 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: 1 or 3 or 4 or 6.
  • a TWIK-6 protein includes a P-loop 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: 1 or 3 or SEQ ID NO:4 or 6.
  • a TWIK-6 protein includes a potassium channel protein 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 or 3 or SEQ ID NO:4 or 6.
  • a TWIK-6 protein includes at least one transmembrane domain and one or more of a TWIK-related ion channel domain, a P-loop domain, and/or a potassium channel protein 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: 1 or 3 or SEQ ID NO:4 or 6.
  • the invention features fragments ofthe protein having the amino acid sequence of SEQ ID NO:2 or 5, wherein the fragment comprises at least 19 amino acids (e.g., contiguous amino acids) ofthe amino acid sequence of SEQ ID NO: 2 or 5, or an amino acid sequence encoded by the DNA insert ofthe plasmid deposited with the ATCC as Accession Number .
  • a fragment comprises at least 19 amino acids (e.g., contiguous amino acids) ofthe amino acid sequence of SEQ ID NO: 2 or 5, or an amino acid sequence encoded by the DNA insert ofthe plasmid deposited with the ATCC as Accession Number .
  • TWIK-6 protein has the amino acid sequence of SEQ ID NO: 2 or 5.
  • the invention features a TWIK-6 protein which is encoded by a nucleic acid molecule consisting of a nucleotide sequence at least about 50%, 55%, 60%, 65%, 70%, 73%, 74%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to a nucleotide sequence of SEQ ID NO: 1 or 3, or SEQ ID NO:4 or 6, or a complement thereof.
  • This invention further features a TWIK-6 protein, which is encoded by a nucleic acid molecule consisting of a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:l or 3, or SEQ ID NO:4 or 6, or a complement thereof.
  • the proteins ofthe present invention or portions thereof, e.g., biologically active portions thereof, can be operatively linked to a non-TWIK-6 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 ofthe invention, preferably TWIK-6 proteins.
  • the TWIK-6 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 TWTK-6 nucleic acid molecule, protein, or polypeptide in a biological sample by contacting the biological sample with an agent capable of detecting a TWIK- 6 nucleic acid molecule, protein, or polypeptide such that the presence of a TWIK-6 nucleic acid molecule, protein or polypeptide is detected in the biological sample.
  • the present invention provides a method for detecting the presence of TWIK-6 activity in a biological sample by contacting the biological sample with an agent capable of detecting an indicator of TWIK-6 activity such that the presence of TWTK-6 activity is detected in the biological sample.
  • the invention provides a method for modulating TWIK-6 activity comprising contacting a cell capable of expressing TWIK-6 with an agent that modulates TWIK-6 activity such that TWIK-6 activity in the cell is modulated.
  • the agent inhibits TWIK-6 activity.
  • the agent stimulates TWIK-6 activity.
  • the agent is an antibody that specifically binds to a TWIK-6 protein.
  • the agent modulates expression of TWIK-6 by modulating transcription of a TWIK-6 gene or translation of a TWIK-6 mRNA.
  • the agent is a nucleic acid molecule having a nucleotide sequence that is antisense to the coding strand of a TWIK-6 mRNA or a TWIK-6 gene.
  • the methods ofthe present invention are used to treat a subject having a disorder characterized by aberrant or unwanted TWIK-6 protein or nucleic acid expression or activity by administering an agent which is a TWIK-6 modulator to the subject.
  • the TWIK-6 modulator is a TWIK-6 protein.
  • the TWIK-6 modulator is a TWIK-6 nucleic acid molecule.
  • the TWIK-6 modulator is a peptide, peptidomimetic, or other small molecule.
  • the disorder characterized by aberrant or unwanted TWTK-6 protein or nucleic acid expression is a CNS disorder, such as a cognitive or neurodegenerative disorder.
  • the disorder characterized by aberrant or unwanted TWIK-6 protein or nucleic acid expression is a cardiovascular disorder. In another preferred embodiment, the disorder characterized by aberrant or unwanted TWIK-6 protein or nucleic acid expression is a muscular disorder. In another embodiment, the disorder characterized by aberrant or unwanted TWIK-6 activity is a cell proliferation, growth, differentiation, or migration disorder.
  • the present invention also provides diagnostic assays 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 TWIK-6 protein; (ii) mis-regulation of the gene; and (iii) aberrant post-translational modification of a TWIK-6 protein, wherein a wild-type form ofthe gene encodes a protein with a TWIK-6 activity.
  • the invention provides methods for identifying a compound that binds to or modulates the activity of a TWIK-6 protein, by providing an indicator composition comprising a TWIK-6 protein having TWIK-6 activity, contacting the indicator composition with a test compound, and detennining the effect ofthe test compound on TWIK-6 activity in the indicator composition to identify a compound that modulates the activity of a TWIK-6 protein.
  • Figure 1 depicts the cDNA sequence and predicted amino acid sequence of human TWIK-6 (A 596 ).
  • the nucleotide sequence corresponds to nucleic acids 1 to 1528 of SEQ ID NO:l.
  • the amino acid sequence corresponds to amino acids 1 to 400 of SEQ ID NO: 2.
  • the coding region without the 3' untranslated region ofthe human TWIK-6 gene is shown in SEQ ID NO: 3.
  • Figure 2 depicts the cDNA sequence and predicted amino acid sequence of human TWIK-6 (G 596 ) containing an A to G polymorphism at nucleotide 596.
  • the nucleotide sequence corresponds to nucleic acids 1 to 1528 of SEQ ID NO: 4.
  • the amino acid sequence corresponds to amino acids 1 to 400 of SEQ ID NO: 5.
  • the coding region without the 3' untranslated region ofthe human TWTK-6 gene is shown in SEQ ID NO: 6.
  • Figure 3 depicts a structural, hydrophobicity, and antigenicity analysis ofthe human TWIK-6 protein.
  • Figure 4 depicts an NBLAST alignment ofthe human TWIK-6 nucleic acid sequence with the nucleic acid sequence of Homo sapiens TWIK-related acid-sensitive K+ channel (TASK) mRNA (Accession Number AF006823) using a score of 100 and a wordlength of 12. The results show that the nucleotide sequence of human TWIK-6 is 72% identical to Accession Number AF006823 over nucleotides 200-1158.
  • TASK acid-sensitive K+ channel
  • Figure 5 depicts a BLASTX alignment ofthe human TWIK-6 predicted amino acid sequence with the amino acid sequence of Homo sapiens TWIK-related acid-sensitive K+ channel (Accession Number AF006823) using a score of 100, a BLOSUM 62 matrix, and a wordlength of 3.
  • the results show that the amino acid sequence of human TWIK-6 is 65% identical to that of Accession Number AF006823 over translated nucleotides 313- 1086, 40% identical over translated nucleotides 1265 to 1354, and 33% identical over translated nucleotides 1253 to 1369.
  • Figure 6 depicts an alignment ofthe human TWIK-6 amino acid sequence with the amino acid sequences of TWIK-related acid-sensitive K+ channel from Homo sapiens (Accession Number AF006823) and TWIK-related acid-sensitive K+ channel from Rattus norvegicus (Accession Number AF031384) using the CLUSTAL W (1.74) multiple sequence alignment program.
  • Figure 7 depicts the results of a search which was performed against the MEMS AT database and which resulted in the identification of four "transmembrane domains" and two "Pore-loops" in the human TWIK-6 protein (SEQ ID NO:2 or SEQ ID NO:5).
  • Figure 8 depicts the results of a search which was performed against the ProDom database and which resulted in the identification of a "potassium channel protein domain" and a TWIK-related ion channel domain in the human TWIK-6 protein (SEQ ID NO:2 or 5).
  • the present invention is based, at least in part, on the discovery of novel molecules, referred to herein as TWIK-6 nucleic acid and protein molecules, which are novel members ofthe TWIK (for Tandem of P domains in a Weak Inward rectifying K + channel) family of potassium channels.
  • novel molecules are capable of, for example, modulating a potassium channel mediated activity in a cell, e.g., a neuronal cell, a muscle cell (e.g., a cardiac muscle), or a thymus cell.
  • a “potassium channel” includes a protein or polypeptide which is involved in receiving, conducting, and transmitting signals in an electrically excitable cell, e.g., a neuronal cell or a muscle cell.
  • Potassium channels are potassium ion selective, and can determine membrane excitability (the ability of, for example, a neuron to respond to a stimulus and convert it into an impulse). Potassium channels can also influence the resting potential of membranes, wave forms and frequencies of action potentials, and thresholds of excitation.
  • Potassium channels are typically expressed in electrically excitable cells, e.g., neurons, muscle, endocrine, and egg cells, and may form heteromultimeric structures, e.g., composed of pore-forming ⁇ and cytoplasmic ⁇ subunits. Potassium channels may also be found in nonexcitable cells (e.g., thymus cells), where they may play a role in, e.g., signal transduction. Examples of potassium channels include: (1) the voltage-gated potassium channels, (2) the ligand-gated potassium channels, e.g. , neurotransmitter-gated potassium channels, and (3) cyclic- nucleotide-gated potassium channels.
  • Noltage-gated and ligand-gated potassium channels are expressed in the brain, e.g., in brainstem monoaminergic and forebrain cholinergic neurons, where they are involved in the release of neurotransmitters, or in the dendrites of hippocampal and neocortical pyramidal cells, where they are involved in the processes of learning and memory formation.
  • brainstem monoaminergic and forebrain cholinergic neurons where they are involved in the release of neurotransmitters, or in the dendrites of hippocampal and neocortical pyramidal cells, where they are involved in the processes of learning and memory formation.
  • 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; cellular proliferation, growth, differentiation, or migration, and emotional, intellectual (e.g., learning and memory), or motor processes.
  • Examples of potassium channel associated disorders include CNS disorders such as cognitive and neurodegenerative disorders, examples of which include, but are not limited to, Alzheimer's disease, dementias related to Alzheimer's disease (such as Pick's disease), Parkinson's and other Lewy diffuse body diseases, senile dementia, Huntington's disease, Gilles de la Tourette's syndrome, multiple sclerosis, amyotrophic lateral sclerosis, progressive supranuclear palsy, epilepsy, and Jakob-Creutzfieldt disease; autonomic function disorders such as hypertension and sleep disorders, and neuropsychiatric disorders, such as depression, schizophrenia, schizoaffective disorder, korsakoff s psychosis, mania, anxiety disorders, or phobic disorders; learning or memory disorders, e.g.
  • CNS disorders such as cognitive and neurodegenerative disorders, examples of which include, but are not limited to, Alzheimer's disease, dementias related to Alzheimer's disease (such as Pick's disease), Parkinson's and other Lewy diffuse body diseases,
  • CNS-related disorders include, for example, those listed in the American Psychiatric Association's Diagnostic and Statistical manual of Mental Disorders (DSM), the most current version of which is incorporated herein by reference in its entirety.
  • Cardiovascular system disorders in which the TWIK-6 molecules of the invention may be directly or indirectly involved 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, Jervell syndrome, Lange-Nielsen syndrome, long-QT syndrome, congestive heart failure, sinus node dysfunction, angina, heart failure, hypertension, atrial fibrillation, atrial flutter, dilated cardiomyopathy, idiopathic cardiomyopathy, myocardial infarction, coronary artery disease, coronary artery spasm, and arrhythmia.
  • TWIK-6-mediated or related disorders also include disorders ofthe musculoskeletal system such as paralysis and muscle weakness, e.g., ataxia, myotonia, and myokymia.
  • Potassium channel disorders also include cellular proliferation, growth, differentiation, or migration disorders.
  • Cellular proliferation, growth, differentiation, or migration disorders include those disorders that affect cell proliferation, growth, differentiation, or migration processes.
  • a "cellular proliferation, growth, differentiation, or migration process" is a process by which a cell increases in number, size or content, by which a cell develops a specialized set of characteristics which differ from that of other cells, or by which a cell moves closer to or further from a particular location or stimulus.
  • the TWIK-6 molecules ofthe present invention are involved in signal iransduction mechanisms, which are known to be involved in cellular growth, differentiation, and migration processes.
  • the TWIK-6 molecules may modulate cellular growth, differentiation, or migration, and may play a role in disorders characterized by aberrantly regulated growth, differentiation, or migration.
  • Such disorders include cancer, e.g., carcinoma, sarcoma, or leukemia; tumor angiogenesis and metastasis; skeletal dysplasia; neuronal deficiencies resulting from impaired neural induction and patterning; 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, Jakob-Creutzfieldt disease, or AIDS related dementia; hepatic disorders; cardiovascular disorders; and hematopoietic and/or myeloproliferative disorders.
  • TWIK-6-associated or related disorders also include disorders of tissues in which TWIK-6 protein is expressed, e.g., thymus, salivary gland, primary cultured osteoblasts, and ovarian epithelium tumor cells. Such disorders include, for example, proliferative disorders.
  • a "potassium channel mediated activity" includes an activity wliich involves a potassium channel, e.g., a potassium channel in a neuronal cell, a muscle cell, or a thymus cell associated with receiving, conducting, and transmitting signals in, for example, the nervous system.
  • Potassium channel mediated activities include release of neurotransmitters, e.g., dopamine or norepinephrine, from cells, e.g., neuronal cells; modulation of resting potential of membranes, wave forms and frequencies of action potentials, and thresholds of excitation; participation in signal transduction pathways, 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 muscle cells.
  • neurotransmitters e.g., dopamine or norepinephrine
  • 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 common structural domain or motif and having sufficient amino acid or nucleotide sequence homology as defined herein.
  • family members can be naturally or non- naturally occurring and can be from either the same or different species.
  • a 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, e.g., monkey proteins.
  • Members of a family may also have common functional characteristics.
  • the family of TWIK-6 proteins comprises at least one "transmembrane domain” and preferably four transmembrane domains.
  • transmembrane domain includes an amino acid sequence of about 15 amino acid residues in length which spans the plasma membrane. More preferably, a transmembrane domain includes about at least 20, 25, 30, 35, 40, or 45 amino acid residues and spans the plasma membrane. Transmembrane domains are rich in hydrophobic residues, and typically have an alpha-helical structure.
  • At least 50%, 60%, 70%, 80%, 90%, 95% or more ofthe amino acids of a transmembrane domain are hydrophobic, e.g., leucines, isoleucines, tyrosines, or tryptophans.
  • Transmembrane domains are described in, for example, Zaeaux W.N. et al, (1996) Annual Rev. Neurosci. 19: 235-263, the contents of which are incorporated herein by reference.
  • Amino acid residues 78-96, 178-195, 229-248, and 290-314 ofthe native TWIK-6 protein are predicted to comprise transmembrane domains (see Figure 7). Accordingly, TWIK-6 proteins having at least 50-60% homology, preferably about 60-70%, more preferably about 70-80%, or about 80-90% homology with a transmembrane domain of human TWIK-6 are within the scope ofthe invention.
  • a TWTK-6 molecule ofthe present invention is identified based on the presence of a Pore loop or P-loop.
  • the term "Pore loop” or "P-loop” includes amino acid sequence of about 15-45 amino acid residues in length, preferably about 15-35 amino acid residues in length, and most preferably about 15-25 amino acid residues in length, which is hydrophobic and which is involved in lining the potassium channel pore.
  • a P-loop is typically found between transmembrane domains of potassium channels and is believed to be a major determinant of ion selectivity in potassium channels.
  • P-loops contain a G- [HYDROPHOBIC AMINO ACIDJ-G sequence, e.g., a GYG, GLG, or GFG sequence.
  • G- [HYDROPHOBIC AMINO ACIDJ-G sequence e.g., a GYG, GLG, or GFG sequence.
  • P-loops are described in, for example, Warrnke et al. (1991) Science 252:1560-1562; Zaelles W.N. et al., (1996) Annual Rev. Neuronsci. 19:235-63 (Pongs, O. (1993) J. Membr. Biol, 136, 1-8; Heginbotham et al. (1994) Biophys. J. 66,1061-1067; Mackinnon, R.
  • Amino acid residues 150-166 and 260-278 of SEQ ID NO: 2 comprise P-loop domains, as do amino acid residues 150-166 and 260-278 of SEQ ID NO: 5.
  • the human TWIK-6 gene contains a nucleotide polymorphism at nucleotide 596 (A to G) which results in an amino acid alteration at amino acid position 165 (E to G).
  • the 165E form ofthe protein is set forth in SEQ ID NO:2, and lacks the "GYG” P-loop motif which the 165G form of the protein (SEQ ID NO:5) possesses at residues 165-167.
  • the 165E form ofthe human TWIK-6 protein (SEQ ID NO:2) has only one "G-[hydrophobic]-G” motif in its two P-loop domains
  • the 165G form ofthe human TWIK-6 protein (SEQ ID NO:5) has two "G-[hydrophobic]-G” motifs in its two P-loop domains.
  • a TWTK-6 molecule ofthe present invention is identified based on the presence of a "potassium channel protein domain" in the protein or corresponding nucleic acid molecule.
  • a “potassium channel protein domain” includes a protein domain having an amino acid sequence of about 150- 250 amino acid residues and having a bit score for the alignment ofthe sequence to the TWIK-related ion channel domain of at least 162.
  • a TWIK-related ion channel domain includes at least about 200-240, or more preferably about 232 amino acid residues, and has a bit score for the alignment ofthe sequence to the TWIK-related ion channel domain of at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160 or higher.
  • the potassium channel protein domain has been assigned ProDom entry 1641.
  • the amino acid sequence ofthe protein is searched against a database of known protein domains (e.g., the ProDom database) using the default parameters (available at http://www.toulouse.inra.fr/prodom.html).
  • a search was performed against the ProDom database resulting in the identification of a potassium channel protein domain in the amino acid sequence of human TWIK-6 (SEQ ID NO: 2 or 5) at about residues 81-312 of SEQ ID NO: 2 or 5.
  • the results ofthe search are set forth in Figure 8.
  • a TWIK-6 molecule ofthe present invention is identified based on the presence of a "TWIK-related ion channel domain" in the protein or corresponding nucleic acid molecule.
  • TWIK-related ion channel domain includes a protein domain having an amino acid sequence of about 25- 125 amino acid residues and having a bit score for the alignment ofthe sequence to the TWIK-related ion channel domain of at least 149.
  • a TWIK-related ion channel domain includes at least about 50-100, or more preferably about 70 amino acid residues, and has a bit score for the alignment ofthe sequence to the TWIK-related ion channel domain of at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145 or higher.
  • the TWIK-related ion channel domain has been assigned ProDom entry 11540.
  • the amino acid sequence ofthe protein is searched against a database of known protein domains (e.g., the ProDom database) using the default parameters (available at http://www.toulouse.inra.fr/prodom.html).
  • a search was performed against the ProDom database resulting in the identification of a TWIK-related ion channel domain in the amino acid sequence of human TWIK-6 (SEQ ID NO: 2 or 5) at about residues 75-144 of SEQ ID NO: 2 or SEQ ID NO:5.
  • the results ofthe search are set forth in Figure 8.
  • the TWIK-6 molecules ofthe invention include at least one transmembrane domain, at least one P-loop, at least one TWIK-related ion channel domain, and at least one potassium channel protein domain.
  • Isolated proteins ofthe present invention preferably TWIK-6 proteins, have an amino acid sequence sufficiently identical to the amino acid sequence of SEQ ID NO: 2 or 5 or are encoded by a nucleotide sequence sufficiently identical to SEQ ID NO:l or 3 or SEQ ID NO:4 or 6.
  • 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% homology, preferably 60% homology, more preferably 70%-80%, and even more preferably 90-95% homology across the amino acid sequences ofthe 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%. homology and share a common functional activity are defined herein as sufficiently identical.
  • a TWIK-6 activity is a direct activity, such as an association with a TWIK-6-target molecule.
  • a "target molecule” or “binding partner” is a molecule with which a TWIK-6 protein binds or interacts in nature, such that TWIK-6-mediated function is achieved.
  • a TWIK-6 target molecule can be a non-TWIK-6 molecule or a TWIK-6 protein or polypeptide ofthe present invention.
  • a TWIK-6 target molecule is a TWIK-6 ligand, e.g., a potassium channel pore-forming subunit or a potassium channel ligand.
  • a TWTK-6 activity is an indirect activity, such as a cellular signaling activity mediated by interaction ofthe TWIK-6 protein with a TWTK-6 ligand. The biological activities of TWIK-6 are described herein.
  • the TWIK-6 proteins ofthe present invention can have one or more ofthe following activities: (1) interacting with a non-TWIK protein molecule; (2) activating a TWIK-dependent signal transduction pathway; (3) modulating the release of neurotransmitters; (4) modulating membrane excitability; (5) influencing the resting potential of membranes, wave forms and frequencies of action potentials, and thresholds of excitation, and (6) modulating processes which underlie learning and memory, such as integration of sub-threshold synaptic responses and the conductance of back-propagating action potentials.
  • another embodiment ofthe invention features isolated TWIK-6 proteins and polypeptides having a TWIK-6 activity.
  • Preferred proteins are TWIK-6 proteins having at least one transmembrane domain, and, preferably, a TWTK-6 activity.
  • Other preferred proteins are TWIK-6 proteins having a TWIK-related ion channel domain and, preferably, a TWIK-6 activity.
  • Other preferred proteins are TWIK-6 proteins having a P-loop domain and, preferably, a TWTK-6 activity.
  • Other preferred proteins are TWIK-6 proteins having a potassium channel protein domain and, preferably, a TWIK-6 activity.
  • Yet other preferred proteins are TWIK-6 proteins having at least one transmembrane domain, a TWIK-related ion channel domain, a P-loop domain, and a potassium channel protein domain and, preferably, a TWIK-6 activity.
  • Additional preferred proteins have at least one transmembrane domain, and one or more of a TWIK-related ion channel domain, a P-loop domain, and/or a potassium channel protein domain, and are, preferably, 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: 1 or 3 or SEQ ID NO:4 or 6.
  • the nucleotide sequence ofthe isolated human TWIK-6 cDNA and the predicted amino acid sequence ofthe human TWIK-6 polypeptide are shown in Figure 1 and in SEQ ID NOs.T and 2, respectively.
  • the nucleotide sequences ofthe human TWTK-6 containing an A to G polymorphism at nucleotide 596 and the predicted amino acid sequence ofthe human TWIK-6 polypeptide containing an E to G substitution at amino acid position 165 are shown in Figure 2 and in SEQ ID NO:s 4 and 6, respectively.
  • a plasmid containing the nucleotide sequence encoding human TWIK-6 was deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, NA 20110-2209, on and assigned Accession Number . This deposit will be maintained under the terms ofthe Budapest Treaty on the International Recognition ofthe Deposit of Microorganisms for the Purposes of Patent Procedure. This deposit was made merely as a convenience for those of skill in the art and is not an admission that a deposit is required under 35 U.S.C. ⁇ 112.
  • the human TWIK-6 gene which is approximately 1528 nucleotides in length, encodes a protein having a molecular weight of approximately 45.3 kD and which is approximately 400 amino acid residues in length. Narious aspects ofthe invention are described in further detail in the following subsections:
  • nucleic acid molecules that encode TWIK-6 proteins or biologically active portions thereof, as well as nucleic acid fragments sufficient for use as hybridization probes to identify TWIK-6-encoding nucleic acid molecules (e.g., TWIK-6 mRNA) and fragments for use as PCR primers for the amplification or mutation of TWIK-6 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 ofthe DNA or RNA generated using nucleotide analogs.
  • the nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.
  • isolated nucleic acid molecule includes nucleic acid molecules which are separated from other nucleic acid molecules which are present in the natural source ofthe nucleic acid.
  • isolated includes nucleic acid molecules which are separated from the chromosome with which the genomic DNA is naturally associated.
  • 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 ofthe organism from which the nucleic acid is derived.
  • the isolated TWIK-6 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 ofthe 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 ofthe present invention e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:l or 3, or SEQ ID NO:4 or 6, or the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC as
  • TWIK-6 nucleic acid molecules can be isolated using standard hybridization and cloning techniques (e.g., as described 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).
  • nucleic acid molecule encompassing all or a portion of SEQ ID NO:l or 3, or SEQ ID NO:4 or 6, or the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC as Accession Number can be isolated by the polymerase chain reaction (PCR) using synthetic oligonucleotide primers designed based upon the sequence of SEQ ID NO:l or 3, or SEQ ID NO:4 or 6, or the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC as Accession Number
  • a nucleic acid ofthe 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 TWIK-6 nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
  • an isolated nucleic acid molecule ofthe invention comprises the nucleotide sequence shown in SEQ ID NO:l or 4.
  • sequences of SEQ ID NO.T and SEQ ID NO:4 correspond to alternate polymorphic forms ofthe human TWIK-6 cDNA.
  • This cDNA comprises sequences encoding the human TWIK-6 protein (i.e., "the coding region", from nucleotides 103-1305), as well as 5' untranslated sequences (nucleotides 1-102) and 3' untranslated sequences (nucleotides 1306-1528).
  • the nucleic acid molecule can comprise only the coding region of SEQ ID NO.T or 4 (e.g., nucleotides 103-1305, corresponding to SEQ ID NO:3 or 6, respectively).
  • an isolated nucleic acid molecule ofthe mvention comprises a nucleic acid molecule which is a complement ofthe nucleotide sequence shown in SEQ ID NO:l or 3, or SEQ ID NO:4 or 6, or the nucleotide sequence of the DNA insert o the plasmid deposited with ATCC as Accession Number , or a portion of any of these nucleotide sequences.
  • a nucleic acid molecule which is complementary to the nucleotide sequence shown in SEQ ID NO:l or 3, or SEQ ID NO:4 or 6, or the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC as Accession Number is one which is sufficiently complementary to the nucleotide sequence shown in SEQ ID NO:l or 3, or SEQ ID NO:4 or 6, or the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC as
  • an isolated nucleic acid molecule ofthe present invention comprises a nucleotide sequence which is at least about 50%, 55%, 60%, 65%, 70%, 73%, 74%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the entire length ofthe nucleotide sequence shown in SEQ ID NOT or 3, or SEQ ID NO:4 or 6, or the entire length ofthe nucleotide sequence ofthe DNA insert of the plasmid deposited with ATCC as Accession Number , or a portion of any of these nucleotide sequences.
  • nucleic acid molecule ofthe invention can comprise only a portion of the nucleic acid sequence of SEQ ID NOT or 3, or SEQ ID NO:4 or 6, or the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC as
  • the nucleotide sequence determined from the cloning of the TWIK-6 gene allows for the generation of probes and primers designed for use in identifying and/or cloning other TWIK-6 family members, as well as TWIK-6 homologues from other species.
  • 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 NOT or 3, or the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC as Accession Number , of an anti-sense sequence of SEQ ID NOT or 3, or SEQ ID NO:4 or 6, or the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC as Accession Number , or of a naturally occurring allelic variant or mutant of SEQ ID NOT or 3, or
  • a nucleic acid molecule ofthe present invention comprises a nucleotide sequence which is greater than 1050, 1051-1100, 1100-1150, 1150-1200, 1200-1250, 1250-1300, 1300-1350, 1350- 1400, 1400-1450, 1450-1500 or more nucleotides in length and hybridizes under stringent hybridization conditions to a nucleic acid molecule of SEQ ID NOT or 3, or SEQ ID NO:4 or 6, or the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC as Accession Number .
  • Probes based on the TWTK-6 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 TWIK-6 protein, such as by measuring a level of a TWIK- 6-encoding nucleic acid in a sample of cells from a subject e.g., detecting TWIK-6 mRNA levels or determining whether a genomic TWIK-6 gene has been mutated or deleted.
  • a nucleic acid fragment encoding a "biologically active portion of a TWTK-6 protein” can be prepared by isolating a portion ofthe nucleotide sequence of SEQ ID NOT or 3, or SEQ ID NO:4 or 6, or the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC as Accession Number , which encodes a polypeptide having a TWIK-6 biological activity (the biological activities ofthe TWIK- 6 proteins are described herein), expressing the encoded portion ofthe TWIK-6 protein (e.g., by recombinant expression in vitro) and assessing the activity ofthe encoded portion ofthe TWIK-6 protein.
  • the invention further encompasses nucleic acid molecules that differ from the nucleotide sequence shown in SEQ ID NO: 1 or 3, or SEQ ID NO:4 or 6, or the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC as Accession Number , due to degeneracy ofthe genetic code and thus encode the same TWIK-6 proteins as those encoded by the nucleotide sequence shown in SEQ ID NOT or 3, or SEQ ID NO:4 or 6, or the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC as Accession Number .
  • an isolated nucleic acid molecule ofthe invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in SEQ ID NO:2 or 5.
  • TWTK-6 nucleotide sequences shown in SEQ ID NO or 3, or SEQ ID NO:4 or 6, or the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC as Accession Number
  • DNA sequence polymorphisms that lead to changes in the amino acid sequences ofthe TWIK-6 proteins may exist within a population (e.g., the human population).
  • Such genetic polymorphism in the TWTK-6 genes may exist among individuals within a population due to natural allelic variation.
  • the terms “gene” and “recombinant gene” refer to nucleic acid molecules which include an open reading frame encoding a TWIK-6 protein, preferably a mammalian TWIK-6 protein, and can further include non-coding regulatory sequences, and introns.
  • Allelic variants of human TWIK-6 include both functional and non-functional TWIK-6 proteins.
  • Functional allelic variants are naturally occurring amino acid sequence variants ofthe human TWIK-6 protein that maintain the ability to bind a TWIK-6 ligand or substrate and/or modulate cell proliferation and/or migration mechanisms.
  • Functional allelic variants will typically contain only conservative substitution of one or more amino acids of SEQ ID NO:2 or 5, or substitution, deletion or insertion of non-critical residues in non-critical regions ofthe protein.
  • Non-functional allelic variants are naturally occurring amino acid sequence variants ofthe human TWIK-6 protein that do not have the ability to either bind a TWIK-6 ligand and/or modulate any ofthe TWIK-6 activities described herein.
  • Nonfunctional allelic variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation ofthe amino acid sequence of SEQ ID NO:2 or 5, or a substitution, insertion or deletion in critical residues or critical regions.
  • the present invention further provides non-human orthologues ofthe human TWTK-6 protein.
  • Orthologues ofthe human TWIK-6 protein are proteins that are isolated from non-human organisms and possess the same TWTK-6 ligand binding and/or modulation of membrane excitability activities ofthe human TWIK-6 protein. Orthologues ofthe human TWIK-6 protein can readily be identified as comprising an amino acid sequence that is substantially identical to SEQ ID NO:2 or 5.
  • nucleic acid molecules encoding other TWIK-6 family members and, thus, which have a nucleotide sequence which differs from the TWIK-6 sequences of SEQ ID NOT or 3, or SEQ ID NO:4 or 6, or the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC as Accession Number are intended to be within the scope ofthe invention.
  • another TWIK-6 cDNA can be identified based on the nucleotide sequence of human TWIK-6.
  • nucleic acid molecules encoding TWIK-6 proteins from different species and which, thus, have a nucleotide sequence which differs from the TWIK-6 sequences of SEQ ID NO: 1 or 3, or SEQ ID NO: 4 or 6, or the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC as Accession Number are intended to be within the scope ofthe invention.
  • a mouse TWTK-6 cDNA can be identified based on the nucleotide sequence of a human TWTK-6.
  • Nucleic acid molecules corresponding to natural allelic variants and homologues ofthe TWTK-6 cDNAs ofthe invention can be isolated based on their homology to the TWIK-6 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. Nucleic acid molecules corresponding to natural allelic variants and homologues ofthe TWIK-6 cDNAs ofthe invention can further be isolated by mapping to the same chromosome or locus as the TWIK-6 gene.
  • an isolated nucleic acid molecule ofthe 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: 1 or 3, or SEQ ID NO:4 or 6, or the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC as Accession Number .
  • the nucleic acid is at least 1050, 1051-1100, 1100-1150, 1150-1200, 1200- 1250, 1250-1300, 1300-1350, 1350-1400, 1400-1450, 1450-1500, or more 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.
  • stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
  • a preferred, non-limiting example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2 X SSC, 0.1% SDS at 50°C, preferably at 55°C, more preferably at 60°C, and even more preferably at 65°C. Ranges intermediate to the above-recited values, e.g., at 60-65 °C or at 55-60 °C are also intended to be encompassed by the present invention.
  • an isolated nucleic acid molecule ofthe invention that hybridizes under stringent conditions to the sequence of SEQ ID NOT or 3, or SEQ ID NO:4 or 6, and 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).
  • a nucleotide sequence that occurs in nature e.g., encodes a natural protein.
  • allelic variants ofthe TWIK-6 sequences that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequences of SEQ ID NOT or 3, or SEQ ID NO:4 or 6, or the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC as Accession Number , thereby leading to changes in the amino acid sequence ofthe encoded TWIK-6 proteins, without altering the functional ability ofthe TWIK-6 proteins.
  • nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in the sequence of SEQ ID NOT or 3, or SEQ ID NO:4 or 6, or the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC as Accession Number .
  • a "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of TWIK-6 (e.g., the sequence of SEQ ID NO:2 or 5) without altering the biological activity, whereas an "essential" amino acid residue is required for biological activity.
  • amino acid residues that are conserved among the TWIK-6 proteins ofthe present invention are predicted to be particularly unamenable to alteration.
  • additional amino acid residues that are conserved between the TWIK-6 proteins ofthe present invention and other members ofthe TWIK family are not likely to be amenable to alteration.
  • another aspect ofthe invention pertains to nucleic acid molecules encoding TWIK-6 proteins that contain changes in amino acid residues that are not essential for activity.
  • Such TWTK-6 proteins differ in amino acid sequence from SEQ ID NO:2 or 5, yet retain biological activity.
  • 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%, 67%, 68%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:2 or 5.
  • An isolated nucleic acid molecule encoding a TWIK-6 protein identical to the protein of SEQ ID NO:2 or 5, can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NOT or 3, or SEQ ID NO:4 or 6, or the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC as Accession Number - such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein.
  • Mutations can be introduced into SEQ ID NOT or 3, or SEQ ID NO:4 or 6, or the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC as Accession Number by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • 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 TWIK-6 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 TWIK- 6 coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for TWIK-6 biological activity to identify mutants that retain activity.
  • the encoded protein can be expressed recombinantly and the activity ofthe protein can be determined.
  • a mutant TWIK-6 protein can be assayed for the ability to (1) interact with a non-TWTK protein molecule; (2) activate a TWIK- dependent signal transduction pathway; (3) modulate the release of neurotransmitters; (4) modulate membrane excitability; (5) influence the resting potential of membranes, wave forms and frequencies of action potentials, and thresholds of excitation, and (6) modulate processes which underlie learning and memory, such as integration of sub- threshold synaptic responses and the conductance of back-propagating action potentials.
  • 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 TWIK-6 coding strand, or to only a portion thereof.
  • an antisense nucleic acid molecule is antisense to a "coding region" ofthe coding strand of a nucleotide sequence encoding TWIK-6.
  • the term “coding region” refers to the region ofthe nucleotide sequence comprising codons which are translated into amino acid residues (e.g., the coding region of human TWIK-6 corresponds to SEQ ID NO: 3 or 6).
  • the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence encoding TWIK-6.
  • noncoding region refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5' and 3' untranslated regions).
  • antisense nucleic acids ofthe 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 TWIK-6 mRNA, but more preferably is an oligonucleotide which is antisense to only a portion ofthe coding or noncoding region of TWIK-6 mRNA.
  • the antisense oligonucleotide can be complementary to the region surrounding the translation start site of TWIK-6 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 ofthe 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 e.g., an antisense oligonucleotide
  • 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.
  • 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 ofthe 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 TWTK-6 protein to thereby inhibit expression ofthe 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 ofthe double helix.
  • An example of a route of administration of antisense nucleic acid molecules ofthe 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 ofthe 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 ofthe 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 ofthe 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 TWIK-6 mRNA transcripts to thereby inhibit translation of TWIK-6 mRNA.
  • a ribozyme having specificity for a TWIK-6-encoding nucleic acid can be designed based upon the nucleotide sequence of a TWIK-6 cDNA disclosed herein (i.e., SEQ ID NOT or 3, or SEQ ID NO:4 or 6, or the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC as Accession Number ).
  • a derivative of a Tetrahymena L- 19 INS R ⁇ A can be constructed in which the nucleotide sequence ofthe active site is complementary to the nucleotide sequence to be cleaved in a TWIK-6-encoding mR ⁇ A. See, e.g., Cech et al. U.S.
  • TWIK-6 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. (1993) Science 261:1411-1418.
  • TWIK-6 gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region ofthe TWIK-6 (e.g., the TWIK-6 promoter and/or enhancers; e.g., nucleotides 1-102 of SEQ ID NOT or 4) to form triple helical structures that prevent transcription ofthe TWIK-6 gene in target cells.
  • nucleotide sequences complementary to the regulatory region ofthe TWIK-6 e.g., the TWIK-6 promoter and/or enhancers; e.g., nucleotides 1-102 of SEQ ID NOT or 4
  • TWIK-6 promoter and/or enhancers e.g., nucleotides 1-102 of SEQ ID NOT or
  • the TWIK-6 nucleic acid molecules ofthe present invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility ofthe molecule.
  • the deoxyribose phosphate backbone ofthe 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 nxxcleic 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 TWIK-6 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 TWTK-6 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.
  • PNAs of TWTK-6 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 TWIK-6 nucleic acid molecules can be generated which may combine the advantageous properties of PNA and DNA.
  • Such chimeras allow DNA recognition enzymes, (e.g., RNAse 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. USA 86:6553-6556; Lemaixre 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. USA 86:6553-6556; Lemaixre 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).
  • an endogenous TWIK-6 gene within a cell line or microorganism may be modified by inserting a heterologous DNA regulatory element into the genome of a stable cell line or cloned microorganism such that the inserted regulatory element is operatively linked with the endogenous TWIK-6 gene.
  • an endogenous TWIK-6 gene which is normally "transcriptionally silent", i.e., a TWIK-6 gene which is normally not expressed, or is expressed only at very low levels in a cell line or microorganism may be activated by inserting a regulatory element which is capable of promoting the expression of a normally expressed gene product in that cell line or microorganism.
  • a transcriptionally silent, endogenous TWIK-6 gene may be activated by insertion of a promiscuous regulatory element that works across cell types.
  • a heterologous regulatory element may be inserted into a stable cell line or cloned microorganism, such that it is operatively linked with an endogenous TWIK-6 gene, using techniques, such as targeted homologous recombination, which are well known to those of skill in the art, and described, e.g. , in Chappel, U.S. Patent No. 5,272,071; PCT publication No. WO 91/06667, published May 16, 1991.
  • TWTK-6 proteins and biologically active portions thereof, as well as polypeptide fragments suitable for use as immunogens to raise anti-TWIK-6 antibodies.
  • native TWIK-6 proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques.
  • TWIK- 6 proteins are produced by recombinant DNA techniques.
  • a TWIK-6 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 TWIK-6 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 TWIK-6 protein in which the protein is separated from cellular components ofthe cells from which it is isolated or recombinantly produced.
  • the language "substantially free of cellular material” includes preparations of TWIK-6 protein having less than about 30% (by dry weight) of non-TWIK-6 protein (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non-TWIK-6 protein, still more preferably less than about 10% of non-TWIK-6 protein, and most preferably less than about 5% non-TWIK-6 protein.
  • non-TWIK-6 protein also referred to herein as a "contaminating protein”
  • the TWIK-6 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% ofthe volume ofthe protein preparation.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of TWIK-6 protein in which the protein is separated from chemical precursors or other chemicals which are involved in the synthesis ofthe protein.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of TWIK-6 protein having less than about 30% (by dry weight) of chemical precursors or non-TWIK-6 chemicals, more preferably less than about 20% chemical precursors or non-TWIK-6 chemicals, still more preferably less than about 10% chemical precursors or non-TWIK-6 chemicals, and most preferably less than about 5% chemical precursors or non-TWIK-6 chemicals.
  • a "biologically active portion" of a TWIK-6 protein includes a fragment of a TWIK-6 protein which participates in an interaction between a TWIK-6 molecule and a non-TWTK-6 molecule.
  • Biologically active portions of a TWIK-6 protein include peptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence ofthe TWIK-6 protein, e.g., the amino acid sequence shown in SEQ ID NO:2 or 5, which include less amino acids than the full length TWIK-6 proteins, and exhibit at least one activity of a TWIK-6 protein.
  • biologically active portions comprise a domain or motif with at least one activity ofthe TWIK-6 protein, e.g., modulating membrane excitability.
  • a biologically active portion of a TWIK-6 protein can be a polypeptide which is, for example, 25, 50, 75, 100, 125, 150, 175, 200 or more amino acids in length.
  • Biologically active portions of a TWIK-6 protein can be used as targets for developing agents which modulate a TWIK-6 mediated activity, e.g., modulation of membrane excitability.
  • a biologically active portion of a TWIK-6 protein comprises at least one transmembrane domain.
  • a preferred biologically active portion of a TWIK-6 protein ofthe present invention may contain at least one transmembrane domain and one or more ofthe following domains: a TWIK-related ion channel domain, a P-loop domain, and a potassium channel protein domain.
  • other biologically active portions, in which other regions ofthe protein are deleted can be prepared by recombinant techniques and evaluated for one or more ofthe functional activities of a native TWIK-6 protein.
  • the TWIK-6 protein has an amino acid sequence shown in SEQ ID NO:2 or 5.
  • the TWIK-6 protein is substantially identical to SEQ ID NO:2 or 5, and retains the functional activity ofthe protein of SEQ ID NO:2 or 5, yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail in subsection I above.
  • the TWIK-6 protein is a protein which comprises an amino acid sequence at least about 50%, 55%, 60%, 65%, 67%, 68%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:2 or 5.
  • 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-identical 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% ofthe length ofthe reference sequence (e.g., when aligning a second sequence to the TWIK-6 amino acid sequence of SEQ ID NO:2 or 5 having 400 amino acid residues, at least 50, preferably at least 100, more preferably at least 150, even more preferably at least 200, and even more preferably at least 300 or more amino acid residues are aligned).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • 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 ofthe 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 ofthe 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 a NWSgapdna.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 xxsing the algorithm of E. Meyers and W. Miller ( Comput. Appl Biosci., 4: 11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the nucleic acid and protein sequences ofthe 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 ofthe respective programs e.g., XBLAST and NBLAST
  • TWIK-6 chimeric or fusion proteins As used herein, a TWIK-6 "chimeric protein” or “fusion protein” comprises a TWIK-6 polypeptide operatively linked to a non-TWTK-6 polypeptide.
  • An "TWIK-6 polypeptide” refers to a polypeptide having an amino acid sequence corresponding to TWIK-6
  • a “non-TWIK-6 polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to the TWIK-6 protein, e.g., a protein which is different from the TWIK-6 protein and which is derived from the same or a different organism.
  • a TWIK-6 fusion protein the TWIK-6 polypeptide can correspond to all or a portion of a TWIK-6 protein.
  • a TWIK-6 fusion protein comprises at least one biologically active portion of a TWIK-6 protein.
  • a TWIK-6 fusion protein comprises at least two biologically active portions of a TWIK-6 protein.
  • the term "operatively linked" is intended to indicate that the TWIK-6 polypeptide and the non-TWIK-6 polypeptide are fused in-frame to each other.
  • the non-TWIK-6 polypeptide can be fused to the N-terminus or C-terminus ofthe TWIK-6 polypeptide.
  • the fusion protein is a GST-TWIK-6 fusion protein in which the TWIK-6 sequences are fused to the C-terminus ofthe GST sequences.
  • Such fusion proteins can facilitate the purification of recombinant TWIK-6.
  • the fusion protein is a TWIK-6 protein containing a heterologous signal sequence at its N-terminus.
  • TWIK-6 a heterologous signal sequence at its N-terminus.
  • expression and/or secretion of TWIK-6 can be increased through use of a heterologous signal sequence.
  • the TWIK-6 fusion proteins ofthe invention can be incorporated into pharmaceutical compositions and administered to a subject in vivo.
  • the TWIK-6 fusion proteins can- be used to affect the bioavailability of a TWIK-6 substrate.
  • Use of TWIK-6 fusion proteins may be useful therapeutically for the treatment of disorders caused by, for example, (i) aberrant modification or mutation of a gene encoding a TWIK-
  • TWIK-6-fusion proteins ofthe invention can be used as immunogens to produce anti-TWIK-6 antibodies in a subject, to purify TWIK-6 ligands and in screening assays to identify molecules which inhibit the interaction of TWIK-6 with a TWIK-6 substrate.
  • a TWIK-6 chimeric or fusion protein ofthe invention is produced by standard recombinant DNA techniques.
  • 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 reampHfied to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).
  • anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reampHfied to generate a chimeric gene sequence
  • many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide).
  • a TWIK-6- encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the TWIK-6 protein.
  • the present invention also pertains to variants ofthe TWIK-6 proteins which function as either TWIK-6 agonists (mimetics) or as TWIK-6 antagonists.
  • Variants of the TWIK-6 proteins can be generated by mutagenesis, e.g., discrete point mutation or truncation of a TWIK-6 protein.
  • An agonist ofthe TWIK-6 proteins can retain substantially the same, or a subset, ofthe biological activities ofthe naturally occurring form of a TWTK-6 protein.
  • An antagonist of a TWIK-6 protein can inhibit one or more ofthe activities ofthe naturally occurring form ofthe TWTK-6 protein by, for example, competitively modulating a TWIK-6-mediated activity of a TWIK-6 protein.
  • treatment of a subject with a variant having a subset ofthe biological activities ofthe naturally occurring form ofthe protein has fewer side effects in a subject relative to treatment with the naturally occurring form ofthe TWIK-6 protein.
  • variants of a TWIK-6 protein which function as either TWIK-6 agonists (mimetics) or as TWIK-6 antagonists can be identified by screening combinatorial libraries of mutants, e.g. , truncation mutants, of a TWIK-6 protein for TWIK-6 protein agonist or antagonist activity.
  • a variegated library of TWIK-6 variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library.
  • a variegated library of TWIK-6 variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential TWIK-6 sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of TWIK-6 sequences therein.
  • methods which can be used to produce libraries of potential TWIK-6 variants from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector.
  • libraries of fragments of a TWIK-6 protein coding sequence can be used to generate a variegated population of TWIK-6 fragments for screening and subsequent selection of variants of a TWIK-6 protein.
  • a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of a TWIK-6 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 ofthe TWIK-6 protein.
  • Recursive 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 TWTK-6 variants (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815; Delgrave et al. (1993) Protein Engineering 6(3): 327-331).
  • cell based assays can be exploited to analyze a variegated TWIK-6 library.
  • a library of expression vectors can be transfected into a cell line, e.g., a neuronal cell line, which ordinarily responds to a TWIK-6 ligand in a particular TWIK-6 ligand-dependent manner.
  • the transfected cells are then contacted with a TWTK-6 ligand and the effect of expression ofthe mutant on, e.g., membrane excitability of TWIK-6 can be detected.
  • Plasmid DNA can then be recovered from the cells which score for inhibition, or alternatively, potentiation of signaling by the TWIK- 6 ligand, and the individual clones further characterized.
  • TWIK-6 protein can be used as an immunogen to generate antibodies that bind TWIK-6 using standard techniques for polyclonal and monoclonal antibody preparation.
  • a full-length TWIK-6 protein can be used or, alternatively, the invention provides antigenic peptide fragments of TWIK-6 for use as immunogens.
  • the antigenic peptide of TWIK-6 comprises at least 8 amino acid residues ofthe amino acid sequence shown in SEQ ID NO:2 or 5 and encompasses an epitope of TWIK-6 such that an antibody raised against the peptide forms a specific immune complex with TWIK-6.
  • 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 TWIK-6 that are located on the surface ofthe protein, e.g., hydrophilic regions, as well as regions with high antigenicity (see, for example, Figure 3).
  • a TWIK-6 immunogen typically is used to prepare antibodies by immunizing a suitable subject, (e.g., rabbit, goat, mouse or other mammal) with the immunogen.
  • An appropriate immunogenic preparation can contain, for example, recombinantly expressed TWIK-6 protein or a chemically synthesized TWIK-6 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 TWIK-6 preparation induces a polyclonal anti-TWIK-6 antibody response. Accordingly, another aspect ofthe invention pertains to anti-TWIK-6 antibodies.
  • 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 TWIK-6.
  • 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 TWIK-6.
  • 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 TWTK-6.
  • a monoclonal antibody composition thus typically displays a single binding affinity for a particular TWIK-6 protein with which it immunoreacts.
  • Polyclonal anti-TWIK-6 antibodies can be prepared as described above by immunizing a suitable subject with a TWIK-6 immunogen.
  • the anti-TWIK-6 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 TWIK-6.
  • ELISA enzyme linked immunosorbent assay
  • the antibody molecules directed against TWIK-6 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) is fused to lymphocytes (typically splenocytes) from a mammal immunized with a TWIK-6 immunogen as described above, and the culture supernatants ofthe resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds TWIK-6.
  • lymphocytes typically splenocytes
  • Any ofthe many well known protocols used for fusing lymphocytes and immortalized cell lines can be applied for the purpose of generating an anti-TWIK-6 monoclonal antibody (see, e.g., G. Galfre et al. (1977) Nature 266:55052; Gefter et al.
  • the immortal cell line e.g., a myeloma cell line
  • murine hybridomas can be made by fusing lymphocytes from a mouse immixnized 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"). 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 Typically, 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 ofthe invention are detected by screening the hybridoma culture supernatants for antibodies that bind TWIK-6, e.g., using a standard ELISA assay.
  • a monoclonal anti-TWIK-6 antibody can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with TWIK-6 to thereby isolate immunoglobulin library members that bind TWIK-6.
  • 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).
  • 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. U.S. Patent No. 5,223,409; Kang et al. PCT International Publication No. WO 92/18619; Dower et al. PCT International Publication No. WO 91/17271; Winter et al. PCT International Publication WO 92/20791 ; Markland et al. PCT International Publication No. WO 92/15679; Breitling et al. PCT International Publication WO 93/01288; McCafferty et al. PCT International Publication No.
  • recombinant anti-TWIK-6 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. U.S. Patent No. 4,816,567; Cabilly et al. European Patent Application 125,023; Better et al. (1988) Science
  • An anti-TWIK-6 antibody (e.g., monoclonal antibody) can be used to isolate
  • TWIK-6 by standard techniques, such as affinity chromatography or immunoprecipitation.
  • An anti-TWIK-6 antibody can facilitate the purification of natural TWIK-6 from cells and of recombinantly produced TWIK-6 expressed in host cells.
  • an anti-TWIK-6 antibody can be used to detect TWIK-6 protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression ofthe TWIK-6 protein.
  • Anti-TWIK-6 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 I, I,
  • vectors preferably expression vectors, containing a nucleic acid encoding a TWIK-6 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 ofthe invention comprise a nucleic acid of the invention in a form suitable for expression ofthe nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis ofthe 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 ofthe 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 include 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 cells and those which direct expression ofthe 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 ofthe expression vector can depend on such factors as the choice ofthe host cell to be transformed, the level of expression of protein desired, and the like.
  • the expression vectors ofthe invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g. , TWIK-6 proteins, mutant forms of TWIK-6 proteins, fusion proteins, and the like).
  • the recombinant expression vectors ofthe invention can be designed for expression of TWIK-6 proteins in prokaryotic or eukaryotic cells.
  • TWIK- 6 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. Expression of proteins in prokaryotes is most often carried out in E.
  • Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus ofthe recombinant protein.
  • Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility ofthe recombinant protein; and 3) to aid in the purification ofthe recombinant protein by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent to purification ofthe fusion protein.
  • enzymes, and their cognate recognition sequences include Factor Xa, thrombin and enterokinase.
  • Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D.B. and Johnson, K.S.
  • GST glutathione S-transferase
  • TWIK-6 activity assays e.g., direct assays or competitive assays described in detail below
  • a TWIK-6 fusion protein expressed in a retro viral expression vector ofthe present invention can be utilized to infect bone marrow cells which are subsequently transplanted into irradiated recipients. The pathology ofthe subject recipient is then examined after sufficient time has passed (e.g., six (6) weeks). Examples of suitable inducible non-fusion E.
  • coli expression vectors include pTrc (Aniann et al, (1988) Gene 69:301-315) and pET l id (Sxudier 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 fusion promoter.
  • Target gene expression from the pET l id vector relies on transcription from a T7 gnlO-lac fusion 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 ofthe lacUV 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 ofthe 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 ofthe invention can be carried out by standard DNA synthesis techniques.
  • the TWIK-6 expression vector is a yeast expression vector.
  • yeast expression vectors for expression in yeast S. cerivisiae 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 (InVitrogen Corp, San Diego, CA).
  • TWIK-6 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 pVL series (Lucklow and Summers (1989) Virology 170:31-39).
  • a nucleic acid ofthe 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) EMBOJ. 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, cytomegalovirus 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, NY, 1989.
  • the recombinant mammalian expression vector is capable of directing expression ofthe 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.
  • 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) EMBOJ.
  • 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 ofthe 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 ofthe DNA molecule) of an RNA molecule which is antisense to TWIK-6 mRNA.
  • Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the continuous expression ofthe 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.
  • Another aspect ofthe invention pertains to host cells into which a TWIK-6 nucleic acid molecule ofthe invention is introduced, e.g., a TWIK-6 nucleic acid molecule within a recombinant expression vector or a TWIK-6 nucleic acid molecule containing sequences which allow it to homologously recombine into a specific site of the host cell's genome.
  • the terms "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 ofthe term as used herein.
  • a host cell can be any prokaryotic or eukaryotic cell.
  • a TWIK-6 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).
  • bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells).
  • CHO Chinese hamster ovary cells
  • COS cells Chinese hamster ovary cells
  • 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, D ⁇ A ⁇ -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 TWIK-6 protein or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drag selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
  • a host cell ofthe invention such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) a TWIK-6 protein.
  • the invention further provides methods for producing a TWTK-6 protein using the host cells ofthe invention.
  • the method comprises culturing the host cell of the invention (into which a recombinant expression vector encoding a TWIK-6 protein has been introduced) in a suitable medium such that a TWIK-6 protein is produced.
  • the method further comprises isolating a TWIK-6 protein from the medium or the host cell.
  • the host cells ofthe invention can also be used to produce non-human transgenic animals.
  • a host cell ofthe invention is a fertilized oocyte or an embryonic stem cell into which TWIK-6-coding sequences have been introduced.
  • Such host cells can then be used to create non-human transgenic animals in which exogenous TWIK-6 sequences have been introduced into their genome or homologous recombinant animals in which endogenous TWIK-6 sequences have been altered.
  • Such animals are useful for studying the function and/or activity of a TWIK-6 and for identifying and/or evaluating modulators of TWIK-6 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 ofthe cells ofthe 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 ofthe mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues ofthe transgenic animal.
  • a "homologous recombinant animal” is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous TWIK-6 gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell ofthe animal, e.g., an embryonic cell ofthe animal, prior to development ofthe animal.
  • a transgenic animal ofthe invention can be created by introducing a TWIK-6- encoding nucleic acid into the male pronuclei of a fertilized oocyte, e.g., by micromjection, retro viral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal.
  • the TWIK-6 cDNA sequence of SEQ ID NOT or 4 can be introduced as a transgene into the genome of a non-human animal.
  • a nonhuman homologue of a human TWTK-6 gene such as a mouse or rat TWIK-6 gene, can be used as a transgene.
  • a TWIK-6 gene homologue such as another TWTK-6 family member, can be isolated based on hybridization to the TWIK-6 cDNA sequences of SEQ ID NO.T or 3, or SEQ ID NO:4 or 6, or the DNA insert ofthe plasmid deposited with ATCC as Accession Number (described ftxrther in subsection I above) and used as a transgene. Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression ofthe transgene.
  • a tissue-specific regulatory sequence(s) can be operably linked to a TWIK-6 transgene to direct expression of a TWIK-6 protein to particular cells.
  • transgenic founder animal can be identified based upon the presence of a TWIK-6 transgene in its genome and/or expression of TWIK-6 mRNA in tissues or cells ofthe animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene encoding a TWIK-6 protein can further be bred to other transgenic animals carrying other transgenes.
  • a vector which contains at least a portion of a TWIK-6 gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the TWIK-6 gene.
  • the TWIK-6 gene can be a human gene (e.g., the cDNA of SEQ ID NO: 3 or 6), but more preferably, is a non-human homologue of a human TWIK-6 gene (e.g., a cDNA isolated by stringent hybridization with the nucleotide sequence of SEQ ID NOT or 4).
  • a mouse TWIK-6 gene can be used to construct a homologous recombination nucleic acid molecule, e.g., a vector, suitable for altering an endogenous TWIK-6 gene in the mouse genome.
  • the homologous recombination nucleic acid molecule is designed such that, upon homologous recombination, the endogenous TWIK-6 gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a "knock out" vector).
  • the homologous recombination nucleic acid molecule can be designed such that, upon homologous recombination, the endogenous TWIK-6 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 ofthe endogenous TWIK-6 protein).
  • the altered portion ofthe TWIK-6 gene is flanked at its 5' and 3' ends by additional nucleic acid sequence ofthe TWIK-6 gene to allow for homologous recombination to occur between the exogenous TWIK-6 gene carried by the homologous recombination nucleic acid molecule and an endogenous TWIK-6 gene in a cell, e.g., an embryonic stem cell.
  • the additional flanking TWIK-6 nucleic acid sequence is of sufficient length for successful homologous recombination with the endogenous gene.
  • homologous recombination nucleic acid molecule typically, several kilobases of flanking DNA (both at the 5' and 3' ends) are included in the homologous recombination nucleic acid molecule (see, e.g., Thomas, K.R. and Capecchi, M. R. (1987) Cell 51 :503 for a description of homologous recombination vectors).
  • the homologous recombination nucleic acid molecule is introduced into a cell, e.g., an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced TWIK-6 gene has homologously recombined with the endogenous TWIK-6 gene are selected (see e.g., Li, E. et al.
  • the selected cells can 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.
  • homologous recombination nucleic acid molecules e.g., vectors, or homologous recombinant animals are described further in Bradley, A. (1991) Current Opinion in Biotechnology 2:823-829 and in PCT International Publication Nos.: WO 90/11354 by Le Mouellec et al; WO 91/01140 by Smithies et al ; WO 92/0968 by Zijlsfra et al. ; and WO 93/04169 by Berns et ⁇ l
  • transgenic non-human animals can be produced which contain selected systems which allow for regulated expression ofthe transgene.
  • a system is the cre/loxP recombinase system of bacteriophage PI .
  • the cre/loxP recombinase system see, e.g., Lakso et ⁇ l. (1992) Proc. N ⁇ tl Ac ⁇ d. Sci. USA 89:6232-6236.
  • Another example of a recombinase system is the FLP recombinase system of S ⁇ cch ⁇ romyces cerevisi ⁇ e (O'Gorman et ⁇ l. (1991) Science 251 :1351-1355.
  • mice 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 ofthe 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
  • the quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal ofthe same species from which the quiescent cell is isolated.
  • the reconstructed oocyte is then cultured such that it develops to morala or blastocyte and then transferred to pseudopregnant female foster animal.
  • the offspring borne of this female foster animal will be a clone ofthe 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.
  • 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.
  • a pharmaceutical composition ofthe 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 ofthe required particle size in the case of dispersion and by the use of surfactants.
  • Prevention ofthe 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 ofthe 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 TWIK-6 protein or an anti-TWIK-6 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.
  • a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder ofthe 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 ofthe composition.
  • the tablets, pills, capsules, troches and the like can contain any ofthe 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 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 fusidic acid derivatives.
  • Transmucosal administration 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 ofthe invention are dictated by and directly dependent on the unique characteristics ofthe 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% ofthe population) and the ED50 (the dose therapeutically effective in 50% ofthe 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.
  • 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.
  • IC50 i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms
  • 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.
  • 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 ofthe ordinarily skilled physician, veterinarian, or researcher.
  • the dose(s) ofthe small molecule will vary, for example, depending upon the identity, size, and condition ofthe 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 ofthe invention.
  • Exemplary doses include milligram or microgram amounts ofthe 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.
  • appropriate doses of a small molecule depend upon the potency ofthe 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 ofthe subject, the time of administration, the route of administration, the rate of excretion, any drag combination, and the degree of expression or activity to be modulated.
  • an antibody or fragment thereof 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, doxorabicin, 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 chlorarnbucil, 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 doxorabicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g
  • the drag moiety is not to be construed as limited to classical chemical therapeutic agents.
  • the drag 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
  • 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 ofthe 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 ofthe 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.
  • the pharmaceutical 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 ofthe 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 prophy lactic).
  • a TWIK-6 protein ofthe invention has one or more ofthe following activities: (1) interacting with a non-TWIK protein molecule; (2) activating a TWIK-dependent signal transduction pathway; (3) modulating the release of neurotransmitters; (4) modulating membrane excitability; (5) influencing the resting potential of membranes, wave forms and frequencies of action potentials, and thresholds of excitation, and (6) modulating processes which underlie learning and memory, such as integration of sub-threshold synaptic responses and the conductance of back- propagating action potentials.
  • the isolated nucleic acid molecules of the invention can be used, for example, to express TWIK-6 protein (e.g. , via a recombinant expression vector in a host cell in gene therapy applications), to detect TWIK-6 mRNA (e.g., in a biological sample) or a genetic alteration in a TWIK-6 gene, and to modulate TWIK-6 activity, as described further below.
  • TWIK-6 proteins can be used to treat disorders characterized by insufficient or excessive production of a TWIK-6 substrate or production of TWIK-6 inhibitors.
  • TWIK-6 proteins can be used to screen for naturally occurring TWIK-6 substrates, to screen for drags or compounds which modulate T WTK- 6 activity, as well as to treat disorders characterized by insufficient or excessive production of TWIK-6 protein or production of TWIK-6 protein forms which have decreased, aberrant or unwanted activity compared to TWIK-6 wild type protein (e.g., proliferative disorders, CNS disorders such as cognitive and 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, senile dementia, Huntington's disease, Gilles de la Tourette's syndrome, multiple sclerosis, amyotrophic lateral sclerosis, progressive supranuclear palsy, epilepsy, and Jakob-Creutzfieldt disease; autonomic function disorders such as hypertension and sleep disorders, and neuropsychiatric disorders, such as depression, schizophrenia, schizoaffective disorder, korsak
  • 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 TWIK-6 proteins, have a stimulatory or inhibitory effect on, for example, TWIK-6 expression or TWIK-6 activity, or have a stimulatory or inhibitory effect on, for example, the expression or activity of TWIK-6 substrate.
  • modulators i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) which bind to TWIK-6 proteins, have a stimulatory or inhibitory effect on, for example, TWIK-6 expression or TWIK-6 activity, or have a stimulatory or inhibitory effect on, for example, the expression or activity of TWIK-6 substrate.
  • the invention provides assays for screening candidate or test compounds which are substrates of a TWIK-6 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 TWTK-6 protein or polypeptide or biologically active portion thereof.
  • the test compounds ofthe present invention can be obtained using any ofthe 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) Anticancer Drug Des. 12:145).
  • Biotechniques 13:412-421 or on beads (Lam (1991) Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner USP 5,223,409), spores (Ladner USP '409), plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage (Scott and Smith (1990) Science 249:386-390); (Devlin (1990) Science 249:404-406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382); (Felici (1991) J. Mol. Biol. 222:301-310); (Ladner supra.).
  • an assay is a cell-based assay in which a cell which expresses a TWIK-6 protein or biologically active portion thereof is contacted with a test compound and the ability ofthe test compound to modulate TWIK-6 activity is determined. Determining the ability ofthe test compound to modulate TWIK-6 activity can be accomplished by monitoring, for example, the release of a neurotransmitter from a cell which expresses TWIK-6.
  • the cell for example, can be of mammalian origin, e.g., a neuronal cell or a thymus cell.
  • the ability ofthe test compound to modulate TWIK-6 binding to a substrate or to bind to TWIK-6 can also be determined.
  • Determining the ability ofthe test compound to modulate TWIK-6 binding to a substrate can be accomplished, for example, by coupling the TWIK-6 substrate with a radioisotope or enzymatic label such that binding ofthe TWIK-6 substrate to TWIK-6 can be determined by detecting the labeled TWIK-6 substrate in a complex.
  • TWIK-6 could be coupled with a radioisotope or enzymatic label to monitor the ability of a test compound to modulate TWIK-6 binding to a TWIK-6 substrate in a complex.
  • Determining the ability ofthe test compound to bind TWIK-6 can be accomplished, for example, by coupling the compound with a radioisotope or enzymatic label such that binding ofthe compound to TWIK-6 can be determined by detecting the labeled TWIK-6 compound in a complex.
  • compounds e.g., TWIK-6 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 microphysiometer can be used to detect the interaction of a compound with TWIK-6 without the labeling of either the compound or the TWIK-6. McConnell, H. M. et ⁇ l. (1992) Science 257:1906-1912.
  • a "microphysiometer” e.g., Cytosensor
  • LAPS light-addressable potentiometric sensor
  • an assay is a cell-based assay comprising contacting a cell expressing a TWTK-6 target molecule (e.g., a TWTK-6 substrate) with a test compound and determining the ability ofthe test compound to modulate (e.g., stimulate or inhibit) the activity ofthe TWIK-6 target molecule. Determining the ability ofthe test compound to modulate the activity of a TWIK-6 target molecule can be accomplished, for example, by determining the ability ofthe TWIK-6 protein to bind to or interact with the TWIK-6 target molecule.
  • Determining the ability ofthe TWIK-6 protein, or a biologically active fragment thereof, to bind to or interact with a TWIK-6 target molecule can be accomplished by one ofthe methods described above for determining direct binding. In a preferred embodiment, determining the ability ofthe TWIK-6 protein to bind to or interact with a TWIK-6 target molecule can be accomplished by determining the activity ofthe target molecule.
  • the activity ofthe target molecule can be determined by detecting induction of a cellular response (i.e., changes in intracellular K levels), detecting catalytic/enzymatic activity ofthe target on 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.
  • a reporter gene comprising a target-responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g., luciferase
  • an assay ofthe present invention is a cell-free assay in which a TWTK-6 protein or biologically active portion thereof is contacted with a test compound and the ability ofthe test compound to bind to the TWIK-6 protein or biologically active portion thereof is determined.
  • Preferred biologically active portions ofthe TWIK-6 proteins to be used in assays ofthe present invention include fragments which participate in interactions with non-TWIK-6 molecules, e.g., fragments with high surface probability scores (see, for example, Figure 3). Binding ofthe test compound to the TWIK-6 protein can be determined either directly or indirectly as described above.
  • the assay includes contacting the TWIK-6 protein or biologically active portion thereof with a known compound which binds TWIK-6 to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability ofthe test compound to interact with a TWIK-6 protein, wherein determining the ability ofthe test compound to interact with a TWIK-6 protein comprises determining the ability ofthe test compound to preferentially bind to TWIK-6 or biologically active portion thereof as compared to the known compound.
  • the assay is a cell-free assay in which a TWIK-6 protein or biologically active portion thereof is contacted with a test compound and the ability ofthe test compound to modulate (e.g. , stimulate or inhibit) the activity ofthe TWIK-6 protein or biologically active portion thereof is determined. Determining the ability of the test compound to modulate the activity of a TWIK-6 protein can be accomplished, for example, by determining the ability ofthe TWTK-6 protein to bind to a TWIK-6 target molecule by one ofthe methods described above for determining direct binding.
  • Determining the ability ofthe TWIK-6 protein to bind to a TWIK-6 target molecule can also be accomplished using a technology such as real-time Biomolecular Interaction Analysis (BIA).
  • BIOA Biomolecular Interaction Analysis
  • BIOA is a technology for studying biospecific interactions in real time, without labeling any ofthe 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 ofthe test compound to modulate the activity of a TWIK-6 protein can be accomplished by determining the ability ofthe TWIK-6 protein to further modulate the activity of a downstream effector of a TWIK-6 target molecule.
  • the activity ofthe effector molecule on an appropriate target can be determined or the binding ofthe effector to an appropriate target can be determined as previously described.
  • the cell-free assay involves contacting a TWIK-6 protein or biologically active portion thereof with a known compound which binds the TWIK-6 protein to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability ofthe test compound to interact with the TWIK- 6 protein, wherein determining the ability of the test compound to interact with the TWTK-6 protein comprises determining the ability ofthe TWIK-6protein to preferentially bind to or modulate the activity of a TWIK-6 target molecule.
  • TWIK-6 or its target molecule it may be desirable to immobilize either TWIK-6 or its target molecule to facilitate separation of complexed from uncomplexed forms of one or both ofthe proteins, as well as to accommodate automation ofthe assay.
  • Binding of a test compound to a TWIK-6 protein, or interaction of a TWIK-6 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-centrifuge tubes.
  • a fusion protein can be provided which adds a domain that allows one or both ofthe proteins to be bound to a matrix.
  • glutathione- S-transferase/TWIK-6 fusion proteins or glutathione-S-transferase/target fusion 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 TWIK-6 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. Alternatively, the complexes can be dissociated from the matrix, and the level of TWIK-6 binding or activity determined using standard techniques.
  • TWIK-6 protein or a TWIK-6 target molecule can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated TWIK-6 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 TWIK-6 protein or target molecules but which do not interfere with binding ofthe TWIK-6 protein to its target molecule can be derivatized to the wells of the plate, and unbound target or
  • TWIK-6 protein trapped in the wells by antibody conj ugation TWIK-6 protein trapped in the wells by antibody conj ugation.
  • Methods for detecting ⁇ such complexes include immunodetection of complexes using antibodies reactive with the TWIK-6 protein or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the TWIK-6 protein or target molecule.
  • modulators of TWIK-6 expression are identified in a method wherein a cell is contacted with a candidate compound and the expression of TWIK-6 mRNA or protein in the cell is determined.
  • the level of expression of TWIK-6 mRNA or protein in the presence ofthe candidate compound is compared to the level of expression of TWIK-6 mRNA or protein in the absence ofthe candidate compound.
  • the candidate compound can then be identified as a modulator of TWIK-6 expression based on this comparison. For example, when expression of TWTK-6 mRNA or protein is greater (statistically significantly greater) in the presence ofthe candidate compound than in its absence, the candidate compound is identified as a stimulator of TWIK-6 mRNA or protein expression. Alternatively, when expression of TWIK-6 mRNA or protein is less (statistically significantly less) in the presence ofthe candidate compoxmd than in its absence, the candidate compound is identified as an inhibitor of TWIK-6 mRNA or protein expression.
  • the level of TWIK-6 mRNA or protein expression in the cells can be determined by methods described herein for detecting TWIK-6 mRNA or protein.
  • the TWIK-6 proteins can be used as "bait proteins" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Patent No.
  • TWIK-6-binding proteins or "TWIK-6-bp"
  • TWIK-6-binding proteins are also likely to be involved in the propagation of signals by the TWIK-6 proteins or TWIK-6 targets as, for example, downstream elements of a TWIK-6-mediated signaling pathway.
  • TWIK-6-binding proteins are likely to be TWIK-6 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 TWIK-6 protein is fused 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 fused to a gene that codes for the activation domain ofthe known transcription factor.
  • the DNA-binding and activation domains ofthe 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 ofthe 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 TWIK-6 protein.
  • a reporter gene e.g., LacZ
  • the invention pertains to a combination of two or more ofthe assays described herein.
  • a modulating agent can be identified using a cell- based or a cell free assay, and the ability ofthe agent to modulate the activity of a
  • TWTK-6 protein can be confirmed in vivo, e.g., in an animal such as an animal model for cellular transformation and/or tumorigenesis.
  • 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 TWIK-6 modulating agent, an antisense TWIK-6 nucleic acid molecule, a TWIK-6-specific antibody, or a TWIK-6-binding partner
  • 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 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 ofthe gene on a chromosome. This process is called chromosome mapping. Accordingly, portions or fragments ofthe TWIK-6 nucleotide sequences, described herein, can be used to map the location ofthe TWIK-6 genes on a chromosome. The mapping ofthe TWIK-6 sequences to chromosomes is an important first step in correlating these sequences with genes associated with disease.
  • TWIK-6 genes can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp in length) from the TWIK-6 nucleotide sequences. Computer analysis ofthe TWTK-6 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 TWIK-6 sequences will yield an amplified fragment. Somatic cell hybrids are prepared by fusing somatic cells from different mammals (e.g., human and mouse cells).
  • Somatic cell hybrids containing only fragments of human chromosomes can also be produced by using human chromosomes with translocations and deletions.
  • 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.
  • TWIK-6 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 TWIK-6 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 ofthe 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.
  • differences in the DNA sequences between individuals affected and unaffected with a disease associated with the TWIK-6 gene can be determined. If a mutation is observed in some or all ofthe affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent ofthe 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 DNA 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 TWTK-6 sequences ofthe 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 DNA 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 ofthe present invention are useful as additional DNA markers for RFLP (described in U.S. Patent 5,272,057).
  • sequences ofthe present invention can be used to provide an alternative technique wliich determines the actual base-by-base DNA sequence of selected portions of an individual's genome.
  • the TWIK-6 nucleotide sequences described herein can be used to prepare two PCR primers from the 5' and 3' ends ofthe 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 ofthe present invention can be used to obtain such identification sequences from individuals and from tissue.
  • the TWIK-6 nucleotide sequences ofthe invention uniquely represent portions ofthe 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 ofthe sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes.
  • the noncoding sequences of SEQ ID NOT or 4 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, such as those in SEQ ID NO: 3 or 6 are used, a more appropriate number of primers for positive individual identification would be 500-2,000.
  • a panel of reagents from TWIK-6 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.
  • positive identification ofthe individual, living or dead can be made from extremely small tissue samples.
  • 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 ofthe origin ofthe biological sample.
  • sequences ofthe 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 of SEQ ID NO or 4 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.
  • polynucleotide reagents include the TWIK-6 nucleotide sequences or portions thereof, e.g., fragments derived from the noncoding regions of SEQ ID NOT or 4 having a length of at least 20 bases, preferably at least 30 bases.
  • the TWTK-6 nucleotide sequences described herein can further 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., thymus or brain tissue. This can be very useful in cases where a forensic pathologist is presented with a tissue of unknown origin.
  • TWIK-6 probes can be used to identify tissue by species and/or by organ type.
  • these reagents e.g., TWIK-6 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 ofthe present invention relates to diagnostic assays for determining TWIK-6 protein and/or nucleic acid expression as well as TWIK-6 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 or unwanted TWIK-6 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 TWIK-6 protein, nucleic acid expression or activity. For example, mutations in a TWIK-6 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 TWIK-6 protein, nucleic acid expression or activity.
  • Another aspect ofthe invention pertains to monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of TWIK-6 in clinical trials.
  • agents e.g., drugs, compounds
  • An exemplary method for detecting the presence or absence of TWIK-6 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 TWIK-6 protein or nucleic acid (e.g., mRNA, or genomic DNA) that encodes TWIK-6 protein such that the presence of TWIK-6 protein or nucleic acid is detected in the biological sample.
  • a preferred agent for detecting TWIK-6 mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to TWIK-6 mRNA or genomic DNA.
  • the nucleic acid probe can be, for example, the TWIK-6 nucleic acid set forth in SEQ ID NOT or 3, or SEQ ID NO:4 or 6, or the DNA insert ofthe plasmid deposited with ATCC as Accession Number , or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to TWIK-6 mRNA or genomic DNA.
  • Other suitable probes for use in the diagnostic assays ofthe invention are described herein.
  • a preferred agent for detecting TWIK-6 protein is an antibody capable of binding to TWIK-6 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 ofthe probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling ofthe 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 ofthe invention can be used to detect TWIK-6 mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo.
  • in vitro techniques for detection of TWTK-6 mRNA include Northern hybridizations and in situ hybridizations.
  • In vitro techniques for detection of TWIK-6 protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence.
  • In vitro techniques for detection of TWIK-6 genomic DNA include Southern hybridizations.
  • in vivo techniques for detection of TWIK-6 protein include introducing into a subject a labeled anti-TWIK-6 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 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 TWIK-6 protein, mRNA, or genomic DNA, such that the presence of TWIK-6 protein, mRNA or genomic DNA is detected in the biological sample, and comparing the presence of TWIK-6 protein, mRNA or genomic DNA in the control sample with the presence of TWIK-6 protein, mRNA or genomic DNA in the test sample.
  • kits for detecting the presence of TWIK-6 in a biological sample can comprise a labeled compound or agent capable of detecting TWIK-6 protein or mRNA in a biological sample; means for determining the amount of TWIK-6 in the sample; and means for comparing the amount of TWIK-6 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 TWTK-6 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 or unwanted TWIK-6 expression or activity.
  • aberrant includes a TWIK-6 expression or activity which deviates from the wild type TWIK-6 expression or activity.
  • Aberrant expression or activity includes increased or decreased expression or activity, as well as expression or activity which does not follow the wild type developmental pattern of expression or the subcellular pattern of expression.
  • aberrant TWIK-6 expression or activity is intended to include the cases in which a mutation in the TWIK-6 gene causes the TWIK-6 gene to be under-expressed or over-expressed and situations in which such mutations result in a non-functional TWIK- 6 protein or a protein which does not function in a wild-type fashion, e.g., a protein which does not interact with a TWIK-6 substrate, e.g., a non-potassium channel subunit or ligand, or one which interacts with a non-TWIK-6 substrate, e.g. a non-potassium channel subunit or ligand.
  • the term "unwanted” includes an unwanted phenomenon involved in a biological response such as cellular proliferation.
  • unwanted includes a TWIK-6 expression or activity which is undesirable in a subject.
  • the assays described herein can be utilized to identify a subject having or at risk of developing a disorder associated with a misregulation in TWTK-6 protein activity or nucleic acid expression, such as a CNS disorder (e.g., a cognitive or neurodegenerative disorder), a cellular proliferation, growth, differentiation, or migration disorder, or a cardiovascular disorder.
  • a CNS disorder e.g., a cognitive or neurodegenerative disorder
  • the prognostic assays can be utilized to identify a subject having or at risk for developing a disorder associated with a misregulation in TWIK-6 protein activity or nucleic acid expression, such as a CNS disorder, a cellular proliferation, growth, differentiation, or migration disorder, or a cardiovascular disorder.
  • the present invention provides a method for identifying a disease or disorder associated with aberrant or unwanted TWIK-6 expression or activity in which a test sample is obtained from a subject and TWIK-6 protein or nucleic ' acid (e.g., mRNA or genomic DNA) is detected, wherein the presence of TWIK-6 protein or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with aberrant or unwanted TWIK-6 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., cerebrospinal fluid or 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 drag candidate) to treat a disease or disorder associated with aberrant or unwanted TWIK-6 expression or activity.
  • an agent e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drag candidate
  • such methods can be used to determine whether a subject can be effectively treated with an agent for a CNS disorder, a muscular disorder, or a cellular proliferation, growth, differentiation, or migration disorder.
  • the present invention provides methods for determining whether a subject can be effectively treated with an agent for a disorder associated with aberrant or unwanted TWTK-6 expression or activity in which a test sample is obtained and TWTK-6 protein or nucleic acid expression or activity is detected (e.g., wherein the abundance of TWIK-6 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 or unwanted TWTK-6 expression or activity).
  • the methods ofthe invention can also be used to detect genetic alterations in a TWIK-6 gene, thereby determining if a subject with the altered gene is at risk for a disorder characterized by misregulation in TWIK-6 protein activity or nucleic acid expression, such as a CNS disorder, a cellular proliferation, growth, differentiation, or migration disorder, or 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 TWIK-6 -protein, or the mis-expression ofthe TWIK-6 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 TWTK-6 gene; 2) an addition of one or more nucleotides to a TWIK-6 gene; 3) a substitution of one or more nucleotides of a TWIK-6 gene, 4) a chromosomal rearrangement of a TWIK-6 gene; 5) an alteration in the level of a messenger RNA transcript of a TWIK-6 gene, 6) aberrant modification of a TWIK-6 gene, such as ofthe methylation pattern ofthe genomic DNA, 7) the presence of a non-wild type splicing pattern of a messenger RNA transcript of a TWIK-6 gene, 8) a non-wild type level of a TWIK-6-protein, 9) allelic loss of a TWTK-6 gene, and 10) inappropriate post-translational modification of a TWIK-6- protein.
  • detection ofthe alteration involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g. , U.S. Patent Nos.
  • PCR polymerase 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 ofthe sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a TWIK-6 gene under conditions such that hybridization and amplification ofthe TWIK-6-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. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any ofthe techniques used for detecting mutations described herein.
  • nucleic acid e.g., genomic, mRNA or both
  • 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. et al. (1988) Bio-Technology 6:1197), or any other nucleic acid amplification method, followed by the detection ofthe amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.
  • mutations in a TWTK-6 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 elecfrophoresis 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 TWIK-6 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 TWIK-6 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 TWIK-6 gene and detect mutations by comparing the sequence ofthe sample TWIK-6 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
  • RNA/RNA or RNA/DNA heteroduplexes Other methods for detecting mutations in the TWIK-6 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).
  • the art technique of "mismatch cleavage" starts by providing heteroduplexes of formed by hybridizing (labeled) RNA or DNA containing the wild-type TWIK-6 sequence with potentially mutant RNA or DNA obtained from a tissue sample.
  • the double-stranded duplexes are treated with an agent which cleaves single-stranded regions ofthe duplex such as which will exist due to basepair mismatches between the control and sample strands.
  • 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 ofthe mismatched regions, the resulting material is then separated by size on denaturing poly acrylamide 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 TWIK-6 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 TWIK-6 sequence e.g., a wild-type TWIK-6 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 elecfrophoresis 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 TWIK-6 genes.
  • single strand conformation polymorphism SSCP
  • SSCP single strand conformation polymorphism
  • Single-stranded DNA fragments of sample and control TWIK-6 nucleic acids will be denatured and allowed to renature.
  • the secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change.
  • the DNA fragments may be labeled or detected with labeled probes.
  • the sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence.
  • the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).
  • the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel elecfrophoresis (DGG ⁇ ) (Myers et al. (1985) Nature 313:495).
  • DGG ⁇ denaturing gradient gel elecfrophoresis
  • 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 et al. (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.
  • allele specific amplification technology which depends on selective PCR amplification may be used in conjunction with the instant invention.
  • Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center ofthe 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 ofthe 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 TWTK- 6 gene.
  • 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 TWTK- 6 gene.
  • any cell type or tissue in which TWIK-6 is expressed may be utilized in the prognostic assays described herein.
  • TWIK-6 protein (e.g., the modulation of cell proliferation and/or migration) can be applied not only in basic drag screening, but also in clinical trials.
  • the effectiveness of an agent determined by a screening assay as described herein to increase TWIK-6 gene expression, protein levels, or upregulate TWIK-6 activity can be monitored in clinical trials of subjects exhibiting decreased TWIK-6 gene expression, protein levels, or downregulated TWIK-6 activity.
  • the effectiveness of an agent determined by a screening assay to decrease TWIK-6 gene expression, protein levels, or downregulate TWIK-6 activity can be monitored in clinical trials of subjects exhibiting increased TWIK-6 gene expression, protein levels, or upregulated TWIK-6 activity.
  • the expression or activity of a TWIK-6 gene and preferably, other genes that have been implicated in, for example, a TWIK-6-associated disorder can be used as a "read out” or markers ofthe phenotype of a particular cell.
  • genes, including TWIK-6, that are modulated in cells by treatment with an agent (e.g., compound, drug or small molecule) which modulates TWIK-6 activity can be identified.
  • an agent e.g., compound, drug or small molecule
  • TWIK-6 activity e.g., identified in a screening assay as described herein
  • cells can be isolated and RNA prepared and analyzed for the levels of expression of TWIK-6 and other genes implicated in the TWIK-6-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 ofthe methods as described herein, or by measuring the levels of activity of TWIK-6 or other genes.
  • the gene expression pattern can serve as a marker, indicative ofthe physiological response ofthe cells to the agent. Accordingly, this response state may be determined before, and at various points during treatment ofthe 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 ofthe agent; (ii) detecting the level of expression of a TWIK-6 protein, mRNA, or genomic DNA in the preadministration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression or activity ofthe TWIK- 6 protein, mRNA, or genomic DNA in the post-administration samples; (v) comparing the level of expression or activity o the TWIK-6 protein, mRNA, or genomic DNA in the pre-administration sample with the TWIK-6 protein, mRNA, or genomic DNA in the post administration sample or samples; and (vi) altering the administration ofthe agent to the
  • TWIK-6 expression or activity may be used as an indicator ofthe 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 or unwanted TWIK-6 expression or activity, e.g. a CNS disorder, a cellular proliferation, growth, differentiation, or migration disorder, or a cardiovascular disorder.
  • a CNS disorder e.g. a CNS disorder, a cellular proliferation, growth, differentiation, or migration disorder, or a cardiovascular disorder.
  • a cardiovascular disorder e.g. a CNS disorder, a cellular proliferation, growth, differentiation, or migration disorder, or a cardiovascular disorder.
  • pharmacogenomics refers to the application of genomics technologies such as gene sequencing, statistical genetics, and gene expression analysis to drags 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 drag (e.g., a patient's "drag response phenotype", or "drug response genotype”).
  • another aspect ofthe invention provides methods for tailoring an individual's prophylactic or therapeutic treatment with either the TWTK-6 molecules of the present invention or TWIK-6 modulators according to that individual's drug 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 or unwanted TWIK-6 expression or activity, by administering to the subject a TWIK-6 or an agent which modulates TWIK- 6 expression or at least one TWIK-6 activity.
  • Subjects at risk for a disease which is caused or contributed to by aberrant or unwanted TWIK-6 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 ofthe TWIK-6 aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression.
  • a TWIK-6, TWIK-6 agonist or TWTK-6 antagonist agent can be used for treating the subject. The appropriate agent can be determined based on screening assays described herein.
  • the modulatory method ofthe invention involves contacting a cell with a TWIK-6 or agent that modulates one or more ofthe activities of TWIK-6 protein activity associated with the cell.
  • An agent that modulates TWIK-6 protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring target molecule of a TWIK-6 protein (e.g., a TWIK-6 substrate), a TWIK-6 antibody, a TWIK-6 agonist or antagonist, a peptidomimetic of a TWTK-6 agonist or antagonist, or other small molecule.
  • the agent stimulates one or more TWIK-6 activities.
  • stimulatory agents include active TWIK-6 protein and a nucleic acid molecule encoding TWTK-6 that has been introduced into the cell.
  • the agent inhibits one or more TWIK-6 activities.
  • inhibitory agents include antisense TWIK-6 nucleic acid molecules, anti-TWIK-6 antibodies, and TWIK-6 inhibitors.
  • the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant or unwanted expression or activity of a TWIK-6 protein or nucleic acid molecule.
  • 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) TWIK-6 expression or activity.
  • the method involves administering a TWIK-6 protein or nucleic acid molecule as therapy to compensate for reduced, aberrant, or unwanted TWIK-6 expression or activity.
  • Stimulation of TWTK-6 activity is desirable in situations in which TWIK-6 is abnormally downregulated and/or in which increased TWIK-6 activity is likely to have a beneficial effect.
  • inhibition of TWIK-6 activity is desirable in situations in which TWTK-6 is abnormally upregulated and/or in which decreased TWIK-6 activity is likely to have a beneficial effect.
  • TWIK-6 molecules ofthe present invention as well as agents, or modulators which have a stimulatory or inhibitory effect on TWIK-6 activity (e.g. , TWIK-6 gene expression) as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) TWIK-6-associated disorders (e.g., proliferative disorders) associated with aberrant or unwanted TWIK-6 activity.
  • TWIK-6-associated disorders e.g., proliferative disorders
  • pharmacogenomics i.e., the study ofthe 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 ofthe pharmacologically active drag.
  • a physician or clinician may consider applying knowledge obtained in relevant pharmacogenomics studies in determining whether to administer a TWIK-6 molecule or TWIK-6 modulator as well as tailoring the dosage and/or therapeutic regimen of treatment with a TWIK-6 molecule or TWIK-6 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 drugs 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, nitrofurans
  • a genome-wide association relies primarily on a high-resolution map ofthe 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 ofthe genome of each of a statistically significant number of patients taking part in a Phase II/III drug trial to identify markers associated with a particular observed drug 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 DNA. For example, 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.
  • 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. According to this method, if a gene that encodes a drags target is known (e.g., a TWIK-6 protein ofthe 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 ofthe gene versus another is associated with a particular drug response.
  • the activity of drag metabolizing enzymes is a major determinant of both the intensity and duration of drug action.
  • the discovery of genetic polymorphisms of drag metabolizing enzymes e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymes CYP2D6 and CYP2C19
  • NAT 2 N-acetyltransferase 2
  • CYP2D6 and CYP2C19 cytochrome P450 enzymes
  • CYP2D6 and CYP2C19 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 C YP2D6- 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 C YP2D6 gene amplification.
  • a method termed the "gene expression profiling” can be utilized to identify genes that predict drag response.
  • the gene expression of an animal dosed with a drag e.g., a TWIK-6 molecule or TWTK-6 modulator ofthe present invention
  • a drag e.g., a TWIK-6 molecule or TWTK-6 modulator ofthe present invention
  • Information generated from more than one ofthe 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 TWIK-6 molecule or TWTK-6 modulator, such as a modulator identified by one ofthe exemplary screening assays described herein.
  • the invention is based, at least in part, on the discovery of a human gene encoding a novel protein, referred to herein as TWIK-6.
  • TWIK-6 novel protein
  • the entire sequence ofthe human clone Fbhl7831 was determined and found to contain an open reading frame termed human "TWIK-6.” It was further determined that this gene possesses an A to G nucleotide polymorphism at nucleotide 596, resulting in an E to G amino acid change at amino acid position 165 (resulting in clones Fbhl7831E and Fbhl7831G).
  • the two alternate nucleotide sequences ofthe human TWIK-6 gene are set forth in Figures 1 and 2 and in the Sequence Listing as SEQ ID NOs: 1 and 3 or 4 and 6.
  • the two alternate amino acid sequences ofthe human TWIK-6 expression product are set forth in Figures 1 and 2 and in the Sequence Listing as SEQ ID NO: 2 or 5.
  • the nucleotide sequence encoding the human TWIK-6 (E 1 5 ) protein is shown in
  • TWIK-6 (G 165 ) protein is shown in Figure 2 and is set forth as SEQ ID NO: 4.
  • the protein encoded by each of these nucleic acids comprises about 400 amino acids and has the amino acid sequence shown in Figures 1 and 2 and set forth as SEQ ID NOs: 2 and i 5, respectively.
  • the coding regions (open reading frames) of SEQ ID NOT and 4 are set forth as SEQ ID NOs:3 and 6.
  • TASK Homo sapiens TWIK-related acid-sensitive K+ channel
  • human TWIK-6 is 69% identical to Rattus norvegicus TWIK- related acid-sensitive K+ channel (TASK) mRNA (Accession Number AF031384) over nucleotides 232 to 1229, and 78% identical over nucleotides 1482 to 1527.
  • human TWIK-6 is 70% identical to Mus musculus TWIK-related acid-sensitive K+ channel (TASK) mRNA (Accession Number AF006824) over nucleotides 324 to 1229, 77% identical over nucleotides 1492 to 1526, 95% identical over nucleotides 1506 to 1527, and 95% identical over nucleotides 1507 to 1528.
  • human TWIK-6 is 70% identical to Mus musculus mRNA for cTBAK (Accession Number AB008537) over nucleotides 303 to 924.
  • human TWIK-6 is 71% identical to Mus musculus putative potassium channel DP4 mRNA (Accession Number AF022821) over nucleotides 318 to 1217.
  • human TWTK-6 is homologous to genomic sequence for Homo sapiens clone 431C18 from chromosome 8 (locus D8S1741) (Accession Number AC007869)
  • human TWIK-6 is 98% identical to tzl5g05.xl NCI_CGAP_Ut2 Homo sapiens cDNA clone IMAGE:2288696 3' similar to TR:O35111 035111 CTBAK (Accession Number AI690321) over nucleotides 415 to 871.
  • This search further revealed that human TWIK-6 is 100% identical to wil7h03.xl NCI_CGAP_Col6 Homo sapiens cDNA clone IMAGE: 2390549 3' similar to TR:O35111 035111 CTBAK (Accession Number AI739096) over nucleotides 460-827.
  • human TWIK-6 is 73% identical to ma07h04.yl Soares mouse p3NMF19.5 Mus musculus cDNA clone IMAGE: 303895 5' similar to
  • TR:O35163 035163 TWIK-RELATED ACID-SENSITIVE K+ CHANNEL (Accession Number AI605559) over nucleotides 324-998.
  • human TWIK-6 is 76%> identical to mf89d07.yl Soares mouse embryo NbME13.5 14.5 Mus musculus cDNA clone IMAGE:421453 5' similar to TR:O35111 035111 MCTBAK (Accession Number AI325858) over nucleotides 508 to 989.
  • human TWIK-6 is 73% identical to EST291996 normalized rat ovary, Bento Soares Rattus sp.
  • human TWIK-6 is 88% identical to UI-M-BH2.2-aos-c-07-0-UI.sl NIH_BMAP_M_S3.2 Mus musculus cDNA clone UI-M-BH2.2-aos-c-07-0-UI (Accession Number AW122298) over nucleotides 764 to 1054.
  • human TWIK-6 is 65% identical to TWIK-related acid-sensitive K+ channel from Homo sapiens (Accession Number AF006823) over translated nucleotides 313 to 1086, 40% identical over translated nucleotides 1265 to 1354, and 33% identical over translated nucleotides 1253 to 1369 (as shown in Figure 6).
  • human TWIK-6 is 64% identical to TWIK-related acid-sensitive K+ channel from Rattus norvegicus (Accession Number AF031384) over translated nucleotides 313 to 1086, 42% identical over translated nucleotides 1253 to 1354, and 33% identical over translated nucleotides 1253 to 1369.
  • human TWIK-6 is 64%) identical to TWIK-related acid-sensitive K+ channel from Mus musculus (Accession Number AF006824) over translated nucleotides 325 to 1086, 44% identical over translated nucleotides 1253 to 1354, and 33% identical over translated nucleotides 1253 to 1369.
  • human TWTK-6 is 65% identical to putative potassium channel DP4 from Mus musculus (Accession Number AAD09338) over translated nucleotides 325 to 1086.
  • the search ftxrther revealed that human TWIK- 6 is 52% identical to putative potassium channel subunit n2P38 from Caenorhabditis elegans (Accession Number AF083652) over translated nucleotides 313 to 1068.
  • the search further revealed that human TWIK-6 is 51% identical to outward rectifier potassium channel homolog twk-23 from Caenorhabditis elegans (Accession Number AF025454) over translated nucleotides 313 to 1068.
  • This search further identified two Pore-loop domains (P-loop domains) at residues 150-166 and 260-278.
  • the results ofthe search are set forth in Figure 8.
  • This example describes the tissue distribution of TWIK-6 mRNA, as was determined by Polymerase Chain Reaction (PCR) on cDNA libraries using oligonucleotide primers based on the human TWIK-6 sequence.
  • the human TWIK-6 gene is expressed in thymus, in salivary gland, in primary cultured osteoblasts, and in ovarian epithelium tumor cells.
  • EXAMPLE 2 EXPRESSION OF RECOMBINANT TWIK-6 PROTEIN IN
  • TWIK-6 is expressed as a recombinant glutathione-S-transferase (GST) fusion polypeptide in E. coli and the fusion polypeptide is isolated and characterized. Specifically, TWIK-6 is fused to GST and this fusion polypeptide is expressed in E. coli, e.g., strain PEB199. Expression ofthe GST-TWIK-6 fusion protein in PEB 199 is induced with IPTG. The recombinant fusion polypeptide is purified from crude bacterial lysates ofthe induced PEB 199 strain by affinity cliromatography on glutathione beads. Using polyacrylamide gel electrophoretic analysis ofthe polypeptide purified from the bacterial lysates, the molecular weight ofthe resultant fusion polypeptide is determined.
  • GST glutathione-S-transferase
  • the pcDNA/Amp vector by Invitrogen Corporation (San Diego, CA) is used.
  • This vector contains an SV40 origin of replication, an ampicillin resistance gene, an E. coli replication origin, a CMV promoter followed by a poiylinker region, and an SV40 intron and polyadenylation site.
  • a DNA fragment encoding the. entire TWIK-6 protein and an HA tag (Wilson et al. (1984) Cell 37:767) or a FLAG tag fused in-frame to its 3' end ofthe fragment is cloned into the poiylinker region ofthe vector, thereby placing the expression ofthe recombinant protein under the control ofthe CMV promoter.
  • the TWIK-6 DNA sequence is amplified by PCR using two primers.
  • the 5' primer contains the restriction site of interest followed by approximately twenty nucleotides ofthe TWIK-6 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 ofthe TWIK-6 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 TWIK-6 gene is inserted in the correct orientation.
  • the ligation mixture is transformed into E. coli cells (strains HB101, DH5 ⁇ , 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 ofthe correct fragment.
  • COS cells are subsequently transfected with the TWIK-6-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 fransfecting 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 ofthe TWIK-6 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. Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988) using an HA specific monoclonal antibody. Briefly, 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 (RIPA 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 TWIK-6 coding sequence is cloned directly into the poiylinker ofthe pCDNA/Amp vector using the appropriate restriction sites.
  • the resulting plasmid is transfected into COS cells in the manner described above, and the expression ofthe TWTK-6 polypeptide is detected by radiolabelling and immunoprecipitation using a TWIK-6 specific monoclonal antibody.

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Abstract

L'invention concerne des molécules isolées d'acides nucléiques désignées sous le nom de molécules d'acides nucléiques TWIK-6 qui codent de nouvelles molécules de sous-unités de canal sodique ayant un rapport aux TWIK. L'invention concerne aussi des molécules antisens d'acides nucléiques, des vecteurs d'expression recombinants contentant des molécules d'acides nucléiques TWIK-6, des cellules hôtes dans lesquelles on a introduit des vecteurs d'expression et des animaux transgéniques non humains dans lesquels on a introduit ou interrompu un gène TWIK-6. L'invention concerne en outre des protéines TWIK-6 isolées, des protéines de fusion, des peptides antigéniques et des anticorps anti-TWIK-6. Elle concerne enfin des procédés diagnostiques employées utilisant des compositions de l'invention.
PCT/US2001/006933 2000-03-03 2001-03-02 Nouvelle molecule de canal sodique et son utilisation WO2001066743A2 (fr)

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AU2001241978A AU2001241978A1 (en) 2000-03-03 2001-03-02 A novel potassium channel molecule and uses therefor

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US09/518,866 2000-03-03

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003066099A1 (fr) * 2002-02-05 2003-08-14 Yamanouchi Pharmaceutical Co., Ltd. Derives de 2,4,6-triamino-1,3,5-triazine

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001046231A2 (fr) * 1999-12-21 2001-06-28 Curagen Corporation Nouvelles proteines et acides nucleiques codant ces dernieres

Patent Citations (1)

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Publication number Priority date Publication date Assignee Title
WO2001046231A2 (fr) * 1999-12-21 2001-06-28 Curagen Corporation Nouvelles proteines et acides nucleiques codant ces dernieres

Non-Patent Citations (7)

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Title
DATABASE EMBL [Online] Entry HSDJ781B1, Acc.no. AL118522, 23 September 1999 (1999-09-23) LOVELL, J.: "Human DNA sequence from clone RP4-781B1 on chromosome 20 Contains a gene encoding the two pore potassium channel KT3.3 (LOC64181), a novel gene similar to myeloblast KIAA0237 and rat protein NIM2, two putative novel genes, 3 CpG islands, ESTs, STSs and GSSs." XP002177782 *
DATABASE EMBL [Online] Entry/Ac.no. O35111, 20 August 2001 (2001-08-20) KIM, D. ET AL.: "POTASSIUM CHANNEL SUBFAMILY K MEMBER 3 (ACID-SENSITIVE POTASSIUM CHANNEL PROTEIN TASK) (TWIK-RELATED ACID-SENSITIVE K+ CHANNEL) (CARDIAC TWO-PORE BACKGROUND K+ CHANNEL) (CTBAK-1)." XP002177784 *
DATABASE EMBL [Online] Entry/Acc.no. AF257081, 20 November 2000 (2000-11-20) VEGA-SAENZ DE MIERA, E. ET AL.: "Homo sapiens two pore potassium channel KT3.3 mRNA, complete cds." XP002177783 -& VEGA-SAENZ DE MIERA, E. ET AL.: "KT3.2 and KT3.3, two novel human two-pore K+ channels closely related to TASK-1." JOURNAL OF NEUROPHYSIOLOGY, vol. 86, no. 1, July 2001 (2001-07), pages 130-142, XP001024414 *
DATABASE EMBL [Online] Entry/Acc.no. AI690321, 28 May 1999 (1999-05-28) STRAUSBERG, R. ET AL.: "tz15g05.x1 NCI_CGAP_Ut2 Homo sapiens cDNA clone IMAGE:2288696 3' similar to TR:O35111 O35111 CTBAK. [2] TR:O54912 ;contains TAR1.t1 TAR1 repetitive element ;, mRNA sequence." XP002177779 *
DATABASE EMBL [Online] Entry/Acc.no. AI739096, 23 June 1999 (1999-06-23) STRAUSBERG, R.: "wi17h03.x1 NCI_CGAP_Co16 Homo sapiens cDNA clone IMAGE:2390549 3' similar to TR:O35111 O35111 CTBAK. [2] TR:O54912 ;contains TAR1.t1 TAR1 repetitive element ;, mRNA sequence." XP002177780 *
DATABASE EMBL [Online] Entry/Acc.no. AI968607, 26 August 1999 (1999-08-26) STRAUSBERG, R. : "wt90e12.x1 NCI_CGAP_GC6 Homo sapiens cDNA clone IMAGE:2514766 3' similar to TR:O14649 O14649 TWIK-RELATED ACID-SENSITIVE K+ CHANNEL. ;, mRNA sequence." XP002177781 *
KIM DONGHEE ET AL: "TASK-5, a new member of the tandem-pore K+ channel family." BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 284, no. 4, 22 June 2001 (2001-06-22), pages 923-930, XP002177778 ISSN: 0006-291X *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003066099A1 (fr) * 2002-02-05 2003-08-14 Yamanouchi Pharmaceutical Co., Ltd. Derives de 2,4,6-triamino-1,3,5-triazine
US7375222B2 (en) 2002-02-05 2008-05-20 Astellas Pharma Inc. 2,4,6-Triamino-1,3,5-triazine derivative

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AU2001241978A1 (en) 2001-09-17

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