WO2000070044A2 - Variants episses de la sous-unite alpha des canaux calcium t dans le cerveau humain - Google Patents

Variants episses de la sous-unite alpha des canaux calcium t dans le cerveau humain Download PDF

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WO2000070044A2
WO2000070044A2 PCT/US2000/012383 US0012383W WO0070044A2 WO 2000070044 A2 WO2000070044 A2 WO 2000070044A2 US 0012383 W US0012383 W US 0012383W WO 0070044 A2 WO0070044 A2 WO 0070044A2
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exon
exons
seq
nos
isolated
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PCT/US2000/012383
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WO2000070044A3 (fr
WO2000070044A9 (fr
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Scott Mittman
William S. Agnew
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The Johns Hopkins University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants

Definitions

  • This invention is related to ion channels.
  • ion channels related to brain function.
  • 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.
  • a subfamily of genes designated Ca- ⁇ T
  • T currents are a diverse class of Ca 2+ 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 Ca 2+ entry and synaptic potentiation.
  • T currents may be the targets of succinimides and related compounds administered in the treatment of absence epilepsy.
  • cDNA sequences of three T x subunits, rat ⁇ 1G and ⁇ u and human ⁇ 1H have been reported.
  • Ca 2+ channel cC j subunits are encoded by at least 10 genes falling into three subfamilies: ABE, SCDF and GHI 1 .
  • Alternative splicing of c ⁇ RNAs generates further molecular diversity.
  • 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.
  • an isolated and purified ⁇ 1G subunit of human brain T calcium channel is provided. The subunit is selected from splice variants 1-64 as shown in Table 1.
  • an isolated and purified nucleic acid encoding the ⁇ 1G subunit is provided.
  • an isolated and purified polypeptide 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 ⁇ ⁇ 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.
  • an isolated and purified nucleic acid 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.
  • inventions are methods to identify candidate drugs for treating epilepsy.
  • a host cell containing a nucleic acid encoding an ⁇ 1G or ⁇ ⁇ 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.
  • Fig. 1 Map of CACNA 1 T and ⁇ u cDNA
  • 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.
  • constitutively-spliced, odd-numbered exons are black and even-numbered exons, gray.
  • Alternatively-processed 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 ⁇ ⁇ 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 ⁇ ⁇ exon with the sequences of the homologous human ⁇ 1G and ⁇ 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; ⁇ ⁇ vs.
  • N-glycosylation and phosphorylation sites and the location of splice sites are indicated by the appropriate symbols.
  • Symbol colors have the following meanings: black - conserved among all human ⁇ . subunits; purple - conserved within 3 aa residues in the multiple sequence alignment of all human a ⁇ subunits; blue - conserved among the human ABE and GHI subfamilies; green - conserved among all human T ⁇ subunits; brown - also present in human ⁇ orange - also present in human ⁇ 1G ; pink - unique to ⁇ ⁇ .
  • 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 ⁇ 1G cDNA
  • 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.
  • cDNA map constitutively-spliced, odd-numbered exons are gray and even-numbered exons, black.
  • Alternatively-processed 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 ⁇ 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 ⁇ 1G exon with the sequences of the homologous human ⁇ 1H (unpublished observations) and (submitted) exons by iterative pairwise use of gap
  • Exon 36 and the C-terminal half of exon 8 had only ⁇ 1H homologues and exon 34 had only an ⁇ ⁇ 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 ⁇ x subunits, purple - conserved within 3 aa residues in the multiple sequence alignment of all human ⁇ 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 ⁇ 1H , pink - unique to ⁇ 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.
  • ID 1-2 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 R ⁇ A processing leading to the 8 ⁇ ⁇ variants.
  • the human brain T calcium channel ⁇ 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 ⁇ G protein products.
  • 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 ⁇ 1G protein products. Only the missing portions of each variant are noted in the description; the symbol “ ⁇ ” denotes deletion of the exon following the symbol. Thus, variant 1 consists of all exons save 14,
  • 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.
  • 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.
  • 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 ⁇ u protein products.
  • Table 4 each exon or portion of an exon is listed.
  • 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 ⁇ ⁇ protein products. Only the missing portions of each variant are noted in the description; the symbol " ⁇ " denotes deletion of the exon following the symbol.
  • 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.
  • 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.
  • FIG. 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
  • 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).
  • exon 36 is divided into 2 parts, 197-nt constitutive exon 36A
  • variable exon 36B (black) and variable exon 36B (red), encoding seven aa before a stop codon.
  • Constititive exon 37 (green) encodes 214 aa before a stop codon.
  • 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
  • Variants 1, 2, 5 and 6 result from the deletion of exon 33 A.
  • 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.
  • 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.
  • exon 36B encodes the final 7 aa of the protein.
  • Isolated and purified polypeptides or proteins comprise at least about 10% by weight of a composition of proteins.
  • 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.
  • 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.
  • 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.
  • 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.
  • mutants are 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.
  • 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.
  • 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.
  • 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.
  • a gap open penalty of 12 a gap open penalty of 12
  • a gap extension penalty of 1 a gap extension penalty of 1.
  • homologous polynucleotide sequences can be confirmed by hybridization under stringent conditions, as is known in the art.
  • homologous sequences can be identified which contain at most about 25-30% basepair mismatches. More preferably, homologous nucleic acid strands contain
  • the nucleic acid can be cloned into a vector, particularly an expression vector.
  • 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 ⁇ . 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.
  • 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.
  • 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).
  • PCRs yielded multiple products (interrupted black bars), indicative of 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).
  • 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
  • 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 ⁇ 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.
  • DI - D4 domains
  • ID 12, ID23, ID34 interdomain loops
  • ⁇ ⁇ is 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).
  • the pore loops and ID34 are similar.
  • the large extracellular loop of DI the N- and C-termini and ID 12 and ID23 differ from their counterparts in ⁇ 1G and ⁇ 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 ⁇ ⁇ .
  • T currents display heterogeneity of biophysical and pharmacological properties and subcellular localization. Identification of multiple T ⁇ . 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 ⁇ 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.
  • the human chromosome 17 genomic DNA of clone hCIT.22_K_21 appeared to include most or all of CACNAIG, a gene encoding the T Ca 2+ channel ⁇ 1G subunit. Thirty-four probable exons were identified by blastn alignment with the rat ⁇ 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
  • 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 pCR ⁇ 2.1 -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.
  • 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 cassette exons 34 (144 nt) and 35 (135 nt) yielded three products ( ⁇ 34 ⁇ 35, +34 ⁇ 35 and +34+35); the ⁇ 34+35 product was not detected.
  • exon 25B 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 ⁇ 25B+26 and +25B ⁇ 26 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 ).
  • 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.
  • ⁇ 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- ( ⁇ ) and carboxyl- (C) termini, reside in the cytoplasm.
  • Regions derived from portions of the R ⁇ A 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 ( ⁇ 14+25B ⁇ 26 ⁇ 34 ⁇ 35 ⁇ 38B) has 2,171 amino-acid (aa) residues; the longest (+14 ⁇ 25B+26+34+35+38B), 2,377 aa residues.
  • the reported rat ⁇ 1G aa sequence corresponds to the human (14+25B ⁇ 26 ⁇ 34 ⁇ 35+38B splice variant and is 93% identical. Additional features of the ⁇ 1G protein product including residue similarity to the other T ⁇ t subunits, comparison of splice sites and sites of potential post-translational modification are shown in Fig. 2 and described in the legend.

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Abstract

Dans cette invention on a déterminé les structures de CACNA1G et de CACNA1I, gènes codant les sous-unités α1G et α1I, respectivement, du canal T Ca2+ dans le cerveau humain, en comparant des ADNc du cerveau amplifiés par PCR et des séquences génomiques. CACNA1G est constitué d'au moins 38 exons recouvrant au moins 66 490 paires de base du chromosome 17q22. L'épissage alternatif de l'ARN a lieu dans six sites: exons de cassette 14, 26, 34 et 35, donneur interne dans l'exon 25 et intron 38B codant des protéines. En outre, l'ARN peut être polyadénylé dans n'importe lequel de ces deux sites. L'épissage alternatif d'ARN CACNA1G peut mener à l'expression de jusqu'à 64 produits protéiques distincts, compris entre les résidus d'acides aminés 2171 et 2377, avec jusqu'à 45 sites potentiels de phosphorylation. CACNA1I consiste d'au moins 37 exons recouvrant au moins 116 390 paires de base du chromosome 22q12.3-13.2. L'épissage alternatif de l'ARN a lieu dans trois sites: exon de cassette 9, accepteur alternatif dans l'exon 33 et introns 36B et 37 de 3' s'excluant mutuellement. L'épissage alternatif d'ARN CACNA1I peut mener à l'expression de jusqu'à 8 produits protéiques distincts, compris entre les résidus d'acides aminés 1968 et 2223, avec jusqu'à 38 sites potentiels de phosphorylation. La diversité moléculaire, générée par l'épissage alternatif et la modification post-traductionnelle de ces membres et d'autres membres de la famille de gènes de sous-unité α¿1G? de T, peut refléter l'hétérogénéité observée des courants T dans les neurones centraux.
PCT/US2000/012383 1999-05-13 2000-05-08 Variants episses de la sous-unite alpha des canaux calcium t dans le cerveau humain WO2000070044A2 (fr)

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EP1224218A1 (fr) * 1999-10-26 2002-07-24 Ortho-McNeil Pharmaceutical, Inc. Adn codant un canal calcium humain de type t alpha1g-c
WO2005058944A2 (fr) * 2003-12-12 2005-06-30 The Government Of The United States, As Represented By The Secretary Of The Department Of Health & Human Services Peptides immunogenes de xage-1
US7157243B1 (en) 1997-02-28 2007-01-02 Neuromed Pharmaceuticals Ltd. DNA encoding mammalian T-type calcium channels

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7157243B1 (en) 1997-02-28 2007-01-02 Neuromed Pharmaceuticals Ltd. DNA encoding mammalian T-type calcium channels
US7501263B2 (en) 1997-02-28 2009-03-10 Neuromed Technologies, Inc. Nucleic acids encoding mammalian T-type calcium channels
US7517672B2 (en) 1997-02-28 2009-04-14 Neuromed Pharmaceuticals Ltd. Nucleic acids encoding mammalian T-type calcium channels
EP1224218A1 (fr) * 1999-10-26 2002-07-24 Ortho-McNeil Pharmaceutical, Inc. Adn codant un canal calcium humain de type t alpha1g-c
EP1224218A4 (fr) * 1999-10-26 2003-06-25 Ortho Mcneil Pharm Inc Adn codant un canal calcium humain de type t alpha1g-c
WO2005058944A2 (fr) * 2003-12-12 2005-06-30 The Government Of The United States, As Represented By The Secretary Of The Department Of Health & Human Services Peptides immunogenes de xage-1
WO2005058944A3 (fr) * 2003-12-12 2006-01-05 Us Gov Health & Human Serv Peptides immunogenes de xage-1

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