WO2000070044A2

WO2000070044A2 - Human brain t calcium channel alpha-subunit splice variants

Info

Publication number: WO2000070044A2
Application number: PCT/US2000/012383
Authority: WO
Inventors: Scott Mittman; William S. Agnew
Original assignee: Johns Hopkins University
Current assignee: Johns Hopkins University
Priority date: 1999-05-13
Filing date: 2000-05-08
Publication date: 2000-11-23
Anticipated expiration: 2001-11-13
Also published as: WO2000070044A3; WO2000070044A9; AU4989500A

Abstract

The structures of CACNA1G and CACNA1I, the genes encoding the human brain T Ca2+ channel α¿1G? and α1I subunits, respectively, were determined by comparison of polymerase chain reaction-amplified brain cDNA and genomic sequences. CACNA1G consists of at least 38 exons spanning at least 66,490 basepairs of chromosome 17q22. Alternative splicing of the RNA occurs at six sites: cassette exons 14, 26, 34 and 35, an internal donor in exon 25 and protein-coding intron 38B. Additionally, the RNA can be polyadenylated at either of two sites. Alternative splicing of CANCA1G RNA may lead to expression of as many as 64 distinct protein products, ranging from 2,171 to 2,377 amino-acids residues, with as many as 45 potential phosphorylation sites. CACNA1I consists of at least 37 exons spanning at least 116,390 basepairs of chromosome 22q12.3-13.2. Alternative splicing of the gene occurs at three sites: cassette exon 9, an alternative acceptor in exon 33 and mutually-exclusive 3' exons 36B and 37. Alternative splicing of CANCA1I RNA may lead to expression of as many as 8 distinct protein products, ranging from 1,968 to 2,223 amino-acids residues, with as many as 28 potential phosphorylation sites. Molecular diversity generated by alternative splicing and post-translation modification of these and other members of the T α1 subunit gene family may account for the observed heterogeneity of T currents in central neurons.

Description

HUMAN BRAIN T CALCIUM CHANNEL ALPHA-SUBUNIT SPLICE VARIANTS

This invention was made using funds from the U.S. government. Under the terms of NIH grants K08NS01939 and P50HL52307, the government may retain certain rights in the invention.

TECHNICAL FIELD OF THE INVENTION

This invention is related to ion channels. In particular, it is related to ion channels related to brain function. BACKGROUND OF THE INVENTION Voltage-dependent calcium channels are involved in both coupling electrical activity to calcium influx and contributing to membrane properties. Low voltage- activated (LVA) calcium channels activate at potentials near the resting membrane potential. LVA participate in spike-induced calcium entry and allow calcium influx at potentials below threshold. LVA calcium channels also are involved in subthreshold membrane fluctuations. LVA calcium channel dysfunction is implicated in epileptiform activity. Moreover, these channels are targets for antiepileptic drugs.

T-type (transient) properties in neurons include low voltage activation, strongly voltage-dependent kinetics, rapid inactivation, slow deactivation, and small single- channel conductance. Recently, a subfamily of genes (designated Ca-_^T) has been discovered encoding a. subunits that are ~ 30% homologous to HVA subunit genes in their putative membrane-spanning regions. T currents are a diverse class of Ca²⁺ current characterized by a low voltage threshold for activation. Proposed functions include generation of low-threshold spikes that lead to bursting, promotion of voltage oscillations, boosting of Ca²⁺ entry and synaptic potentiation. T currents may be the targets of succinimides and related compounds administered in the treatment of absence epilepsy. Recently, cDNA sequences of three T _x subunits, rat α_1G and α_u and human α_1H, have been reported.

Ca²⁺ channel cC_j subunits are encoded by at least 10 genes falling into three subfamilies: ABE, SCDF and GHI¹. Alternative splicing of c^ RNAs generates further molecular diversity. There is a need in the art for identifying the different splice forms of the calcium channel subunits, so that they can be used as targets in drug discovery and development programs. SUMMARY OF THE INVENTION

It is an object of the present invention to provide an isolated and purified α_1G subunit of human brain T calcium channel.

It is an object of the present invention to provide an isolated and purified nucleic acid encoding the α_1G subunit.

It is an object of the present invention to provide an isolated and purified an subunit of human brain T calcium channel. It is an object of the present invention to provide an isolated and purified nucleic acid encoding the α_π subunit.

It is another object of the present invention to provide an isolated and purified nucleic acid comprising an exon of a human brain T calcium channel alpha subunit. Another object of the invention is to provide an isolated and purified polypeptide which comprises a translated exon of a human brain T calcium channel.

1

The gene encoding the subunit α_ιx, where X is A - 1, or S, is denoted CACNA1X. Alternative names for the SCDF and GHI subfamilies are L and T, respectively. Another object of the invention is to provide expression vectors and host cells for expressing the subunits of human brain T calcium channel.

Another object of the invention is to provide a method to identify candidate drugs for treating epilepsy. These and other objects of the invention are achieved by one or more of the embodiments described below. In one embodiment an isolated and purified α_1G subunit of human brain T calcium channel is provided. The subunit is selected from splice variants 1-64 as shown in Table 1.

According to another object of the invention an isolated and purified nucleic acid encoding the α_1G subunit is provided.

According to still another object of the invention an isolated and purified polypeptide is provided which comprises a translated exon selected from the group consisting of 1-38D as shown in Table 2.

Another embodiment of the invention is an isolated and purified nucleic acid which comprises an exon selected from the group consisting of 1-38D as shown in Table 2.

Still another embodiment of the invention is an isolated and purified α_π subunit of human brain T calcium channel selected from splice variants 1-8 as shown in Table 3.

The present invention also provides an isolated and purified polypeptide which comprises a translated exon selected from the group consisting of 1-37 as shown in Table 4.

According to another aspect of the invention an isolated and purified nucleic acid is provided which comprises an exon selected from the group consisting of 1-37 as shown in Table 4.

Vectors and host cells which contain and/or express any of the nucleic acids, polypeptides or proteins described above are also contemplated as part of the present invention.

Other embodiments of the invention are methods to identify candidate drugs for treating epilepsy. A host cell containing a nucleic acid encoding an α_1G or α_π subunit or exon is contacted with a test substance. Uptake by the cell of calcium ions is measured. A test substance which inhibits the uptake by the cell of calcium ions is identified as a candidate drug for treating epilepsy. These and other embodiments of the invention which will be described in more detail below, and which will be evident to those of ordinary skill in the art upon reading the disclosure, provide the art with new drug discovery targets which can form the basis of a drug screening program. BRTEF DESCRIPTION OF THE DRAWINGS

Fig. 1. Map of CACNA 1 T and α_u cDNA In the genomic map (bottom), exons are indicated by vertical bars and introns by the connecting horizontal line. The smallest exons are not to scale due to the minimum line thickness required for printing. In the cDNA map (middle), constitutively-spliced, odd-numbered exons are black and even-numbered exons, gray. Alternatively-processed exons (or portions of exons) are colored as follows: 9 - red, 33 A - orange, 36B - green. The thinner portions of exons 1 and 36 represent the 5' and 3' untranslated regions, respectively. Selected exons are labeled to facilitate counting. Black bars above this cDNA map indicate relative PCR product locations. Four of the bars are interrupted by a thin line to indicate portions deleted by alternative splicing. Exon 37 (blue) is mutually exclusive with exon 36, requiring a separate representation of the 3^' end of the cDNA at the top. Only a small portion of the exon 37 3' UTR has been amplified and sequenced. Two of the PCR products containing portions of exon 37 are represented as black bars above the partial cDNA map. The starred scale bar equals 1 kb for PCR products and the cDNA maps and 15 kb for the genomic map.

Fig. 2. Schematic of the predicted α_π protein. Each aa residue is represented by a small circle. In the large cytoplasmic and extracellular loops, a full up-down cycle measures 100 residues. Main features of the topology are labeled in large type and described in the text. Portions of the protein derived from odd-numbered exons are labeled in small type. A similarity score was computed for each residue from alignments of the aa sequence of each α_π exon with the sequences of the homologous human α_1G and α_1H exons by iterative pairwise use of gap with default parameters. Pipe, colon, period and space similarity symbols were assigned numerical values of 3, 2, 1 and 0, respectively; α_π vs. α_1G and α_π vs. α_1H scores for an individual α_π residue were added to yield a final score of 0 to 6. Residue identity in all three proteins produced a score of 6; pairing of an a_n residue with unrelated amino acids in both alignments produced a score of 0. Exons 9, 34 and 35 had no apparent <χ._G or α_1H homologues; these residues are uncolored. Exon 16 had only an α_1H homologue and exons 33 and 37 had only α_1G homologues; the maximum possible similarity score for these exons is 3. Portions of the protein deleted by alternative splicing have a light blue background. Mutually exclusive exons 36B (7 aa) and 37 (214 aa) are side-by-side. Extracellular cysteines, potential

N-glycosylation and phosphorylation sites and the location of splice sites (mapped to the protein product) are indicated by the appropriate symbols. Symbol colors have the following meanings: black - conserved among all human α. subunits; purple - conserved within 3 aa residues in the multiple sequence alignment of all human a_λ subunits; blue - conserved among the human ABE and GHI subfamilies; green - conserved among all human T _γ subunits; brown - also present in human α^ orange - also present in human α_1G; pink - unique to α_π. PKA: cyclic-nucleotide-dependent protein kinase phosphorylation site, PKC: protein kinase C phosphorylation site, CKII: casein kinase II phosphorylation site, Tyr: tyrosine kinase phosphorylation site. One residue in the C-terminus was identified as a potential site for phosphorylation by both PKA and CKH; another was identified as a potential site for phosphorylation by both PKA and PKC.

Fig. 3. Map ofCACNAIG and the α_1G cDNA In the genomic map (bottom), exons are indicated by vertical bars and introns by the connecting horizontal line. The smallest exons are not to scale due to the minimum line thickness required for printing. In the cDNA map (middle), constitutively-spliced, odd-numbered exons are gray and even-numbered exons, black. Alternatively-processed exons (or portions of exons) are colored as follows: 14 - olive, 25B - red, 26 - blue, 34 - light green, 35 - orange, 38B - dark green, 38D - purple. The thinner portions of exons 1 and 38 represent the 5 ' and 3 ' untranslated regions, respectively. Selected exons are labeled to facilitate counting. Black bars at the top of the figure indicate PCR product locations relative to the cDNA map. Nine of the bars are interrupted by a thin line to indicate portions deleted by alternative splicing. Red bars (labeled with GenBank accession numbers) represent infant brain cDNA clone ESTs. For one clone, only a 3 ^' EST has been reported. Thin lines indicate portions deleted by alternative splicing and dashed lines indicate unsequenced portions The starred scale bar equals 1 kb for PCR products and the cDNA map and 10 kb for the genomic map.

Fig. 4. Schematic of predicted α_1G proteins. Each aa residues is represented by a small circle. In the large cytoplasmic and extracellular loops, a full up-down cycle measures 100 residues. Main features of the topology are labeled in large type and described in the text. Portions of the protein involved in alternative splicing have a blue background. These and portions derived from odd-numbered exons are labeled in small type. A similarity score was computed for each residue from alignments of the aa sequence of each α_1G exon with the sequences of the homologous human α _1H (unpublished observations) and (submitted) exons by iterative pairwise use of gap

(Genetics Computer Group, Wisconsin Package Version 9.0) with default parameters. Pipe, colon, period and space similarity symbols were assigned numerical values of 3, 2, 1 and 0, respectively; α_1G vs. α_1H and α_1Gv.s. _u scores for an individual α_1G residue were added to yield a final score of 0 to 6. Residue identity in all three proteins produced a score of 6; pairing of an α_1G residue with unrelated amino acids in both alignments produced a score of 0. Exons 14, 16, 26, 35 and 38 had no apparent α_1H or α_u homologues; these residues are uncolored. Exon 36 and the C-terminal half of exon 8 had only α_1H homologues and exon 34 had only an α_π homologue; the maximum possible similarity score for these regions is 3. Splice sites, extracellular cysteines and potential N-glycosylation and phosphorylation sites identified with PROSITE are indicated by the appropriate symbols. Symbol colors have the following meanings: black - conserved among all human α_x subunits, purple - conserved within 3 aa residues in the multiple sequence alignment of all human α_t subunits, blue - conserved among the human ABE and GHI subfamilies, green - conserved among all human T a subunits, brown - also present in human an, orange - also present in human α_1H, pink - unique to α_1G. PKA: cyclic-nucleotide-dependent protein kinase phosphorylation site, PKC: protein kinase C phosphorylation site, CKH: casein kinase II phosphorylation site, Tyr: tyrosine kinase phosphorylation site. One residue in ID 1-2 was identified as a potential site for phosphorylation by both PKA and PKC.

Fig. 5 is a schematic diagram of the RΝA processing leading to the 8 α_π variants. DETAILED DESCRIPTION OF THE INVENTION

The human brain T calcium channel α_1G subunit gene, CACNA1G, has now been discovered to consist of 38 protein-coding exons. Alternative processing of the gene transcript allows this single gene to code for sixty-four distinct αι_G protein products. In Table 2, each exon or portion of an exon is listed. In Table 1, the component exons of individual splice variants are described. These two tables are sufficient for a complete description of the newly discovered compositions.

Table 1 lists the component exons of the 64 α_1G protein products. Only the missing portions of each variant are noted in the description; the symbol " Δ" denotes deletion of the exon following the symbol. Thus, variant 1 consists of all exons save 14,

25B, 26, 34, 35 and 38B; in other words, exons 1 - 13, 15- 24, 25A 27 - 33, 36 - 37, 38A and 38C are concatenated to form the protein. The final column lists the number of aa residues in each variant.

Table 1. α_1G Splice Variants

vo

-N>

υ-

+

+ +

n 00 co

<1

CO

VO •o For each exon, the nucleotide sequence and the corresponding amino-acid (aa) sequence are listed in single-letter IUPAC code. Lower case letters in the aa sequences indicate that only two nucleotides of the codon belong to the exon (the codon is interrupted). A dash indicates a stop codon.

Table 2.

(SEQ ID NOs: 1 and 82. Exon 1 (constitutive) atggacgaggaggaggatggagcgggcgccgaggagtcgggacagccccggagcttcatgcggctcaa cgacctgtcgggggccgggggccggccggggccggggtcagcagaaaaggacccgggcagcgcggact ccgaggcggaggggctgccgtacccggcgctggccccggtggttttcttctacttgagccaggacagc cgcccgcggagctggtgtctccgcacggtctgtaaccc

MDEEEDGAGAEESGQPRSFMRLND SGAGGRPGPGSAEKDPGSADSEAEGLPYPA-LAPWFFYLSQDS RPRSWCLRTVCNp

(SEQ ID NOs: 2 and 83) Exon 2 (constitutive) ctggtttgagcgcatcagcatgttggtcatccttctcaactgcgtgaccctgggcatgttccggccat gcgaggacatcgcctgtgactcccagcgctgccggatcctgcag

WFERISMLVILLNCVTLGMFRPCEDIACDSQRCRI Q

(SEQ ID NOs: 3 and 84) Exon 3 (constitutive) gcctttgatgacttcatctttgccttctttgccgtggagatggtggtgaagatggtggccttgggcat ctttgggaaaaag gttacctgggagacacttggaaccggcttgactttttcatcgtcatcgcagg

AFDDFIFAFFAVEMWKMVA GIFGKKCYLGDTW RLDFFIVIAg

(SEQ ID NOs: 4 and 85) Exon 4 (constitutive) gatgctggagtactcgctggacctgcagaacgtcagcttctcagctgtcaggacagtccgtgtgctgc gaccgctcagggccattaaccgggtgccca

MLEYSLDLQNVSFSAVRTVRVLRPLRAINRVP

(SEQ ID NOs: 5 and 86) Exon 5 (constitutive) gcatgcgcatccttgtcacgttgctgctggatacgctgcccatgctgggcaacgtcctgctgctctgc ttcttcgtcttcttcatcttcggcatcgtcggcgtccagctgtgggcagggctgcttcggaaccgatg cttcctacctgagaatttcagcct

SMRILVTLLLDTLPMLGNVLL CFFVFFIFGIVGVQLWAGLLRNRCFLPENFS1 (SEQ ID NOs: 6 and 87) Exon 6 (constitutive) ccccctgagcgtggacctggagcgctattaccagacagagaacgaggatgagagcccct catctgct cccagccacgcgagaacggcatgcggtcctgcagaagcgtgcccacgctgcgcggggacgggggcggt ggcccaccttgcggtctggactatgaggcctacaacagctccagcaacaccacctgtgtcaactggaa ccagtac acaccaactgctcagcgggggagcacaaccccttcaagggcgccatcaact-ttgacaaca ttggctatgcctggatcgccatcttccag

PLSVDLERYYQTENEDESPFICSQPRENGMRSCRSVPTLRGDGGGGPPCGLDYEAYNSSSNTTCVNWN QYYTNCSAGEHNPFKGAINFDNIGYAWIAIFQ

(SEQ ID NOs: 7 and 88) Exon 7 (constitutive) gtcatcacgctggagggctgggtcgacatcatgtactttgtgatggatgctcattccttctacaattt ca ctacttcatcctcctcatcatc

VITLEGWVDIMYFVMDAHSFYNFIYFILLII

(SEQ ID NOs: 8 and 89) Exon 8 (constitutive) gtgggctccttcttcatgatcaacctgtgcctggtggtgattgccacgcagttctcagagaccaagca gcgggaaagccagctgatgcgggagcagcgtgtgcggttcctgtccaacgccagcaccctggctagct tctctgagcccggcagctgctatgaggagctgctcaagtacctggtg acatccttcgtaaggcagcc cgcaggctggctcaggtctctcgggcagcaggtgtgcgggttgggctgctcagcagcccagcacccct cgggggccaggagacccagcccagcagcagctgctctcgctcccaccgccgcc a ccgtccaccacc tggtgcaccaccaccaccaccatcaccaccactaccacctgggcaatgggacgctcagggccccccgg gccagcccggagatccaggacagggatgccaatgggtcccgcaggctcatgctgccaccaccctcgac gcctgccctctccggggccccccctggtggcgcagagtctgtgcacagcttctaccatgccgactgcc acttagagccagtccgctgccaggcgccccctcccaggtccccatctgaggcatccggcaggactgtg ggcagcgggaagg g a cccaccg gcacaccagccctccaccggagacgctgaaggagaaggcact agtagaggtggctgccagctctgggcccccaaccctcaccagcctcaacatcccacccgggccctaca gctccatgcacaagctgctggagacacagagtacag

VGSFFMINLCLWIATQFSETKQRESQ M-REQRVRFLSNASTLASFSEPGSCYEELLKYLVYILRKAA RRLAQVSRAAGVRVGLLSSPAPLGGQETQPSSSCSRSHRRLSVHHLVHHHHHHHHHYHLGNGTLRAPR ASPEIQDRDA GSRR MLPPPSTPALSGAPPGGAESVHSFYHADCHLEPVRCQAPPPRSPSEASGRTV GSGKVYPTVHTSPPPETLKEKALVEVAASSGPPTLTSLNIPPGPYSSMHKLLETQST

(SEQ ID NOs: 9 and 90) Exon 9 (constitutive) gtgcctgccaaagctcttgcaagatctccagcccttgcttgaaagcagacagtggagcctgtggtcca gacagctgcccctactgtgcccgggccggggcaggggaggtggagctcgccgaccgtgaaatgcctga ctcagacagcgaggcagtttatgagttcacacaggatgcccagcacagcgacctccgggacccccaca gccggcggcaacggagcctgggcccagatgcagagcccagctctgtgctggccttctggaggctaatc tgtgacaccttccgaaagattgtggacagcaagtactttggccggggaatcatgatcgccatcctggt caacacactcagcatgggcatcgaataccacgagcag gACQSSCKISSPCLKADSGACGPDSCPYCARAGAGEVELADRE-MPDSDSEAVYEFTQDAQHSDLRDPH S-RRQRSLGPDAEPSSVLAFWR ICDTFRKIVDSKYFGRGIMIAILVNTLSMGIEYHEQ

(SEQ ID NOs: 10 and 91) Exon 10 (constitutive) cccgaggagcttaccaacgccctagaaatcagcaacatcgtcttcaccagcctctttgccctggagat gctgctgaagctgcttgtgtatggtccctttggctacatcaagaatccctacaacatcttcgatggtg tcattgtggtcatcag

PEELTNALEISNIVFTSLFA EMLLKLLVYGPFGYIKNPYNIFDGVIWIs

(SEQ ID NOs: 11 and 92) Exon 11 (constitutive) cgtgtgggagatcgtgggccagcaggggggcggcctgtcggtgctgcggaccttccgcctgatgcgtg tgctgaagctggtgcgcttcctgccggcgctgcagcggcagctggtggtgctcatgaagaccatggac aacgtggccaccttctgcatgctgcttatgctcttcatcttcatcttcag

VWE I VGQQGGG-LS VLRTFRIJrøVl,KLVRF PALQRQLVVI--^T-ffi-WATFC-^l--MLF I F I F S

(SEQ ID NOs: 12 and 93) Exon 12 (constitutive) ca cctgggcatgcatctcttcggctgcaagtttgcctctgagcgggatggggacaccctgccagacc ggaagaattttgactccttgctctgggccatcgtcactgtctttcag

ILGMHLFGCKFASERDGDTLPDRKNFDSLLWAIVTVFQ

(SEQ ID NOs: 13 and 94) Exon 13 (∞nstitutive) atcctgacccaggaggactggaacaaagtcctctacaatgg atggcctccacg cgtcctgggcggc cctttatttcattgccctcatgaccttcggcaactacgtgctcttcaatttgctggtcgccattctgg tggagggcttccaggcggag

ILTQEDWNKVLYNG-MASTSSWAA YFIALMTFGNYVLFNLLVAILVEGFQAE

(SEQ ID NOs: 14 and 95) Exon 14 (variable) gaaatcagcaaacgggaagatgcgagtggacagttaagctgtattcagctgcctgtcgactcccaggg g

EISK-REDASGQLSCIQLPVDSQG

(SEQ ID NOs: 15 and 96) Exon 15 (constitutive) ggagatgccaacaagtccgaatcagagcccgatttcttctcacccagcctggatggtgatggggacag gaagaagtgcttggcct

GDANKSESEPDFFSPSLDGDGDRKKCLA

(SEQ ID NOs: 16 and 97) Exon 16 (constitutive) tggtgtccctgggagagcacccggagctgcggaagagcctgctgccgcctctcatcatccacacggcc gccacacccatgtcgctgcccaagagcaccagcacgggcctgggcgaggcgctgggccctgcgtcgcg ccgcaccagcagcagcgggtcggcagagcctggggcggcccacgagatgaagtcaccg

1VSLGEHPELRKSL PPLIIHTAATPMSLPKSTSTG GEALGPAS-RRTSSSGSAEPGAAHEMKSP

(SEQ ID NOs: 17 and 98) Exon 17 (constitutive) cccagcgcccgcagctctccgcacagcccctggagcgctgcaagcagctggaccagcaggcgctccag ccggaacagcctcggccgtgcacccagcctgaagcggagaagcccaagtggagagcggcggtccctgt tgt-cgggagaaggccaggagagccaggatgaagaggagagctcagaagaggagcgggccagccctgcg ggcagtgaccatcgccacagggggtccctggagcgggaggccaagagttcctttgacctgccagacac actgcaggtgccagggctgcatcgcactgccagtggccgagggtctgcttctgagcaccaggactgca atggcaagtcggcttcagggcgcctggcccgggccctgcggcctgatgaccccccactggatggggat gacgccgatgacgagggcaacctg

PSARSSPHSP SAASSWTSRRSSRNSLGRAPSLKRRSPSGERRSL SGEGQESQDEEESSEEERASPA GSDHRHRGS E-REAKSSFD-PDTLQVPGLHRTASGRGSASEHQDCNGKSASGRLARALRPDDPPLDGD DADDEGNL

(SEQ ID NOs: 18 and 99) Exon 18 (∞nstitutive) agcaaaggggaacgggtccgcgcgtggatccgagcccgactccctgcctgctgcctcgagcgagactc ctggtcagcctacatcttccctcctcagtccag

SKGERVRAWIRARLPACC-LERDSWSAYIFPPQSr

(SEQ ID NOs: 19 and 100) Exon 19 (∞nstitutive) gttccgcctcctgtgtcaccggatcatcacccacaagatgttcgaccacgtggtccttgtcatcatct tccttaactgcatcaccatcgccatggagcgccccaaaattgacccccacagcgct

FR CHRIITHKMFDHWLVIIFLNCITIAMERPKIDPHSA

(SEQ ID NOs: 20 and 101) Exon 20 (∞nstitutive) gaacgcatcttcctgaccctctccaattacatcttcaccgcagtctttctggctgaaatgacagtgaa g ERI T SNYIFTAVFLAEMTVK

(SEQ ID NOs: 21 and 102) Exon 21 (constitutive) gtggtggcactgggctggtgcttcggggagcaggcgtacctgcggagcagttggaacgtgctggacgg gctgttggtgctcatctccgtcatcgacattctggtgtccatggtctctgacagcggcaccaagatcc tgggcatgctgagggtgctgcggctgctgcggaccctgcgcccgctcag

VV- iGWCFGEQAYLRSSWNVLDGLLV ISVIDILVSMVSDSGTKILGMLRVLRLLRTLRPLr

(SEQ ID NOs: 22 and 103) Exon 22 (constitutive) ggtgatcagccgggcgcaggggctgaagctggtggtggagacgctgatgtcctcactgaaacccatcg gcaacattgtagtcatctgctgtgccttcttcatcattttcggcatcttgggggtgcag

VISRAOGLKLWETLMSS KPIGNIWICCAFFIIFGILGVQ

(SEQ ID NOs: 23 and 104) Exon 23 (constitutive) ctcttcaaagggaagtttttcgtgtgccagggcgaggataccaggaacatcaccaataaatcggactg tgccgaggccagttaccggtgggtccggcacaagtacaactttgacaaccttggccag

LFKGKFFVCς2GEDT-RNITNKSDCAEASYRWVRHKYNFDN GQ

(SEQ ID NOs: 24 and 105) Exon 24 (constitutive) gccctgatgtccctgttcgttttggcctccaaggatggtggg ggacatcatgtacgatgggctgga tgctgtgggcgtggaccagcag

A MSLFV-LASK-DGWVDIMYDGLDAVGVDQQ

(SEQ ID NOs: 25 and 106) Exon 25A (∞nstitutive) cccatcatgaaccacaacccctggatgctgctgtact ca ctcgttcctgctcattgtggccttctt tgtcctgaacatgtttgtgggtgtggtggtggagaacttccacaagtgtcggcagcaccaggaggaag aggaggcccggcggcgggaggagaagcgcctacgaagactggagaaaaagagaagga

PIMNHNPWM LYFISF IVAFFVLNMFVGVVVENFHKCRQ

(SEQ ID NOs: 26 and 107) Exon 25B (variable) gtaaggagaagcagatggctg

sKEKQMA

(SEQ ID NOs: 27 and 108 and 162) Exon 26 (variable) atctaatgctggacgatgtaattgcttccggcagctcagccagcgctgcgtcag n MLDDVIASGSSASAAS (when follows exon 25A) d MLDDVIASGSSASAAS (when follows exon 25B)

(SEQ ID NOs: 28 and 109 and 163) Exon 27 (constitutive) aagcccagtgcaaaccttactactccgactactcccgcttccggctcctcgtccaccacttgtgcacc agccactacctggacctcttcatcacaggtgtcatcgggctgaacgtggtcaccatggccatggagca ctaccagcagccccag eAQCKPYYSDYSRFRLLVHHLCTSHY DLFITGVIGLNWTMAMEHYCQPQ (when it follows exon 25B or exon 26 )

- AQCKPYYSDYSRFRLLVHHLCTSHY D FITGVIGLNWTMAMEHYQQPQ (when it follows exon 25A)

(SEQ ID NOs: 29 and 110) Exon 28 (∞nstitutive) attctggatgaggctctgaagatctgcaactacatcttcactg ca ctttgtcttggagtcagtttt caaacttgtggcctttggtttccgtcggttcttccaggacag

ILDEA KICNYIFTVIFVLESVFK VAFGFRRFFODr

(SEQ ID NOs: 30 and 111) Exon 29 (∞nstitutive) gtggaaccagctggacctggccattgtgctgctgtccatcatgggcatcacgctggaggaaatcgagg tcaacgcctcgctgcccatcaaccccaccatcatccgcatcatgagggtgctgcgcattgcccgag

WNQLDLAIVLLSIMGITLEEIEVNASLPINPTIIRI-MRV RIAR

(SEQ ID NOs: 31 and 112) Exon 30 (∞nstitutive) tgctgaagctgctgaagatggctgtgggcatgcgggcgctgctggacacggtgatgcaggccctgccc cag vLKLLKMAVGMRAL-LDTVMOALPQ

(SEQ ID NOs: 32 and 113) Exon 31 (∞nstitutive) gtggggaacctgggacttctcttcatgttgttgtttttcatctttgcagctctgggcgtggagctctt tggagacctgg

VGN GLL FMLLFF I FAALGVE FGDL

(SEQ ID NOs: 33 and 114) Exon 32 (constitutive) agtgtgacgagacacacccctgtgagggcctgggccgtcatgccacctttcggaactttggcatggcc ttcctaaccctcttccgagtctccacaggtgacaattggaatggcattatgaag eCDETHPCEGLGRHATFRNFGMAFLTLFRVSTGDNWNGIMK

(SEQ ID NOs: 34 and 115) Exon 33 (constitutive) gacaccctccgggactgtgaccaggagtccacctgctacaacacgg ca ctcgccta ctactttgt gtccttcgtgctgacggcccagttcgtgctagtcaacgtggtgatcgccgtgctgatgaagcacctgg aggagagcaacaaggaggccaaggaggaggccgagctagaggctgagctggagctggagatgaagacc ctcagcccccagccccactcgccactgggcagccccttcctctggcctggggtcgagggccccgacag ccccgacagccccaagcctggggctctgcacccagcggcccacgcgagatcagcctcccacttttccc tggagcaccccacg

DTL---^CDQESTCYNTVISPIYFVSF\n-,TAQFVL\-TfVAtIAVI-MKHLEESNKEAKEEAELEAELELE-MKT SPQPHSP GSPFLWPGVEGPDSPDSPKPGALHPAAHARSASHFSLEHPT

(SEQ ID NOs: 35 and 116) Exon 34 (variable) gacaggcagctgtttgacacca ccctgctgatccagggctccctggagtgggagctgaagctgat ggacgagctggcaggcccagggggccagccctctgccttcccttctgcccccagcctgggaggctccg acccacag

DRQ FDTISLLIOGS EWELK-LMDELAGPGGQPSAFPSAPSLGGSDPQ

(SEQ ID NOs: 36 and 117) Exon 35 (variable) atccctctagctgagatggaggctctgtctctgacgtcagagattgtgtctgaaccgtcctgctctct agctctgacggatgactctttgcctgatgacatgcacacactcttacttagtgccctggagagcaat

IP AEMEALSLTSEIVSEPSCSLALTDDSLPDDMHTLL SA ESN

(SEQ ID NOs: 37 and 118) Exon 36 (∞nstitutive) atgcagccccaccccacggagctgccaggaccagacttactgactgtgcggaagtctggggtcagccg aacgcactctctgcccaatgacagctacatgtgtcggcatgggagcactgccgaggggcccctgggac acaggggctgggggctccccaaagctcagtcag

MQPHPTELPGPDLLTVRKSGVSRTHS PNDSYMCRHGSTAEGPLGHRGWGLPKAQS

(SEQ ID NOs: 38 and 119) Exon 37 (∞nstitutive) gctccgtcttgtccgttcactcccagccagcagataccagctacatcctgcagcttcccaaagatgca cctcatctgctccagccccacagcgccccaacctggggcaccatccccaaactgcccccaccaggacg ctcccctttggctcagaggccactcaggcgccag gSVLSVHSQPADTSYILQ PKDAPHLLQPHSAPTWGTIPKLPPPGRSPLAQRPLRRQ

(SEQ ID NOs: 39 and 120) Exon 38A (∞nstitutive) gcagcaataaggactgactccttggacgttcagggtctgggcagccgggaagacctgctggcagag

AAIRTDS DVOGLGSREDLLAE

(SEQ ID NOs: 40 and 121) Exon 38B (variable) gtgagtgggccctccccgcccctggcccgggcctactctttctggggccagtcaagtacccaggcaca gcagcactcccgcagccacagcaagatctccaagcacatgaccccgccagccccttgcccaggcccag aacccaactggggcaagggccctccagagaccagaagcagcttagagttggacacggagctgagctgg atttcaggagacctcctgccccctggcggccag

VSGPSPPLARAYSFWGQSSTQAQQHSRSHSKISKHMTPPAPCPGPEP GKGPPETRSSLELDTE S ISGDLLPPGGQ

(SEQ ID NOs: 41 and 122) Exon 38C (constitutive) gaggagcccccatccccacgggacctgaagaagtgctacagcgtggaggcccagagctgccagcgccggcctacgt tctattttattaaattaattgaatctagta

EEPPSPRDLKKCYSVEAQSCQ-RRPTSW DEQRRHSIAVSC DSGSQPHLGTDPSNLGGQPLGGPGSRP ---α LSPPSITIDPPESQGPRTPPSPGICLRR-RAPSSDSKDP ASGPPDSMAASPSPKKDV SLSGLSS DPAD DP-

(SEQ ID NO: 42) Exon 38D (variable) tatgcgggatgtacgacattttgtgactgaagagacttgtttccttctacttttatgtgtctcagaat atttttgaggcgaaggcgtctgtctcttggctattttaacctaaaataacagtctagttatattccct cttcttgcaaagcacaagctgggaccgcgagcacattgcagccccaacggtggcccatcttcagcgga gagcgagaaccattttggaaactgtaatgtaacttattttttcctttaacctcgtcatcattttctgt agggaaaaaaaaaagaaaaagaaaaaatgagattttacaagtgaaatggaacctttttatatatacat acatacatatctatctatctatctatatatatataaaataaagtaattttcctaaataaaaa

Non-coding

The calcium channel an subunit gene, CACNA1I, consists of 37 protein-coding exons. Alternative processing of the gene transcript allows this single gene to code for eight distinct α_u protein products. In Table 4, each exon or portion of an exon is listed. In Table 3, the component exons of individual splice variants is described. These two tables are sufficient for a complete description of composition. The presumed RNA processing mechanisms giving rise to these variants are discussed below. Table 3 lists the composition of the 8 α_π protein products. Only the missing portions of each variant are noted in the description; the symbol "Δ" denotes deletion of the exon following the symbol. Thus, variant 1 consists of all exons save 9, 33A and 36B; in other words, exons 1 - 8, 10 - 32, 33B, 34 - 35, 36A and 37 are concatenated to form the protein. The final column lists the number of aa residues in each variant.

Table 3. a Splice Variants

For each exon, the nucleotide sequence and the corresponding amino-acid (aa) sequence are listed in single-letter IUPAC code. Lower case letters in the aa sequences indicate that only two nucleotides of the codon belong to the exon (the codon is interrupted). A dash indicates a stop codon.

TABLE 4

(SEQ ID NOs: 43 and 123) Exon 1 (constitutive) atggctgagagcgcctccccgccctcctcatctgcagcagccccagccgctgagccaggagtcaccac ggagcagcccggaccccggagccccccatcctccccgccaggcctggaggagcctctggatggagctg atcctcatgtcccacacccagacctggcgcctattgccttcttctgcctgcgacagaccaccagcccc cggaactggtgcatcaagatggtgtgcaaccc

MAESASPPSSSAAAPAAEPGVTTEQPGPRSPPSSPPGLEEPLDGADPHVPHPDLAPIAFFCLRQTTSP RNWCIKMVCNp

(SEQ ID NOs: 44 and 124) Exon 2 (constitutive) gtggtttgaatgtgtcagcatgctggtgatcctgctgaactgcgtgacacttggcatgtaccagccgt gcgacgacatggactgcctgtccgaccgctgcaagatcctgcag

WFECVSMLVI LNCVTLGMYQPCDDMDCLSDRCKI Q

(SEQ ID NOs: 45 and 125) Exon 3 (constitutive) gtctttgatgacttcatctttatcttctttgccatggagatggtgctcaagatggtggccctggggat ttttggcaagaagtgctacctcggggacacatggaaccgcctggatttcttcatcgtcatggcagg

VFDDFIFIFFAMEMVLKMVALGIFGKKCYLGDTWNRLDFFIVMAg

(SEQ ID NOs: 46 and 126) Exon 4 (constitutive) gatggtcgagtactccctggaccttcagaacatcaacctgtcagccatccgcaccgtgcgcgtcctga ggcccctcaaagccatcaaccgcgtgccca

MVEYSLDLQNINLSAIRTVRVLRPLKAINRVP

(SEQ ID NO?: 47 and 127) Exon 5 (constitutive) gtatgcggatcctggtgaacctgctcctggacacactgcccatgctggggaatgtcctgctgctctgc ttctttgtcttct ca ctttggcatcataggtgtgcagctctgggcgggcctgctgcgtaaccgctg cttcctggaggagaacttcaccat aMRI VNLLLDTLPMLGNVLL CFFVFFIFGIIGVQL AGLLRNRCFLEENFTi (SEQ ID NOs: 48 and 128) Exon 6 (constitutive) acaaggggatgtggccttgcccccatactaccagccggaggaggatgatgagatgcccttcatctgct ccctgtcgggcgacaatgggataatgggctgccatgagatccccccgctcaaggagcagggccgtgag tgctgcctgtccaaggacgacgtctacgactttggggcggggcgccaggacctcaatgccagcggcct ctgtgtcaactggaaccgttactacaatgtgtgccgcacgggcagcgccaacccccacaagggtgcca tcaactttgacaacatcggttatgcttggattgtcatcttccag

QGDVA PPYYQPEEDDEMPFICSLSGDNGIMGCHEIPPLKEQGRECCLSKDDVYDFGAGRQDLNASGL CVNWNRYYNVCRTGSANPHKGAINFDNIGYAWIVIFQ

(SEQ ID NOs: 49 and 129) Exon 7 (constitutive) gtgatcactctggaaggctgggtggagatcatgtactacgtgatggatgctcactccttctacaactt catctacttcatcctgcttatcata

VITLEGWVEIMYYVMDAHSFYNFIYFILLII

(SEQ ID NOs: 50 and 130) Exon 8 (constitutive) gtgggctccttcttcatgatcaacctgtgcctcgttgtcatagcgacccagttctcggagaccaagca acgggagcaccggctgatgctggagcagcggcagcgctacctgtcctccagcacggtggccagctacg ccgagcctggcgactgctacgaggagatcttccagtatgtctgccacatcctgcgcaaggccaagcgc cgcgccctgggcctctaccaggccctgcagagccggcgccaggccctgggcccggaggccccggcccc cgccaaacctgggccccacgccaaggagccccggcactacc

VGSFFMINLC WIATQFSETKQREHRLMLEQRQRYLSSSTVASYAEPGDCYEEIFQYVCHILRKAKR RALGLYQALQSRRQALGPEAPAPAKPGPHAKEPRHY

(SEQ ID NOs: 51 and 131) Exon 9 (variable) atgggaagactaagggtcagggagatgaagggagacatctcggaagccggcattgccagactttgcat gggcctgcctcccctggaaatgatcactcgggaagag hGKTKGQGDEGRHLGSRHCQTLHGPASPGNDHSGR

(SEQ ID NOs: 52 and 132 and 164) Exon 10 (constitutive) agctgtgcccgcaacatagccccctggatgcgacgccccacaccctggtgcagcccatccccgccacg ctggcttccgatcccgccagctgcccttgctgccagcatgaggacggccggcggccctcgggcctggg cagcaccgactcgggccaggagggctcgggctccgggagctccgctggtggcgaggacgaggcggatg gggacggggcccggagcagcgaggacggagcctcctcagaactggggaaggaggaggaggaggaggag caggcggatggggcggtctggctgtgcggggatgtgtggcgggagacgcgagccaagctgcgcggcat cgtggacagcaagtacttcaaccggggcatcatgatggccatcctggtcaacaccgtcagcatgggca tcgagcaccacgagcag eLCPQHSPLDATPHT VQPIPATLASDPASCPCCQHEDGRRPSGLGSTDSGQEGSGSGSSAGGEDEAD GDGARSSEDGASSELG----EEEEEEQADGAVV^CGDVWRETRAK RGIVDSKYFNRGIMMAILVNTVSMG IEHHE ( when it follows exon 9 ) qLCPQHSPLDATPHTLVQPIPATLASDPASCPCCQHEDGRRPSGLGSTDSGQEGSGSGSSAGGEDEAD GDGARSSEDGASSELGKEEEEEEQADGAVWLCGDλ-TTOETRAKLRGIVDSKYFNRGIMMAILVNTVSMG IEHHEQ ( when if follows exon 8 )

(SEQ ID NOs: 53 and 133) Exon 11 (constitutive) ccggaggagctgaccaacatcctggagatctgcaatgtggtcttcaccagcatgtttgccctggagat gatcctgaagctggctgcatttgggctcttcgactacctgcgtaacccctacaacatcttcgacagca tcattgtcatcatcag

PEELTNILEICNWFTSMFA EMI KLAAFGLFDYLRNPYNIFDS11 IIs

(SEQ ID NOs: 54 and 134) Exon 12 (∞nstitutive) catctgggagatcgtggggcaggcggacggtgggctgtcggtgctgcggaccttccggctgctgcgcg tgctgaaactggtgcgcttcatgcctgccctgcggcgccagctcgtggtgc ca gaagaccatggac aacgtggccaccttctgcatgctgctcatgctcttcatcttcatcttcag

IWEIVGQADGGLSVLRTFRLI--RV K VRFMPALRRQLVVLMKT-MDNVATFCM LMLFIFIFs

(SEQ ID NOs: 55 and 135) Exon 13 (∞nstitutive) catccttgggatgcatatttttggctgcaagttcagcctccgcacggacactggagacacggtgcccg acaggaagaacttcgactccctgctgtgggccatcgtcactgtgttccag

ILGMHIFGCKFSLRTDTGDTVPDRKNFDSLLWAIVTVFQ

(SEQ ID NOs: 56 and 136) Exon 14 (∞nstitutive) atcctcacccaggaggactggaacgtcgttctctacaatggcatggcctccacttctccctgggcctc cctctactttgtcgccctcatgaccttcggcaactatgtgctcttcaacctgctggtggccatcctgg tggagggcttccaggcggag

ILTQEDWNWLYNGMASTSPWASLYFVALMTFGNYVLFNLLVAILVEGFQAE

(SEQ ID NOs: 57 and 137) Exon 15 (constitutive) ggtgacgccaatcgctcctactcggacgaggaccagagctcatccaacatagaagagtttgataagct ccaggaaggcctggacagcagcggag

GDANRSYSDEDQSSSNIEEFDKLQEGLDSSG

(SEQ ID NOs: 58 and 138) Exon 16 (∞nstitutive) atcccaagctctgcccaatccccatgacccccaatgggcacctggaccccagtctcccactgggtggg cacctaggtcctgctggggctgcgggacctgccccccgactctcactgcagccggaccccatgctggt ggccctgggctcccgaaagagcagtgtcatgtctctagggaggatgagctatgaccagcgctccctg dPK CPIPMTPNGH-DPSLPLGGH GPAGAAGPAPRLSLQPDPMLVALGSRKSSVMSLGRMSYDQRSL

(SEQ ID NOs: 59 and 139) Exon 17 (constitutive) tccagctcccggagctcctact cgggccatggggccgcagcgcggcctgggccagccgtcgctccag ctggaacagcctcaagcacaagccgccgtcggcggagcatgagtccctgctctctgcggagcgcggcg gcggcgcccgggtctgcgaggttgccgcggacgaggggccgccgcgggccgcacccctgcacacccca cacgcccaccacattcatcacgggccccatctggcgcaccgccaccgccaccaccgccggacgctgtc cctcgacaacagggactcggtggacctggccgagctggtgcccgcggtgggcgcccacccccgggccg cctggagggcggcaggcccggcccccgggcatgaggactgcaatggcaggatgcccagcatcgccaaa gacgtcttcaccaagatgggcgaccgcggggatcgcggggaggatgaggaggaaatcgactac

SSSRSSYYGPWGRSAAWASRRSSWNSLKHKPPSAEHESL-SAERGGGARVCEVAADEGPPRAAPLHTP

-H-AHHIHHGPHLAH-RH-RH-HRRTLSLDNRDSλ-ΦI-AELVPAVGAHPRAAW-RAAGPAPGHEDCNG-RMPSI

DVFTKMGDRGDRGEDEEEIDY

(SEQ ID NOs: 60 and 140) Exon 18 (∞nstitutive) accctgtgcttccgcgtccgcaagatgatcgacgtctataagcccgactggtgcgaggtccgcgaaga ctggtctgtctacctcttctctcccgagaacag

TLCFRVRKMIDVYKPDWCEVREDWSVYLFSPENr

(SEQ ID NOs: 61 and 141) Exon 19 (constitutive) gttccgggtcctgtgtcagaccattattgcccacaaactcttcgactacgtcgtcctggccttcatct ttctcaactgcatcaccatcgccctggagcggcctcagatcgaggccggcagcacc

FRVLCQTIIAHK FDYW AFIFLNCITIA ERPQIEAGST

(SEQ ID NOs: 62 and 142) Exon 20 (constitutive) gaacgcatctttctcaccgtgtccaactacatcttcacggccatcttcgtgggcgagatgacattgaa g

ERIFLTVSN IFTAIFVGEMTLK

(SEQ ID NOs: 63 and 143) Exon 21 (constitutive) gtagtctcgctgggcctgtacttcggcgagcaggcgtacctacgcagcagctggaacgtgctggatgg ctttcttgtcttcgtgtccatcatcgacatcgtggtgtccctggcctcagccgggggagccaagatct tgggggtcctccgagtcttgcggctcctgcgcaccctacgccccctgcg VVS GLYFGEQAYLRSSWNV DGFLVFVSIIDIWSLASAGGAKILGVLRVL LRTLRPLr

(SEQ ID NOs: 64 and 144) Exon 22 (constitutive) tg catcagccgggcgccgggcctgaagctggtggtggagacactcatctcctccctcaagccca cg gcaacatcgtgctcatctgctgtgccttcttcatcatctttggcatcctgggagtgcag

VISRAPGLKLWETLISSLKPIGNIVLICCAFFIIFGILGVQ

(SEQ ID NOs: 65 and 145) Exon 23 (constitutive) ctcttcaagggcaagttctaccactgtctgggcgtggacacccgcaacatcaccaaccgctcggactg ca ggccgccaactaccgctgggtccatcacaaatacaacttcgacaacctgggccag

LFKGKFYHC GVDTRNITNRSDCMAANYRWVHHKYNFDNLGQ

(SEQ ID NOs: 66 and 146) Exon 24 (constitutive) gc ctgatgtccctctttgtcctggcatccaaggatggttgggtgaacatcatgtacaatggactgga tgctgttgctgtggaccagcag

A MSLFVLASKDGWVNIMYNGLDAVAVDQQ

(SEQ ID NOs: 67 and 147) Exon 25 (constitutive) cctgtgaccaaccacaacccctggatgctgctgtacttcatctccttcctgctcatcgtcagcttctt tgtgctcaacatgtttgtgggtgtcgtggtggagaacttccacaagtgccggcagcaccaggaggctg aagaggcacggcggcgtgaggagaagcggctgcggcgcctggagaagaagcgccgga

PVTNHNPWML YFISF IVSFFVLNMFVGVV\-^NFHKCRQHQEAEEARRREEK-RL-R--^EKKRR

(SEQ ID NOs: 68 and 148) Exon 26 (constitutive) aggcccagcggctgccctactatgccacctattgtcacacccggctgctcatccactccatgtgcacc agccactacctggacatcttcatcaccttcatcatctgcctcaacgtggtcaccatgtccctggagca ctacaatcagcccacg kAQRLPYYATYCHTRL IHSMCTSHYLDIFITFIICLNW MSLEHYNQPT

(SEQ ID NOs: 69 and 149) Exon 27 (∞nstitutive) tccctggagacagccctcaagtactgcaactatatgttcaccactgtctttgtgctggaggctgtgct gaagctggtggcatttggtctgaggcgcttcttcaaggaccg

SLETALKYCNYMFTTVFVLEAV KLVAFGLRRFFKDr

(SEQ ID NOs: 70 and 150) Exon 28 (constitutive) atggaaccagctggacctggccattgtgctactgtcagtcatgggcatcaccctggaggagatcgaga tcaatgcggccctgcccatcaatcccaccatcatccgcatcatgagggttctgcgcattgcccgag

WNQLD AIV LSVMGITLEEIEINAALPINPTIIRIMRV RIAR

(SEQ ID NOs: 71 and 151) Exon 29 (constitutive) tgctgaagctgttgaagatggccacaggaatgcgggccctgctggacacggtggtgcaagctttgccc cag vLK LKMATGMRA LDTWQA PQ

(SEQ ID NOs: 72 and 152) Exon 30 (constitutive) gtgggcaacctgggcctcctcttcatgctgctcttct ca ctatgctgctctcggggtggagctctt tgggaagctgg

VGNLGLLFMLLFFIYAALGVELFGKL

(SEQ ID NOs: 73 and 153) Exon 31 (∞nstitutive) tctgcaacgacgagaacccgtgcgagggcatgagccggcatgccaccttcgagaacttcggcatggcc ttcctcacactcttccaggtctccacgggtgacaactggaacgggatcatgaag vCNDENPCEGMSRHATFENFGMAFLTLFQVSTGDNWNGIMK

(SEQ ID NOs: 74 and 154) Exon 32 (∞nstitutive) gacacgctgcgggactgcacccacgacgagcgcagctgcctgagcagcctgcagtttgtgtcgccgct gtacttcgtgagcttcgtgctcaccgcgcagttcgtgctcatcaacgtggtggtggctgtgctcatga agcacctggacgacagcaacaaggaggcgcaggaggacgccgagatggatgccgagctcgagctggag atggcccatggcctgggccctggcccgaggctgcctaccggctccccgggcgcccctggccgagggcc gggaggggcgggcggcgggggcgacaccgagggcggcttgtgccggcgctgc actcgcctgcccag

DTL-RDCTHDERSC SSLQFVSPLYFVSFVI-,TAQE-^INVVVA\^MKHLDDSNKEAQEDAEMDAELELE MAHGLGPGPRLPTGSPGAPGRGPGGAGGGGDTEGGLCRRCYSPAQ

(SEQ ID NOs: 75 and 155) Exon 33A (variable) gagaacctgtggctggacagcgtctctttaatcatcaag ENLW DSVS IIK

(SEQ ID NOs: 76 and 156) Exon 33B (∞nstitutive) gactccttggagggggagctgaccatcatcgacaacctgtcgggctccatcttccaccac ac cctc gcctgccggctgcaagaagtgtcaccacgacaagcaagag

DSLEGE TIIDN SGSIFHHYSSPAGCKKCHHDKQE (SEQ ID NOs: 77 and 157) Exon 34 (∞nstitutive) gtgcagctggctgagacggaggccttctccctgaactcagacaggtcctcgtccatcctgctgggtga cgacctgagtctcgaggaccccacagcctgcccacctggccgcaaagacagcaag

VQLAETEAFSLNSDRSSSILLGDDLS EDPTACPPGRKDSK

(SEQ ID NOs: 78 and 158) Exon 35 (constitutive) ggtgagctggacccacctgagcccatgcgtgtgggagacctgggcgaatgcttcttccccttgtcctc tacggccgtctcgccggatccagagaacttcctgtgtgagatggaggagatcccattcaaccctgtcc ggtcctggctgaaacatgacagcagtcaag

GELDPPEPMRVGDLGECFFPLSSTAVSPDPENFLCEMEEIPFNPVRSWLKHDSSQ

(SEQ ID NOs: 79 and 159) Exon 36A (constitutive) cacccccaagtcccttctccccggatgcctccagccctctcctgcccatgccagccgagttcttccac cctgcagtgtctgccagccagaaaggcccagaaaagggcactggcactggaaccctccccaagattgc gctgcagggctcctgggcatctctgcggtcaccaagggtcaactgtaccctcctccggcag aPPSPFSPDASSPLLPMPAEFFHPAVSASQKGPEKGTGTGTLPKIA QGSWASLRSPRVNCTL RQ

(SEQ ID NOs: 80 and 160) Exon 36B (mutually exclusive with Exon 37) g accgacacctcccaggccctagagcactggtctgtgggcaaggggcaggatctaagccaggcct ggaagtccaagggactgggaggggaaggacccaaccaaaggccgagggcaccaccgtgcaaggggg tttgggaacgctggggtgacgctgagactggagggggaggtggcactggggcggatggagtgggcg gggctgggtcctggggacagcagagtgtggggaggaccccaaggcgggtctggaagaggcctgtga tccctagcttgaggggaggggaggagaggaggaggagtactggaggttttgcagggtggcggggtg ctggcagtggggaggacaccctgggtgctctgggtgggtgtgagtgggggcttgat c aggaat ggaggtgggagggcgggtctggtggatgagaagcctcgggctgcagggtcccccgtactggattgg ccagggccacagccctcctacccacgggcacacagaggtctgaagcactgagggctccgctgtggg ggtggggaaatggggccgggccggctcccacagtgagtgcagttgattcactgggtgactgtctga cccgtcacaccaggctgtgtgctctggcgggcaggacacaaactccctgcctgccgggctcactgt ttagtgctgagagtgagctgcctgggtgcaggagggatgataaccaaaataaa

VPTPPRP-

(SEQ ID NOs: 81 and 161) Exon 37 (mutually exclusive with exon 36B) gccaccgggagcgacacgtcgctggacgccagccccagcagctccgcgggcagcctgcagaccacgct cgaggacagcctgaccctgagcgacagcccccggcgtgccctggggccgcccgcgcctgctccaggac cccgggccggcctgtcccccgccgctcgccgccgcctgagcctgcgcggccggggcctcttcagcctg cgggggctgcgggcgcatcagcgcagccacagcagcgggggctccaccagcccgggctgcacccacca cgactccatggacccctcggacgaggagggccgcggtggcgcgggcggcgggggcgcgggcagcgagc ac cggagaccctcagcagcctctcgctcacctccctcttctgcccgccgcccccgccgccagccccc ggcctcacgcccgccaggaagttcagcagcaccagcagcctggccgcccccggccgcccccacgccgc cgccctggcccacggcctggcccggagcccctcgtgggccgcggaccgcagcaaggacccccccggcc gggcaccgctgcccatgggcctgggccccttggcgcccccgccgcaaccgctccccggagagctggag ccgggagacgccgccagcaagaggaagagatgagggtcgcaggggcccccggccgcccaccgcccgcc ccgtctcaccttctttacctcaggagccaggagcagacagcaatacttcgtccacacctgggatcgcg cagggcccgcagggcacaggcgcccgacagccgggctgagcggagtctgggttagccaggcctgcgtg gcccatggtggcccttccagtgcatatacatacatatatatatatatatgcatatatatatatatata atatatatgtgtatacacacacacatagacagacatatatatat t tttattttttttactgagag cttatgacttc

ATGSDTSLDASPSSSAGSLQTT EDSLT SDSPRRALGPPAPAPGPRAGLSPAARRRLSLRGRGLFSL

RGLRAHQRSHSSGGSTSPGCTHHDSMDPSDEEGRGGAGGGGAGSEHSETLSS S TSLFCPPPPPPAP

GLTPA-RKFSSTSSLAAPGRPHAAALAHG-LARSPSWAADRSKDPPGRAPLPMGLGPLAPPPQP PGELE

PGDAASKRKR-

RNA processing mechanisms

Figure 5 is a schematic diagram of the RNA processing leading to the 8 variants. The portion of the Figure above the scale bar represents the CACNAII gene. The three sections of the gene involved in alternative processing are drawn to scale. At the left, variable exon 9 (olive) is flanked by constitutive exons 8 (black) and

10 (purple). The black lines between exons represent introns.

In the middle, constitutive exon 32 is black. Exon 33 is divided into 2 parts, 39-nucleotide (nt) variable exon 33A (orange) and 108-nt constitutive exon 33B (blue). At the right, exon 36 is divided into 2 parts, 197-nt constitutive exon 36A

(black) and variable exon 36B (red), encoding seven aa before a stop codon. Constititive exon 37 (green) encodes 214 aa before a stop codon.

Exons 1 - 7, 11 -31 and 34 - 35 are not represented.

The blue and red lines and red arrow above and below the exons represent alternative RNA processing reactions.

Below the scale bar are representations of the 8 α_π protein products. The portions of the protein derived from exons 1 - 7, 11 -31 and 34 - 35 are uncolored. Portions derived from the other exons are color-coded as in the gene map, above. Note that exon 36B encodes only 7 aa. The thin blue and red lines above the protein products correspond to the lines around the gene map and represent the type of RNA processing reactions that resulted in the particular variant. a. Alternative splicing of exon 9

Variants 1 - 4 result from the deletion of exon 9. In the blue reaction, splicing takes place between the donor 3' to exon 8 and the acceptor 5' to exon 10. Variants

5 - 8 result from RNAs subjected to the red reactions. In this case, two splicing reactions take place. The donor 3' of exon 8 and the acceptor 5' of exon 9 are joined as are the donor 3' of exon 9 and the acceptor 5' of exon 10. The portion encoded by exon 9 is retained. b. Selection of the splice acceptor preceding exon 33A or 33B

Variants 1, 2, 5 and 6 result from the deletion of exon 33 A. In the blue reaction, splicing takes place between the donor 3' of exon 32 and the acceptor internal to exon 33. Variants 3, 4, 7 and 8 result from RNAs subjected to the red reaction. In this case, splicing takes place between the donor 3' of exon 32 and the acceptor 5' of exon 33. The portion encoded by exon 33 A is retained. c. Processing of the 3' end

Variants 1, 3, 5 and 7 result from the deletion of exon 36B. In the blue reaction, splicing takes place between the donor internal to exon 36 and the acceptor 5' of exon 37. Exon 37 encodes the final 214 aa of the protein in these variants. Variants 2, 4, 6 and 8 result from RNAs subjected to the red reaction. In this case, the RNA is cleaved and polyadenylated just 3 ' of exon 36. In these variants, exon 36B encodes the final 7 aa of the protein.

Isolated and purified polypeptides or proteins, according to the present invention comprise at least about 10% by weight of a composition of proteins. Preferably the composition contains at least 25%, 50%, 75%, 85%, or 90% by weight of the particular polypeptide or protein. Any purification method can be applied, either to naturally expressing cells, such as neurons, or to cells which have been engineered to express a recombinant form of the polypeptide or protein. Purification methods known in the art which can be used without limitation include affinity chromatography, immunoprecipitation, immunoaffinity chromatography, molecular sieves, and ion exchange chromatography. Non-naturally occurring variants which retain substantially the same biological activities as naturally occurring protein variants, such as calcium channel function, are also included here. Preferably, naturally or non-naturally occurring variants have amino acid sequences which are at least 85%, 90%, or 95% identical to the amino acid sequences shown in the SEQUENCE LISTING found at the end of the application. More preferably, the molecules are at least 98% or 99% identical. Percent identity is determined using the Smith- Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 1. The Smith- Waterman homology search algorithm is taught in Smith and Waterman, Adv. Appl. Math. (1981) 2:482-489.

Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological or immunological activity can be found using computer programs well known in the art, such as DNASTAR software. Preferably, amino acid changes in secreted protein variants are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. A conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains. Naturally occurring amino acids are generally divided into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cystine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids.

It is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the biological properties of the resulting variant. Whether an amino acid change results in a functional calcium channel subunit protein or polypeptide can readily be determined by testing the altered protein or polypeptide in a functional assay. Variants of the calcium channel subunit proteins disclosed herein include glycosylated forms, aggregative conjugates with other molecules, and covalent conjugates with unrelated chemical moieties. Covalent variants can be prepared by linking functionalities to groups which are found in the amino acid chain or at the N- or C-terminal residue, as is known in the art. Variants also include allelic variants, species variants, and muteins. Truncations or deletions of regions, particularly exons, which do not affect functional activity of the proteins are also variants.

A subset of mutants, called muteins, is a group of polypeptides in which neutral amino acids, such as serines, are substituted for cysteine residues which do not participate in disulfide bonds. These mutants may be stable over a broader temperature range than native proteins or have other beneficial changes in physicochemical properties.

Any coding sequence can be used to generate a recombinant form of the protein which results in the proper amino acids being used. However, the natural human nucleic acid sequences are preferred. The coding sequence can be fused, for example, to expression control sequences, signal sequences, and/or to other coding sequences to form a fusion protein. All of the exons of a particular subunit can be used in such constructs. Alternatively one or more isolated exons can be used.

Nucleic acids which are isolated and purified are separated from the rest of the chromosome on which they reside in human cells. Preferably the particular nucleic acid is the predominant molecular species in a composition. More preferably the nucleic acid comprises at least 75%, 80%, 85%, 90%, or 95% of the molecular species (including only nucleic acids) in the composition.

Degenerate polynucleotide sequences which encode amino acid sequences of the proteins and variants, as well as homologous nucleotide sequences which are at least 65%, 75%, 85%, 90%, 95%, 98%, or 99% identical to the nucleotide sequences shown in the Sequence Listing are also polynucleotide molecules of the invention.

Percent sequence identity is determined using computer programs which employ the Smith- Waterman algorithm, such as the MPSRCH program (Oxford Molecular), using an affine gap search with the following parameters: a gap open penalty of 12 and a gap extension penalty of 1. Typically, homologous polynucleotide sequences can be confirmed by hybridization under stringent conditions, as is known in the art. For example, using the following wash conditions— 2 x SSC (0.3 M NaCl, 0.03 M sodium citrate, pH 7.0), 0.1% SDS, room temperature twice, 30 minutes each; then 2 x SSC, 0.1% SDS, 50 °C once, 30 minutes; then 2 x SSC, room temperature twice, 10 minutes each—homologous sequences can be identified which contain at most about 25-30% basepair mismatches. More preferably, homologous nucleic acid strands contain

15-25% basepair mismatches, even more preferably 5-15% basepair mismatches.

The nucleic acid can be cloned into a vector, particularly an expression vector. Any suitable expression vector as is known in the art may be used without limitation. Host cells are preferably used which are human, although other host cells including yeast, bacteria, insect, plant and mammalian cells can be used. The cells can be selected for their desired properties. Typically these are selected for their interaction with a vector, or for a property which renders nucleic acids or proteins easily obtainable from the cells.

Host cells which express an α. subunit according to the present invention or an C-_! polypeptide can be used to test compounds or compositions for their possible beneficial effect for treating epilepsy. Thus, a test substance can be contacted with such a host cell and the calcium ion uptake by the cell can be measured. A test substance which blocks calcium ion uptake by the cell is identified as a candidate drug for treating or preventing epilepsy. Methods for measuring calcium uptake are known in the art, and any such method may be used for drug identification. See for example, Lee et al, J. Neuroscience 19: 1912-21, 1999.

The following examples are provided to demonstrate how the invention was made. However, the subject matter of the invention is not limited to any particular method of making the claimed polypeptides, proteins, vectors, and host cells. EXAMPLES

Example 1

Analysis of sequence produced by the Human Chromosome 22 Sequencing Group at the Sanger Centre revealed putative exons of a T α . subunit gene in three overlapping clones of a human genomic DNA library mapping to 22ql2.3-13.2: dJl 104E15 (AL022312), dJ206C7 (AL008716) and (U172B20 (AL022319). tblastn alignment with the t-^G (AF027984) or α₁H (AF051946) amino-acid (aa) sequence identified 26 exons; FEX analysis, another six; and inspection of upstream sequence, a candidate exon encoding the N-terminus. Potential polyadenylation signals were located with POLYAH. Putative exons were assembled into a provisional cDNA sequence and primers for polymerase chain reaction (PCR)-amplification of overlapping portions of the cDNA were designed with OLIGO (National

Biosciences).

PCR screening of a multiple-tissue cDNA panel (Clontech #K 14201) revealed brain as the most abundant cDNA source. Hence, human brain cDNA (Clontech #74001) served as template in subsequent PCRs. The predominant (and in some cases secondary) product of each PCR was recovered on a spin-column (Qiagen

#28704) after agarose gel electrophoresis, eluted in water and submitted for sequencing. Exon boundaries were determined by comparison of the cDNA and genomic sequences; ambiguity was resolved by matching potential donors and acceptors to consensus sequences. Fig. 1 shows 28 of the 49 overlapping PCR products (top) that contributed to the cDNA sequence. Also pictured are exon maps of the cDNA (middle) and the gene (bottom). CACNA1I consists of at least 37 exons distributed over at least 116,390 basepairs (bp). Most PCRs yielded a single product suggesting constitutive splicing of 33 exons (colored gray or black in the cDNA and genomic maps). Certain PCRs, however, yielded multiple products (interrupted black bars), indicative of alternative splicing. PCRs spanning the 105-nucleotide (nt) exon 9 (red), for example, yielded two products, (14 and +14; thus, exon 9 is a cassette exon subject to type A alternative splicing. Sequencing of PCR products spanning exon 33 revealed that exon 33 harbors an internal acceptor that leads to type C alternative splicing and deletion of 39 nt at the 5' end of the exon defined as exon 33 A (orange).

Sequence analysis suggested the possibility of alternative 3' exons. Indeed, PCR-amplification of brain cDNA followed by sequencing showed two forms with substantially different 3' termini. In the first form, both exon 36A and 36B (green) are part of the mature mRNA. Exon 37 (blue) is presumably lost as a result of polyadenylation and cleavage at a site 686 bp downstream of the stop codon in exon

36B. In the second form, splicing between an alternative donor internal to exon 36 and the acceptor 5' of exon 37 leads to substitution of exon 36B with exon 37. The polyadenylation signal of exon 37 has not been identified.

Introns 2 - 8 and 11 - 35 are common U2type GTAG introns. The donors of introns 9 and 10 begin with the dinucleotide GC. Intron 1, like its counterparts in CACNAIG, CACNA1H (unpublished observations), and CACNA1A, is a rare

U12type AT AC intron. Exon 1 includes at least 709 bp of 5' untranslated region and the putative start codon.

Fig. 2 shows a schematic of the deduced protein product. Sequence alignment with other members of the α_x subunit family suggests a transmembrane topology with four domains (DI - D4), each consisting of six membrane-spanning segments, a pore loop and cytoplasmic and extracellular connecting loops. The domains are linked by interdomain loops (ID 12, ID23, ID34), which, along with the amino- (N) and carboxyl- (C) termini, reside in the cytoplasm. Six of the 35 α splice sites (black bars) are conserved in the other α_t subunits studied to date, α_1A, α_lc, α_1D, α_1F, α_ιs, α_1G and α_1H and another three are located within nine nucleotides in the multiple sequence alignment (purple bars). Seventeen of the splice sites (green bars) are in identical locations in the other T subunits, but are not conserved in non-T subunits. Only nine splice sites (pink bars) are unique to _yϊ; these sites join exons that contribute to the cytoplasmic ID 1-2, ID2-3 and C-terminus. As indicated by residue color-coding, α_πis quite similar to the two other human T _x subunits in its membrane-spanning segments — 84% of residues are identical and 92% have similarity scores ( 4 (see legend). Likewise, the pore loops and ID34 are similar. Apart from islands of similarity, the large extracellular loop of DI, the N- and C-termini and ID 12 and ID23 differ from their counterparts in α_1Gand α_1H. Five potential N-glycosylation sites in putative extracellular portions of the protein and 28 potential phosphorylation sites in putative cytoplasmic portions were identified with PROSITE. Although some of the potential phosphorylation sites are conserved among the T a_x subunits, the majority are unique to α_π. Seventeen extracellular cysteines, including six conserved in all ten reported human α_x subunits (black and purple hooks) and nine conserved among T α. subunits (green hooks), may play a role in maintaining proper conformation of the extracellular portions of the protein. Regions derived from portions of the RNA subject to alternative processing are highlighted with a blue background. The shortest predicted product (Δ9Δ33AΔ37) has 1,968 aa residues; the longest (Δ36B), 2,223 aa residues. The reported rat orthologue corresponds to the human Δ9Δ36B variant with a few differences. Exon 32 of the human gene lacks an 18-aa stretch of cysteines, glycines and prolines found in rat (arrow). In addition, 40 nt of exon 34 are deleted in the rat sequence. This leads to a frameshift and early termination of the rat aa sequence. In addition, the published rat sequence contains sequencing errors in exon 35.

T currents display heterogeneity of biophysical and pharmacological properties and subcellular localization. Identification of multiple T α. subunit genes reveals one likely source of heterogeneity. Indeed, heterologous expression experiments demonstrate biophysical differences among the isoforms. The molecular diversity generated by alternative splicing of T α_x subunit genes has the potential to yield additional functional diversity. CACNA1I is subject to alternative splicing in at least two exons while CACNAIG undergoes alternative splicing in at least six (unpublished observations). Variation in channel phosphorylation and isoform-specific interactions with other proteins may also contribute to diversity. Knowledge of the ec aa sequence and its variants will allow explicit tests of these ideas.

EXAMPLE 2

The human chromosome 17 genomic DNA of clone hCIT.22_K_21 (AC004590, Whitehead Institute/MIT Center for Genome Research) appeared to include most or all of CACNAIG, a gene encoding the T Ca²⁺ channel α_1G subunit. Thirty-four probable exons were identified by blastn alignment with the rat α_1G cDNA sequence (AF027984). Four potential polyadenylation signals were located by blastn alignment with sequences (R40146, R43876, R43935, R46109) derived from the 3 ' end of infant brain cDNA clones. A provisional cDNA sequence was assembled and primers for polymerase chain reaction (PCR)-amplification of overlapping portions of human brain cDNA (Clontech #74001) were designed with

OLIGO (National Biosciences). PCR products were fractionated by agarose-gel electrophoresis. When adequately resolved, individual products were cut from the gel, recovered on a spin-column (Qiagen #28704), eluted in water and submitted for sequencing. When resolution was incomplete, DNA was recovered from the gel for cloning into pCRΔ2.1 -TOPO (Invitrogen #K4500-01). Insert DNA was PCR-amplified from overnight cultures of white colonies, purified by agarose-gel electrophoresis and submitted for sequencing. Exon boundaries were determined by comparison of the cDNA and genomic sequences; ambiguity was resolved by matching potential donors and acceptors to consensus sequences. All reported splice variants were observed in at least two independent PCRs.

Fig. 3 shows 25 of the 83 overlapping PCR products (top, black bars) that contributed to the cDNA sequence (AF134985, AF134986). Also pictured are exon maps of the cDNA (middle) and the gene (bottom). CACNAIG consists of at least 38 exons distributed over at least 66,490 basepairs (bp). Thirty-four exons have conterparts in the rat cDNA sequence ; exons 14, 26, 34 and 35 are newly-identified.

Most PCRs yielded a single product suggesting constitutive splicing of 32 exons (colored gray or black in the cDNA and genomic maps). Certain PCRs, however, yielded multiple products (interrupted black bars), indicative of alternative splicing. PCRs spanning the 69-nucleotide (nt) exon 14 (brown), for example, yielded two products, Δ14 and +14; thus, exon 14 is a cassette exon subject to type A alternative splicing. PCRs spanning cassette exons 34 (144 nt) and 35 (135 nt) yielded three products (Δ34Δ35, +34Δ35 and +34+35); the Δ34+35 product was not detected. Sequencing of PCR products spanning exons 25 and 26 revealed that exon 25 harbors an internal donor that leads to type D alternative splicing and deletion of 21 nt at the 3 ' end of the exon (defined as exon 25B, red); the 54-nt exon 26 (blue) is a cassette exon. Exons 25B and 26 appear to be mutually exclusive in that only Δ25B+26 and +25BΔ26 variants were detected. Sequence data also demonstrated that a 237-nt, protein-coding portion of exon 38 (defined as exon 38B, green) could be excised as an intron (type E alternative splicing ). Additional evidence for alternative processing of the human α_1G RNA comes from four clones of a normalized, oligo(dT)-primed infant brain cDNA library. Sequence derived from these clones (red bars), suggests two polyadenylation sites: an upstream site 321 nt 3' to the stop codon and a downstream site 719 nt 3 No the stop codon. Cleavage at the upstream site would delete 398 nt of the mRΝA, defined as exon 38D (purple). Exon 1 includes at least 432 bp of 5' untranslated region and the putative start codon. Introns 2 - 37 are common U2type GTAG introns. Intron 1, like its counterparts in CACNA1H (unpublished observations), CACNA II (submitted), and CACNA1A, is a rare U12type ATAC intron.

Fig. 4 shows a schematic of the deduced protein products encoded by CACNAIG. Like other members of the α. subunit family, α_1G has a proposed transmembrane topology with four domains (D 1 - D4), each consisting of six membrane-spanning segments, a pore loop and cytoplasmic and extracellular connecting loops. The domains are linked by interdomain loops (ID12, ID23, ID34), which, along with the amino- (Ν) and carboxyl- (C) termini, reside in the cytoplasm. Regions derived from portions of the RΝA subject to alternative splicing are highlighted with a blue background, with mutually-exclusive exons 25B and 26 placed side-by-side. The shortest predicted product (Δ14+25BΔ26Δ34Δ35Δ38B) has 2,171 amino-acid (aa) residues; the longest (+14Δ25B+26+34+35+38B), 2,377 aa residues. The reported rat α_1G aa sequence corresponds to the human (14+25BΔ26Δ34Δ35+38B splice variant and is 93% identical. Additional features of the α_1G protein product including residue similarity to the other T α_t subunits, comparison of splice sites and sites of potential post-translational modification are shown in Fig. 2 and described in the legend.

Six CACNAIG exons undergo alternative splicing, leading to a possible 64 splice variants. Analysis of full-length PCR products is underway to determine relative splice- variant abundance. Of note, all potential variants maintain the open reading frame, leave the transmembrane topology intact and, hence, could be translated into plausible protein products. Individual α_1G isoforms may play distinct cellular roles by virtue of differences in biophysical behavior, protein-protein interactions, second-messenger-dependent regulation or other isoform-specific properties.

Claims

Claims:

1. An isolated and purified α_1G subunit of human brain T calcium channel selected from splice variants 1-64 as shown in Table 1.

2. An isolated and purified nucleic acid encoding the α_1G subunit of claim 1.

3. The isolated and purified nucleic acid of claim 2 which comprises a human coding sequence as described in Table 1.

4. An isolated and purified polypeptide which comprises a translated exon selected from the group consisting of 1-38D as shown in Table 2.

5. An isolated and purified nucleic acid which comprises an exon selected from the group consisting of 1-38D as shown in Table 2.

6. An isolated and purified α_π subunit of human brain T calcium channel selected from splice variants 1-8 as shown in Table 3.

7. An isolated and purified nucleic acid encoding the α_π subunit of claim 6.

8. The isolated and purified nucleic acid of claim 7 which comprises a human coding sequence as described in Table 3.

9. An isolated and purified polypeptide which comprises a translated exon selected from the group consisting of 1-37 as shown in Table 4.

10. An isolated and purified nucleic acid which comprises an exon selected from the group consisting of 1-37 as shown in Table 4.

11. An expression vector comprising the nucleic acid of claim 2.

12. An expression vector comprising the nucleic acid of claim 3.

13. An expression vector comprising the nucleic acid of claim 7.

14. An expression vector comprising the nucleic acid of claim 8.

15. A host cell comprising an expression vector according to claim 11.

16. A host cell comprising an expression vector according to claim 12.

17. A host cell comprising an expression vector according to claim 13.

18. A host cell comprising an expression vector according to claim 14.

19. A method to identify candidate drugs for treating epilepsy, comprising the steps of: contacting a cell according to claim 15 with a test substance; measuring uptake by the cell of calcium ions, wherein a test substance which inhibits the uptake by the cell of calcium ions is identified as a candidate drug for treating epilepsy.

20. A method to identify candidate drugs for treating epilepsy, comprising the steps of: contacting a cell according to claim 16 with a test substance; measuring uptake by the cell of calcium ions, wherein a test substance which inhibits the uptake by the cell of calcium ions is identified as a candidate drug for treating epilepsy.

21. A method to identify candidate drugs for treating epilepsy, comprising the steps of: contacting a cell according to claim 17 with a test substance; measuring uptake by the cell of calcium ions, wherein a test substance which inhibits the uptake by the cell of calcium ions is identified as a candidate drug for treating epilepsy.

22. A method to identify candidate drugs for treating epilepsy, comprising the steps of: contacting a cell according to claim 18 with a test substance; measuring uptake by the cell of calcium ions, wherein a test substance which inhibits the uptake by the cell of calcium ions is identified as a candidate drug for treating epilepsy.