Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/08—Antiepileptics; Anticonvulsants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/20—Hypnotics; Sedatives
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/06—Antiarrhythmics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/12—Antihypertensives
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the invention relates to T-type calcium channel encoding sequences, expression of these sequences, and methods to screen for compounds which antagonize calcium channel activity.
• the invention is also related to molecular tools derived from knowledge of the molecular structure of T-type calcium channels.
• Calcium channels are a heterogeneous class of molecules that respond to depolarization by opening a calcium-selective pore through the plasma membrane.
• the entry of calcium into cells mediates a wide variety of cellular and physiological responses including excitation-contraction coupling, hormone secretion and gene expression.
• In neurons, calcium entry directly affects membrane potential and contributes to electrical properties such as excitability, repetitive firing patterns and pacemaker activity. Miller, R.J. (1987) "Multiple calcium channels and neuronal function.” Science 235:46-52.
• Calcium entry further affects neuronal functions by directly regulating calcium-dependent ion channels and modulating the activity of calcium-dependent enzymes such as protein kinase C and calmodulin-dependent protein kinase II.
• An increase in calcium concentration at the presynaptic nerve terminal triggers the release of neuro transmitter.
• Calcium entry also plays a role in neurite outgrowth and growth cone migration in developing neurons and has been implicated in long-term changes in neuronal activity.
• T-type (or low voltage- activated) channels describe a broad class of molecules that activate at negative potentials and are highly sensitive to changes in resting potential.
• the L, N, P and Q-type channels activate at more positive potentials and display diverse kinetics and voltage-dependent properties. There is some overlap in biophysical properties of the high voltage-activated channels, consequently pharmacological profiles are useful to further distinguish them.
• L-type channels are sensitive to dihydropyridine (DHP) agonists and antagonists
• DHP dihydropyridine
• N-type channels are blocked by the Conus geographus peptide toxin
• ⁇ -conotoxin GVIA and P-type channels are blocked by the peptide ⁇ -agatoxin IVA from the venom of the funnel web spider, Agelenopsis aperta.
• Q-type high voltage-activated Ca channel
• the ci ! subunit is the major pore-forming subunit and contains the voltage sensor and binding sites for calcium channel antagonists.
• the mainly extracellular ⁇ 2 subunit is disulphide- linked to the transmembrane ⁇ subunit and both are derived from the same gene and are proteolytically cleaved in vivo.
• the ⁇ subunit is a non-glycosylated, hydrophilic protein with a high affinity of binding to a cytoplasmic region of the ⁇ i subunit.
• a fourth subunit, ⁇ is unique to L-type Ca channels expressed in skeletal muscle T- tubules. The isolation and characterization of ⁇ -subunit-encoding cDNAs is described in U.S. Patent No. 5,386,025 which is incorporated herein by reference.
• the ⁇ i subunits are generally of the order of 2000 amino acids in length, ranging from 1873 amino acids in ⁇ is derived from rabbit to 2424 amino acids in ⁇ i A derived from rabbit.
• these subunits contain 4 internal homologous repeats (I-IV) each having six putative alpha helical membrane spanning segments (S1-S6) with one segment (S4) having positively charged residues every 3rd or 4th amino acid. There are a minority of a splice variant exceptions.
• domains II and III there is a cytoplasmic domain which is believed to mediate excitation-contraction coupling in ⁇ is and which ranges from 100- 400 amino acid residues among the subtypes.
• the domains I-IV make up roughly 2/3 of the molecule and the carboxy terminus adjacent to the S6 region of domain IV is believed to be on the intracellular side of the calcium channel.
• the high threshold a ⁇ subunits alone can form functional calcium channels although their electrophysiological and pharmacological properties can be differentially modulated by coexpression with any of the four ⁇ subunits.
• the reported modulatory affects of ⁇ subunit coexpression were to mainly alter kinetic and voltage- dependent properties. More recently it has been shown that ⁇ subunits also play crucial roles in modulating channel activity by protein kinase A, protein kinase C and direct G-protein interaction. (Bourinet, et al. (1994) "Voltage-dependent facilitation of a neuronal ⁇ lC L-type calcium channel.” EMBO J. 13: 5032-5039; Stea, et al.
• the present invention provides sequences for a novel mammalian calcium channel subunits of T-type calcium channels, which we have labeled as ( IG , oti ⁇ and ⁇ subunits. Knowledge of the sequences of these calcium channel subunits may be used in the development of probes for mapping the distribution and expression of the subunits in target tissues. In addition, as the molecular structure of the ⁇ i subunits of these T-type calcium channels has been elucidated, it is possible to identify those portions which reside extracellularly and thus to design peptides to elicit antibodies which can be employed to assess the location and level of expression of T-type calcium channels.
• these subunits can produce functional calcium channels, which can be evaluated in model cell lines to determine the properties of the channels containing the subunits of the invention.
• These cell lines can be used to evaluate the effects of pharmaceuticals and/or toxic substances on calcium channels incorporating ⁇ , ⁇ X I H and ⁇ subunits.
• the resulting identified compounds are useful in treating conditions where undesirable T-type calcium channel activity is present. These conditions include epilepsy, sleep disorders, mood disorders, cardiac hypertrophy and arrythmia and hypertension, among others.
• antisense and triplex nucleotide sequences can be designed to inhibit the production of T-type calcium channels.
• the subunits are other than those encoded by SEQ ID NO: 17; or, alternatively, are other than those encoded by SEQ ID NO: 17 and by the full length sequences of which SEQ ID NO: 19 and 21 are part.
• Other embodiments of the methods and products of this invention exclude probes representing portions of or all of SEQ ID NO: 13-21; or, alternatively, exclude probes representing portions of or all of SEQ ID NO: 1-22.
• Figs. 1 A and B show a comparison of the waveforms and current voltage relationship for ⁇ j G i
• Figs. 2 A and B show a comparison of the waveforms and current voltage relationship for ⁇ calcium channels.
• Fig. 3 shows a comparison of the steady state inactivation profiles of the ⁇ io and ⁇ calcium channels.
• FIGs. 4A-C show a comparison of the inactivation kinetics of the ⁇ io and ⁇ calcium channels.
• Figures 5 A and 5B show the construction of the human ⁇ io cDNA complete sequence from partial clones.
• Figure 6 shows the nucleotide and deduced amino acid sequence of human T- type calcium channel ⁇ iG.
• Figure 7 shows a comparison of the waveforms and current voltage relationship for human ⁇ j G calcium channel.
• Figure 8 shows the characteristic pore pattern for T-type channels.
• the present invention includes the following aspects for which protection is sought:
• the invention encompasses an at least partially purified DNA molecule comprising a sequence of nucleotides that encodes an ⁇ i subunit of a T-type calcium channel, and such aj subunits per se.
• polymorphic variations may be made or may exist in the DNA of some individuals leading to minor deviations in the DNA or amino acids sequences from those shown which do not lead to any substantial alteration in the function of the calcium channel. Such variations, including variations which lead to substitutions of amino acids having similar properties are considered to be within the scope of the present invention.
• the present application claims DNA molecules which encode ⁇ subunits of mammalian T-type calcium channels, and which hybridize under conditions of medium (or higher) hybridization stringency with one or another of the specific sequences disclosed in this application.
• This level of hybridization stringency is generally sufficient given the length of the sequences involved to permit recovery of the subunits within the scope of the invention from mammalian DNA libraries.
• the T-type calcium channels of the invention are recognized by their functional characteristic of low voltage gating along with defined structural characteristics which classify them as ⁇ j calcium channel subunits and also characterize them as of the T-type.
• figure 8 shows that in sodium (Na) channels the P-region of the four domains contains the residues: aspartate/glutamate/lysine/alanine (single letter amino acid code: DEKA), while high threshold calcium channels such as the L-type channel contain the residues: glutamate/glutamate/glutamate/glutamate (single letter amino acid code: EEEE).
• DEKA sodium
• L-type channel contains the residues: glutamate/glutamate/glutamate/glutamate/glutamate (single letter amino acid code: EEEE).
• the ⁇ !G , 0C1 .H and ⁇ T-type channels are also distinct in this region compared to other types of ion channels including the C. elegans CI 1D2.6 and C27F2.3 and the rat NIC- channel ( Figure 8).
• a second distinguishing characteristic of the OL ⁇ G , X J H and ⁇ T-type channels compared to other types of calcium channels is that they do not contain a ⁇ subunit binding consensus sequence in the cytoplasmic linker separating domains I and II.
• all high threshold calcium channels contain a consensus sequence (single letter amino acid code: QQ-E— L-GY--WI — E) shown to physically interact with the calcium channel ⁇ subunit (Pragnell, M., De Waard, M., Mori, Y., Tanabe, T., Snutch, T.P. & Campbell, K.P., 1994, Nature 368:67-70).
• QQ-E— L-GY--WI — E single letter amino acid code
• a third distinguishing characteristic of the ( ⁇ ic am and ⁇ T-type channels is that they do not possess an EF-hand calcium binding motif in the region carboxyl to domain IV S6.
• all high threshold calcium channels contain a consensus sequence that is closely related to the EF-hand domain found in certain calcium binding proteins (de Leon, M., Wang, Y., Jones, L., Perez-Reyes, E., Wei, X., Soong, T.W., Snutch, T.P. & Yue, D.T., 1995, Science270: 1502-1506).
• T-type calcium channel ⁇ i subunits refers to subunits which contain these structural characteristics.
• the T-type ⁇ i subunit molecules can be defined by homology to the human and rat nucleotide and amino acid sequences described herein.
• T- type ⁇ ] subunits will typically have at least 50% and preferably 70% homology in terms of amino acid sequence or encoding nucleotide sequence to the sequences set forth in SEQ ID NOS. 23-28 herein or those shown in Figure 6.
• the homology will be at least 80%, more preferably 90%, and most preferably 95%, 97%, 98% or 99%.
• Relative homology may also be defined in terms of specific regions; as set forth above, certain regions of T-type channel ⁇ i subunits have very high homologies while other regions, such as the cytoplasmic region between domains II and III have less homology.
• T-type ⁇ i. subunits will have over 75% homology, preferably over 85% or over 95% homology, more preferably over 98% homology in domains I- IV to those of SEQ ID NO: 23-28 or Figure 6.
• the degree of homology in the cytoplasmic region between domains II and III may be substantially less, e.g., only 25%o homology, preferably 50% homology or more preferably 60% homology.
• the intracellular region downstream of domain IV may be less homologous than those within domains I-IV.
• T-type subunits have particular patterns of amino acids in the pore forming units as set forth in Figure 8.
• multiple probes might be used to distinguish other subunits, such as probes which represent the ⁇ -binding domain missing from the T-type ⁇ i subunits combined with a probe representing a consensus sequence for calcium channel ⁇ subunits in general.
• cell lines can be used to evaluate compounds as pharmacological modifiers of the function of the novel calcium channel subunits.
• These diseases include, but are not limited to; epilepsy, migraine, ataxia, schizophrenia, hypertension, arrhythmia, angina, depression and Parkinson's disease; characterization of such associations and ultimately diagnosis of associated diseases can be carried out with probes which bind to the wild-type or defective forms of the novel calcium channels.
• T-type channels in particular are responsible for rebound burst firing in central neurons and are implicated in normal brain functions such as slow-wave sleep and in neurological disorders such as epilepsy and mood disorders. They are also important in pacemaker activity in the heart, hormone secretion and fertilization, and are associated with disease states such as cardiac hypertrophy and hypertension.
• T-type calcium channel refers to a voltage-gated calcium channel having a low activation voltage, generally less than -50 mV, and most commonly less than -60 mV. T-type calcium channels also exhibit comparatively negative steady-state inactivation properties and slow deactivation kinetics.
• ⁇ i subunit or " ⁇ i calcium channel” refer to a protein subunit of a calcium channel which is responsible for pore formation and contains the voltage sensor and binding sites for calcium channel agonists and antagonists. Such subunits may be independently functional as calcium channels or may require the presence of other subunit types for complete functionality.
• the phrase "at least partially purified” refers to DNA or protein preparations in the which the specified molecule has been separated from adjacent cellular components and molecules with which it occurs in the natural state, either by virtue of performing a physical separation process or by virtue of making the DNA or protein molecule in a non- natural environment in the first place.
• the term encompasses cDNA molecules and expression vectors.
• mammalian DNA sequences which code for novel T-type calcium channel ⁇ j subunits. These subunits are believed to represent new types of ⁇ -, subunits of mammalian voltage- dependent calcium channels which have been designated as types ⁇ , am and ⁇ .
• BAC sequence (bK206c7) was identified from sequences in Sanger Genome Sequencing Center (Cambridge, U.K.) and the Washington University Genome Sequencing Center (St. Louis. MO) that contains a nucleotide sequence encoding the ⁇ subunit of human T-type calcium channel. The rationale for this identification is set forth in WO 98/38301, incorporated herein by reference. The relevant nucleotide sequence and the translated amino acid sequence containing 1854 amino acids are set forth in SEQ ID NO: 17 and 18.
• cDNA clones homologous to the probe are identified by autoradiography. Positive clones are purified by sequential rounds of screening. Following this protocol, most purified cDNA's are likely to be partial sequence clones due the nature of the cDNA library synthesis. Full length clones are constructed from cDNA's which overlap in DNA sequence. Restriction enzyme sites which overlap between cDNAs are used to ligate the individual cDNA's to generate a full-length cDNA.
• the full-length cDNA is subcloned directly into an appropriate vertebrate expression vector, such as pcDNA-3 (Invitrogen, San Diego, CA) in which expression of the cDNA is under the control of a promoter such as the CMV major intermediate early promoter/enhancer.
• pcDNA-3 Invitrogen, San Diego, CA
• suitable expression vectors include, for example, pMT2, pRC/CMV, pcDNA3.1 and pCEP4.
• ⁇ , and ⁇ calcium channel subunit cDNAs were isolated by using the 567 base pair human fragment (Seq. ID No. 19) to screen a rat brain cDNA library. Sequencing of the recovered sequences identified the three distinct classes of calcium channel subunits which have been denominated herein as ⁇ io, a and ⁇ subunits. For each class of subunit, complete sequencing of the largest cDNA confirmed that it represented only a portion of the predicted calcium channel coding region. Complete sequences for the three new subunits were obtained by rescreening the rat brain cDNA library with probes derived from the partial length cDNAs to obtain overlapping segments.
• a host cell for expression.
• Suitable host cells include Xenopus oocytes or mammalian cells such as human embryonic kidney cells as described in International Patent Publication No. WO 96/39512 which is incorporated herein by reference and Ltk cells as described in US Patent No. 5,386,025 which is incorporated herein by reference.
• Transfection into host cells may be accomplished by microinjection, lipofection, glycerol shock, electroporation calcium phosphate or particle-mediated gene transfer.
• the vector may also be transfected into host cells to provide coexpression of the novel ⁇ i subunits with other subunits, such as an ⁇ 2 ⁇ subunit or a ⁇ subunit.
• the ⁇ jo and ⁇ cDNAs were transiently transfected into human embryonic kidney cells and evaluated using electrophysiological recording techniques. The results are consistent with a role of these subunits in native T-type channels in nerve, muscle and endocrine cells. Similarly, a full length clone encoding human ⁇ i G T-type subunit was recovered and verified to have the characteristic properties of T-type channels.
• the resulting cell lines expressing functional calcium channels including the novel ⁇ i subunits of the invention can be used test compounds for pharmacological activity with respect to these calcium channels.
• the cell lines are useful for screening compounds for pharmaceutical utility.
• Such screening can be carried out using several available methods for evaluation of the interaction, if any, between the test compound and the calcium channel.
• One such method involves the binding of radiolabeled agents that interact with the calcium channel and subsequent analysis of equilibrium binding measurements including but not limited to, on rates, off rates, K ⁇ j values and competitive binding by other molecules.
• Another such method involves the screening for the effects of compounds by electrophysiological assay whereby individual cells are impaled with a microelectrode and currents through the calcium channel are recorded before and after application of the compound of interest.
• Another method, high-throughput spectrophotometric assay utilizes the loading the cell lines with a fluorescent dye sensitive to intracellular calcium concentration and subsequent examination of the effects of compounds on the ability of depolarization by potassium chloride or other means to alter intracellular calcium levels.
• Compounds to be tested as agonists or antagonists of the novel ⁇ calcium channel subunits are combined with cells that are stably or transiently transformed with a DNA sequence encoding the ⁇ iG, am and ⁇ calcium channel subunits of the invention and monitored using one of these techniques.
• Compounds which are shown to modulate the activity of calcium channels can then be used in pharmaceutical compositions for the treatment associated with inappropriate T-type calcium channel activity. Such conditions may also include those with inappropriate calcium channel activity in general since such activity may be modified by enhancing or decreasing T-type channel activity. Conditions appropriate for such treatment include those set forth above.
• the compounds identified are formulated in conventional ways as set forth in Remington's "Pharmaceutical Sciences," latest edition, Mac Publishing Co., Easton, PA. Modes of administration are those appropriate for the condition to be treated and are within the ordinary skill of the practitioner.
• T-type calcium channels can be achieved by constructing expression systems encoding antisense sequences or sequences designed for triplex binding to interrupt the expression of nucleotide sequences encoding the T-type calcium channels of the invention.
• DNA fragments with sequences given by SEQ ID Nos. 13-17 and 19, or polynucleotides with the complete coding sequences as given by Sequence ED Nos. 23, 25 and 27 or Figure 6, or distinctive portions thereof which do not exhibit non- discriminatory levels of homology with other types of calcium channel subunits may also be used for mapping the distribution of ⁇ 1G , ⁇ 1H and ⁇ calcium channel subunits within a tissue sample.
• This method follows normal histological procedures using a nucleic acid probe, and generally involves the steps of exposing the tissue to a reagent comprising a directly or indirectly detectable label coupled to a selected DNA fragment, and detecting reagent that has bound to the tissue.
• Suitable labels include fluorescent labels, enzyme labels, chromophores and radio-labels.
• Host cells such as human embryonic kidney cells, HEK 293 (ATCC# CRL 1573) are grown in standard DMEM medium supplemented with 2 mM glutamine and 10%o fetal bovine serum.
• HEK 293 cells are transfected by a standard calcium- phosphate-DNA co-precipitation method using a full-length mammalian ⁇ i T-type calcium channel cDNA (for example, Seq. ID. No. 27) in a vertebrate expression vector (for example see Current protocols in Molecular Biology).
• the ⁇ calcium channel cDNA may be transfected alone or in combination with other cloned subunits for mammalian calcium channels, such as ⁇ 2 ⁇ and ⁇ or ⁇ subunits, and also with clones for marker proteins such the jellyfish green fluorescent protein.
• Electrophysiological Recording After an incubation period of from 24 to 72 hrs the culture medium is removed and replaced with external recording solution (see below).
• Whole cell patch clamp experiments are performed using an Axopatch 200B amplifier (Axon Instruments, Burlingame, CA) linked to an IBM compatible personal computer equipped with pCLAMP software.
• Microelectrodes are filled with 3 M CsCl and have typical resistances from 0.5 to 2.5 M ohms.
• the external recording solution is 2 mM BaCl 2 , 1 mM MgCl 2 , 10 mM HEPES, 40 mM TEAC1, 10 mM Glucose, 92 mM CsCl, (pH 7.2).
• the internal pipette solution is 105 mM CsCl, 25 mM TEAC1, 1 mM CaCl 2 , 11 mM EGTA, 10 mM HEPES (pH 7.2).
• Currents are typically elicited from a holding potential of -100 mV to various test potentials. Data are filtered at 1 kHz and recorded directly on the harddrive of a personal computer. Leak subtraction is carried out on-line using a standard P/5 protocol. Currents are analyzed using pCLAMP versions 5.5 and 6.0.
• Xenopus Oocytes Stage V and VI Xenopus oocytes are prepared as described by Dascal et al (1986), Expression and modulation of voltage-gated calcium channels after RNA injection into Xenopus oocytes. Science 231 :1147- 1150. After enzymatic dissociation with collagenase, oocytes nuclei are microinjected with the human an calcium channel cDNA expression vector construct (approximately 10 ng DNA per nucleus) using a Drummond nanoject apparatus. The ⁇ calcium channel may be injected alone, or in combination with other mammalian calcium channel subunit cDNAs, such as the ⁇ 2- ⁇ and ⁇ lb and ⁇ subunits.
• Endogenous Ca (and Ba) -activated CI currents are suppressed by systematically injecting 10-30 nl of a solution containing lOOmM BAPTA-free acid, lOmM HEPES (pH titrated to 7.2 with CsOH) using a third pipette connected to a pneumatic injector. Leak currents and capacitive transients are subtracted using a standard P/5 procedure.
• Mammalian cell lines stably expressing human ⁇ calcium channels are constructed by transfecting the ⁇ calcium channel cDNA into mammalian cells such as HEK 293 and selecting for antibiotic resistance encoded for by an expression vector. Briefly, a full-length mammalian T-type calcium channel ⁇ i subunit cDNA (for example Seq. ID No.
• a vertebrate expression vector with a selectable marker such as the pcDNA3 (InvitroGen, San Diego, CA)
• pcDNA3 InvitroGen, San Diego, CA
• the ⁇ calcium channel may be transfected alone, or in combination with other mammalian calcium channel subunit cDNAs, such as the ⁇ 2- ⁇ and ⁇ lb subunits, either in a similar expression vector or other type of vector using different selectable markers.
• the medium After incubation for 2 days in nonselective conditions, the medium is supplemented with Geneticin (G418) at a concentration of between 600 to 800 ug/ml. After 3 to 4 weeks in this medium, cells which are resistant to G418 are visible and can be cloned as isolated colonies using standard cloning rings. After growing up each isolated colony to confluency to establish cell lines, the expression of ⁇ calcium channels can be determined at with standard gene expression methods such as Northern blotting, RNase protection and reverse-transcriptase PCR.
• the functional detection of ⁇ calcium channels in stably transfected cells can be examined electrophysiologically, such as by whole patch clamp or single channel analysis (see above).
• Other means of detecting functional calcium channels include the use of radiolabeled 45 Ca uptake, fluorescence spectroscopy using calcium sensitive dyes such as FURA-2, and the binding or displacement of radiolabeled ligands that interact with the calcium channel.
• Partial Rat and Human Subunits In order to recover mammalian sequences for novel calcium channels, the 567 base pair partial length human brain ⁇ cDNA described in WO 98/3801 was gel- purified, radio-labelled with 32 P dATP and dCTP by random priming (Feinberg et al., 1983, Anal. Biochem. 132: 6-13) and used to screen a rat brain cDNA library constructed in the phase vector Lambda Zapp II. (Snutch et al., 1990, Proc Natl Acad Sci (USA) 87: 3391-3395).
• Double stranded DNA sequencing on the recombinant phagemids was performed using a modified dideoxynucleotide protocol (Biggin et al, 1983, Proc Natl Acad Sci (USA) 80:3963-3965) and Sequenase version 2.1 (United States Biochemical Corp.).
• DNA sequencing identified three distinct classes of calcium channel ⁇ i subunits: designated as ⁇ ic am and ⁇ calcium channel subunits.
• the largest cDNA was completely sequenced and determined to represent only a portion of the predicted calcium channel coding region.
• the ⁇ i G clone was digested with Hindlll and Spel.
• the resulting 540 base pair fragment was gel purified, radiolabeled with 32 P dATP and dCTP by random priming and used to rescreen the rat brain cDNA library as described above.
• the sequence of the 540 base pair ⁇ screening probe used is given by Seq. ID No. 29. Other sequences spanning regions of distinctiveness within the sequences for the subunits could also be employed.
• Double-stranded DNA sequencing of the purified recombinant phagemids showed that additional ⁇ , am and ⁇ calcium channel subunit cDNAs overlapped with the original partial length cDNAs and together encoded complete protein coding regions as well as portions of their respective 5' and 3' non-coding untranslated regions.
• the 567 base pair partial length human brain ⁇ cDNA (Seq. 19) was radio- labelled with P dATP and dCTP by random priming and used to screen a commercial human thalamus cDNA library (Clontech). Hybridization was performed overnight at 65 ° C in 6 X SSPE; 0.3% SDS; 5X Denhardt's. Filters were washed at 65 ° C in 0.1 X SSPE/ 0.3% SDS. After four rounds of screening and plaque purification, positive phages were selected, DNA prepared and the insert cDNA excised from the lambda vector by digestion with Eco Rl restriction enzyme.
• the excised cDNA was subcloned into the plasmid Bluescript KS (Stratagene, La Jolla, CA) and the DNA sequence determined using a modified dideoxynucleotide protocol and Sequence version 2.1.
• the partial length ⁇ cDNA isolated consisted of 2212 base pairs of which 279 base pairs were 5' noncoding and 1,933 base pairs were coding region representing 644 amino acids (Seq. ID Nos. 30, 31).
• the partial am cDNA isolated consisted of 1,608 base pairs of which 53 base pairs were 5' noncoding and 1,555 were coding region representing 518 amino acids (Seq. ID Nos. 32, 33).
• the full-length rat brain ⁇ cDNA (Seq. 27; see example 2) was radio-labelled P dATP and dCTP by random priming and used to screen a commercial human hippocampus cDNA library (Stratagene). Hybridization was performed overnight at 65°C in 6 X SSPE; 0.3% SDS; 5X Denhardt's. Filters were washed at 65° C in 0.1 X SSPE/ 0.3% SDS. After four rounds of screening and plaque purification, positive phages were transformed into Bluescript phagemids (Stratagene, LA Jolla, CA) by in vitro excision.
• the excised cDNA DNA sequence was determined using a modified dideoxynucleotide protocol and Sequenase version 2.1.
• the partial ⁇ cDNA isolated consisted of 1,080 base pairs of coding region representing 360 amino acids (Seq. ID Nos. 34, 35).
• Double-stranded DNA sequencing of the purified recombinant phagemids from rat brain showed that additional ⁇ jc and ⁇ calcium channel cDNAs overlapped with the original partial length cDNAs and together encoded complete protein coding regions as well as portions of their respective 5' and 3' non-coding untranslated regions. (Seq. ID Nos. 23 and 27, respectively)
• DNA sequencing of the recombinant phagemids also identified a third class of calcium channel ⁇ ] subunit: designated as the a calcium channel subunit.
• HEK-tsa201 transiently-transfected human embryonic kidney cells prepared using the general protocol above. Transfection was carried out by standard calcium phosphate precipitation. (Okayama et al., 1991, Methods in Molec. Biol, Vol. 7, ed. Murray, E.J.). For maintenance, HEK-tsa201 cells were cultured until approximately 70% confluent, the media removed and cells dispersed with trypsin and gentle trituration.
• Cells were then diluted 1:10 in culture medium (10% FBS, DMEM plus L-glutamine, pen-strp) warmed to 37°C and plated onto tissue culture dishes.
• 0.5 mM CaCl 2 was mixed with a total of 20 ⁇ g of DNA (consisting of 3 ⁇ g of either rat brain ⁇ -, 0 or ⁇ calcium channel cDNA, 2 ⁇ g of CD8 plasmid marker, and 15 ⁇ g of Bluescript plasmid carrier DNA).
• I-V current- voltage
• Figs. 1-4 show the results obtained for HEK cells transfected with and expressing the cDNA of sequences ID Nos. 23 and 27, which correspond to the subunits designated as ⁇ tG and ⁇ - Figs. 1 A and B and 2A and B shows a comparison of the waveforms and current- voltage relationship for the two channel subunit types.
• both the ⁇ j G and ⁇ channel subunits exhibit activation properties consistent with native T-type calcium currents.
• Figs 1 A and 2A show the subunit calcium current from a cell held at -120 mV and depolarized to a series of test potentials.
• Figs IB and 2B show the magnitude of the calcium current.
• both channel first activate at approximately -70 mV and peak currents are obtained between -40 and -50 mV.
• the current waveforms of the ⁇ io and ⁇ calcium channels exhibit an overlapping pattern characteristic of native T-type channels in nerve, muscle and endocrine cells.
• Fig. 3 shows steady-state inactivation profiles for the ⁇ io and ⁇ calcium channels in HEK 293 cells transiently transformed with full length cDNAs (SEQ ID Nos 23 or 27) for ⁇ or ⁇ subunits.
• Steady state inactivation properties were determined by stepping from -120 mV to prepulse holding potentials between -120 mV and -50 mV for 15 sec. prior to a test potential of -30 mV.
• the data are plotted as normalized whole cell current versus prepulse holding potential and show that ⁇ jc exhibits a V 50 of approximately -85 mV and ⁇ a V 50 of approximately -93 mV.
• Figs. 4A-C show a comparison of the voltage-dependent deactivation profiles of the ⁇ !G and ⁇ calcium channels.
• HEK 293 cells were transiently transfected with either an ⁇ j G or ⁇ subunit cDNA (Seq. ID No. 23 or 27).
• the deactivation properties of ⁇ !G were determined by stepping from a holding potential of -100 mV to -40mV for 9 msec, and then to potentials between -120 mV and -45 mV.
• the deactivation properties of ⁇ were determined by stepping from a holding potential of -100 mV to -40 mV for 20 msec, and then to potentials between -120 mV and -45 mV. Both channels exhibit slow deactivation kinetics compared to typical high-threshold channels, and is consistent with the ⁇ >, G and ⁇ subunits being subunits for T-type calcium channels
• Example 3 Cloning of a Full Length cDNA for the Human ⁇ lg T-Type Calcium Channel Subunit Materials and Methods: A full length cDNA encoding the human ⁇ jo subunit was constructed from 5 overlapping clones ( Figure IB) isolated from a human thalamus cDNA library constructed in ⁇ gtl 1 vector (Clontech, Cat#HL5009b).
• ⁇ gtl 1 cDNA clones were isolated by conventional filter hybridization.
• Clone 1 was identified by hybridization to a 567 bp cDNA probe (SEQ ID NO: 19) containing the transmembrane region S4 to S6 of domain I of the previously cloned human neuronal ⁇ T-type calcium channel subunit.
• Clones HG10-1112 and HG5-1211 were identified by hybridization to a 1265 bp cDNA probe of the rat am T-type calcium channel subunit spanning domain II and part of the II-III intracellular loop.
• cDNA probes were P-dCTP labelled by random priming using a Multiprime DNA labeling system (Amersham Pharmacia).
• Plaque lifts using H-bond nylon membranes were done in duplicate following the standard protocols supplied by manufacturer (Amersham Pharmacia). Hybridization was performed for at least 16hrs at 65°C for clone 1 and for at least 16hrs at 58° C, clones HG10-1112 and HG5-1211. Membranes were washed in 0.1X SSC/0.3% SDS at 62°C for clone 1 and 0.2X SSC/0.1% SDS at 58°C clones HG10-1112 and HG5-1211. Blots were exposed to BioMax MS Kodak film with Kodak HE intensifying screens for at least 48hrs at -80°C. Double positive plaques were isolated and re-screened to isolate single clones according to the procedure above.
• Bacteriophage DNAs were then isolated according to the ⁇ gtl 1 library User Manual (Clontech). Clone 1 cDNA insert was excised with EcoRI (NEB) and subcloned into pBluescriptKS (Stratagene). Clones HGlO-1112 and HG5-1211 cDNA inserts were excised from ⁇ DNA with Not I (NEB) and subcloned into the Not I site of pBluescriptKS. Plasmids with cDNA inserts were transformed by electroporation into XL-I E.Coli host strain bacteria and sequenced using universal reverse and forward primers according to Sanger double stranded DNA sequencing method in combination with automatic sequencing ABI 100 PRISM model 377 Version3.3 (PE Biosystems).
• Clone 1 was identified as a human ⁇ subunit containing the 5'UTR and 1933 bp of the in-frame coding region, including part of the intracellular I-II loop.
• Clone HG10-1112 was identified as a human ⁇ subunit of 3915 bp, spanning Domain I (IS5-IS6) to the III-IV loop.
• Clone HG5-1211 was identified as human ⁇ subunit of 3984 bp containing the I-II linker and C-terminus.
• the remaining region of the 3' ⁇ ic subunit cDNA was obtained using the PCR method on a human thalamus cDNA library with primers MD19-sense (5'GCG TGG AGC TCT TTG GAG 3 ') and G26- antisense (5 ' GCA CCC AGT GGA GAA AGG TG 3').
• the PCR protocol used was 94°C -30 sec, 58°C -30 sec, 72°C -30 sec for 25 cycles (Bio-rad Gene Cycler).
• a cDNA fragment of 1617 bp was subcloned into p- Gem-T-Easy plasmid vector (Promega) and sequenced.
• the 3 'PCR cDNA was identified as a human ⁇ io subunit spanning from Domain IV-S5 to the carboxyl terminus including the stop codon.
• the intrapipette solution contained (in mM): 105 CsCl, 25 CsCl, 1 CaCl 2 , 11 EGTA, 10 HEPES, pH 7.2.
• the extracellular solution contained (in mM): 40 TEA-C1, 2 CaCl 2 , 1 MgCl 2 , 92 CsCl, 10 glucose, 10 HEPES, pH 7.2.
• Figure 7 shows that the human ⁇ cDNA encodes a calcium channel with typical properties of a T-type current.
• the left panel illustrates representative current traces obtained from a holding potential of -100 mV to test pulses potentials of -90 mV to +20 mV. The traces show a typical crossover pattern and considerable inactivation during the test pulse, both of which are consistent with native T-type channels.
• the right panel shows a plot of the peak whole current at various test potentials and indicates that the human ⁇ ic cDNA first activates near -60 mV with maximal current near —40 mV, which is also consistent with native low-threshold T- type calcium channels.

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