| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | April 2, 2002 |
| PATENT TITLE |
Genes encoding neuronal voltage-gated calcium channel gamma subunits |
| PATENT ABSTRACT | Disclosed are mammalian nucleic acid sequences encoding a neuronal-specific subunit of a voltage-gated calcium channel. Specifically disclosed are .gamma..sub.2, .gamma..sub.3 and .gamma..sub.4 subunits. In other aspects, the disclosure relates to expression vectors which encode neuronal-specific subunits, as well as cells containing such vectors. In other aspects, the disclosure relates to antigenic fusion proteins comprising at least a portion of a mammalian neuronal-specific subunit of a voltage-gated calcium channel. Such fusion proteins are useful, for example, in the production of antibodies specifically reactive with the subunits of the invention. The nucleic acid sequences of the invention find application, for example, in screening for compounds which modulate the activity of neuronal voltage-gated calcium channels and also in diagnostic methods for diagnosing the autoimmune disease Lambert-Eaton Syndrome, as well as diagnosing defects in .gamma. subunit genes of a patient with a neuronal disease such as epilepsy. An additional application of the nucleic acid sequences of the invention is in therapeutic methods of treatment for such disorders |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | July 27, 1998 |
| PATENT REFERENCES CITED |
Jay et al., Science 248: 490-492 (1990). Ludwig et al., J. Neurosci. 17: 1339-1349 (1997). Walker and DeWaard, Trends Neurosci.21: 148-154 (1998). Smart et al., Neuron 20: 809-819 (1998). Homanics et al.., Proc. Natl. Acad. Sci. USA 94: 4143-4148 (1997). Brusa et al., Science 270: 1677-1680 (1995). Fletcher et al., Cell 87: 607-617 (1996). Burgess et al., Cell 88: 385-392 1997. Noebels et al., Epilepsy Res. 7: 129-135 (1990). Sweet et al., Mouse Genome 89: 552-553 (1991). Letts et al., Genomics 43:62-68 (1997). Moon and Friedman, Genomics 42: 152-156 (1997). DeWaard and Campbell, J. Physiol. (Land) 485: 619-634 (1995). Ikeda, S.R., Nature 380: 255-258 (1996). Herlitze et al., Nature 380: 258-262 (1996). Pragnell et al., Nature 368: 67-70 (1994). Niidome et al., Biochem. Biophys. Res. Commun. 203: 1821-1827 (1994). |
| PATENT CLAIMS |
What is claimed is: 1. An isolated nucleic acid which encodes a .gamma..sub.2 subunit of a voltage-gated calcium channel with the amino acid sequence of SEQ ID NO: 8. 2. The isolated nucleic acid of claim 1 which is nucleotide 390-1358 of SEQ ID NO: 7. 3. An isolated genomic DNA encoding a .gamma..sub.2 subunit of a voltage-gated calcium channel with the amino acid sequence of SEQ ID NO: 8. 4. A DNA expression vector comprising a nucleic acid sequence which encodes a .gamma..sub.2 subunit of a voltage-gated calcium channel with the amino acid sequence of SEQ ID NO: 8. 5. The DNA expression vector of claim 4 wherein the nucleic acid sequence is nucleotide 390-1358 of SEQ ID NO: 7. 6. A cell transformed with a DNA expression vector comprising a nucleic acid sequence which encodes a .gamma..sub.2 subunit of a voltage-gated calcium channel with the amino acid sequence of SEQ ID NO: 8. 7. The cell of claim 6 wherein the nucleic acid sequence is SEQ ID NO: 7. 8. The cell of claim 6 which is prokaryotic. 9. The cell of claim 6 which is eukaryotic. 10. The cell of claim 6 which is also transformed with DNA expression vectors encoding additional calcium channel subunits necessary and sufficient for assembly of a functional voltage-gated calcium channel. 11. A recombinant DNA expression vector comprising a nucleic acid sequence which encodes an antigenic fusion-protein comprising a C-terminal portion of a .gamma..sub.2 subunit of a voltage-gated calcium channel with the amino acid sequence of SEQ ID NO: 8. 12. A cell transformed with a recombinant DNA expression vector comprising a nucleic acid sequence which encodes an antigenic fusion protein comprising a C-terminal portion of a .gamma..sub.2 subunit of a voltage-gated calcium channel with the amino acid sequence of SEQ ID NO: 8. 13. An isolated cDNA which encodes a .gamma..sub.2 subunit of a voltage-gated calcium channel with the amino acid sequence of SEQ ID NO: 8. 14. An isolated nucleic acid comprising SEQ ID NO: 7. 15. The recombinant DNA expression vector of claim 11 wherein the C-terminal portion of the .gamma..sub.2 subunit encoded by the nucleic acid is amino acid 210-323 of SEQ ID NO: 8. 16. A method for identifying candidate compounds for modulating the activity of human neuronal voltage-gated calcium channels, comprising: a) providing a cell culture model system comprising cells transformed with a DNA expression vector comprising a nucleic acid sequence which encodes a .gamma..sub.2 subunit of a voltage-gated calcium channel with the amino acid sequence of SEQ ID NO: 8, said cell comprising additional calcium channel subunits necessary and sufficient for assembly of a functional voltage-gated calcium channel; b) contacting the cell culture model system of step a) with a test compound; and c) measuring calcium channel currents in at least one cell of the cell culture model system, wherein an increase or decrease in the calcium channel currents in the presence of the test compound, compared to calcium channel currents measured in the absence of the test compound, identifies the test compound as a candidate compound for modulating the activity of human neuronal voltage-gated calcium channels. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
BACKGROUND OF THE INVENTION Voltage-gated calcium channels are a diverse family of proteins which have a variety of biological functions, including presynaptic neurotransmitter release and protein signaling within the cell (Bito et al. (1997) Curr. Opin. Neurobiol. 7, 419-429; Dunlap et al. (1995) Trends Neurosci. 18, 89-98). The calcium currents produced by these channels are classified into P/Q-, N-, L-, R-, and T-type based on their pharmacological and biophysical properties, and all are expressed in brain (Dunlap et al. (1995) Trends Neurosci. 18, 89-98; Varadi et al. (1995) Trends Pharmacol. Sci. 16, 43-49; Nooney et al. (1997) Trends Pharmacol. Sci. 18, 363-371; Perez-Reyes et al. (1998) Nature 391, 896-900). Except for the T-type, whose molecular structure is unknown, all voltage-gated calcium channels are composed of at least three subunits, .alpha..sub.1, .alpha..sub.2.delta. and .beta. (De Waard et al. Structural and functional diversity of voltage-activated calcium channels. In Ion Channels, (ed. T. Narahashi) 41-87 (Plenum Press, New York, 1996)). A fourth subunit, .gamma., is associated with skeletal muscle calcium channels. The mRNA for this .gamma. subunit is abundant in skeletal muscle, but has not been detected in brain (Jay, S. D et al. (1990) Science 248, 490-492; Ludwig et al. (1997) J. Neurosci. 17, 1339-1349). Whether .gamma. subunits specific for brain calcium channels exist, remains to be determined. The .alpha..sub.1 subunit forms the membrane pore and voltage-sensor and is a major determinant for current classification. Several isoforms of .alpha..sub.1, arising from different genes, have been identified. The other subunits modulate the voltage-dependence and kinetics of activation and inactivation, and the current amplitude (Walker et al. (1998) Trends Neurosci. 21, 148-154). There is currently only one known gene and isoform for the .alpha..sub.2.delta. subunit, and four different .beta. subunit genes encoding the distinct .beta. isoforms. P/Q- and N-type channels purified from brain contain .alpha..sub.1A and .alpha..sub.1B subunits, respectively. These .alpha..sub.1 subunits are associated with various proportions of the four separately encoded .beta. subunit proteins, indicating that considerable subunit complexity exists (Scott et al. (1996) J. Biol. Chem. 271, 3207-3212; Liu et al. (1996) J. Biol. Chem. 271, 13804-13810). A number of compounds useful in treating various diseases in animals, including humans, are thought to exert their beneficial effects by modulating functions of voltage-gated calcium channels. Many of these compounds bind to calcium channels and block, or reduce the rate of influx of calcium into cells in response to depolarization of the inside and outside of the cells. An understanding of the pharmacology of compounds that interact with calcium channels, and the ability to rationally design compounds that will interact with calcium channels to have desired therapeutic effects, depends upon the understanding of the structure of channel subunits and the genes that encode them. The identification and study of tissue specific subunits allows for the development of therapeutic compounds specific for pathologies of those tissues. Cellular calcium homeostasis plays an essential part in the physiology of nerve cells. The intracellular calcium concentration is about 0.1 uM compared with 1 mM outside the nerve cell. This steep concentration gradient (.times.10,000) is regulated primarily by voltage-gated calcium channels. Several pathologies of the central nervous system involve damage to or inappropriate function of voltage-gated calcium channels. In cerebral ischaemia (stroke) the channels of neurons are kept in the open state by prolonged membrane depolarisations, producing a massive influx of calcium ions. This, in turn activates various calcium/calmodulin dependent cellular enzyme systems, e.g. kinases, proteases and phospholipases. Such prolonged activation leads to irreversible damage to nerve cells. Certain diseases, such as Lambert-Eaton Syndrome, involve autoimmune interactions with calcium channels. The availability of the calcium channel subunits makes possible immunoassays for the diagnosis of such diseases. An understanding of them at the molecular level will lead to effective methods of treatment. Epilepsies are a heterogeneous group of disorders characterized by recurrent spontaneous seizures affecting 1% of the population. In recent years several human genes have been identified, including the most recently identified potassium channels KCNQ2 and KCNQ3 for benign familial neonatal convulsions (Charlier et al. (1998) Nature Genet. 18, 53-55; Singh et al. (1998) Nature Genet. 18, 25-29; Biervert et al. (1998) Science 279, 403-406). To date, the involvement of voltage-gated calcium channels in epilepsies has been poorly characterized. A number of mouse mutants have generalized tonic-clonic seizures, mostly resulting from gene knockouts. Ion channels are involved in many of these cases, including potassium (Smart et al. (1998) Neuron 20, 809-819), GABA (Homanics et al. (1997) Proc. Natl. Acad. Sci. USA 94, 4143-4148) and glutamate receptor channels (Brusa, et al. (1995) Science 270, 1677-1680). Comparatively fewer mouse models have been described with absence seizures, (also known as petit-mal or spike-wave), although this may be due to ascertainment bias as these seizures are associated with only a brief loss of consciousness. It has thus required a systematic electrocorticographic screen of known mutants to uncover mouse absence models. The mouse mutants ducky, lethargic, mocha, slow-wave epilepsy, stargazer and tottering, each show some form of spike-wave discharge associated with behavioral arrest which is characteristic of absence epilepsy (Noebels, J. L. In Basic Mechanisms of the Epilepsies: Molecular and Cellular Approaches., (ed. A. V. Delgado-Escueta, A. A. Ward, D. M. Woodbury and R. J. Porter) 44, 97-113 (Raven Press, New York, 1986); Noebels et al. (1990) Epilepsy Res. 7, 129-135; Cox et al. (1997) Cell 91, 1-20). The underlying genes are described in most of these models, and in two--tottering and lethargic--the defect is in a gene encoding a neuronal calcium channel subunit (Fletcher et al. (1996) Cell 87, 607-617; Burgess et al. (1997) Cell 88, 385-392). Because of the overlap in expression of voltage-gated calcium channel subunits and a limited understanding of tissue differences, it has not been straightforward to study the specific function of neuronal channels in vivo. The study of mouse mutations has begun to allow a dissection of this problem. For example, the neurological mutants tottering and lethargic have defects in genes encoding .alpha..sub.1A and .beta..sub.4 subunits, respectively (Fletcher et al. (1996) Cell 87, 607-617; Burgess et al. (1997) Cell 88, 385-392). Their phenotypes are very similar, each exhibiting spike-wave seizures and moderate cerebellar ataxia without obvious neuronal damage. The nature of the mutation in each is commensurate with the respective roles of major and auxiliary calcium channel subunits: tottering has an amino acid substitution in the structural .alpha..sub.1A subunit, whereas lethargic is not likely to express any functional .beta..sub.4 protein. The phenotype of the lethargic mouse shows that defects in regulatory subunits can also lead to the same neuronal malfunctions as observed for structural subunit mutations. Continued study of these mouse mutants will give further insight into neuronal calcium channel function in vivo. The stargazer mutation arose spontaneously at The Jackson Laboratory on the A/J inbred mouse line (Noebels et al. (1990) Epilepsy Res. 7, 129-135), and was initially detected for its distinctive head-tossing and ataxic gait. Subsequent electrocorticography revealed recurrent spike-wave seizures when the animal was still, characteristic of absence epilepsy. The seizures were notably more prolonged and frequent than in tottering or lethargic mice, lasting on average six seconds and recurring over one hundred times an hour. The ataxia and head-tossing are presumed to be pleiotropic consequences of the mutation in the cerebellum and inner ear, respectively; the latter also distinguishes stargazer from the other mutants. The waggler mutant arose independently on the MRL/MpJ strain and was subsequently found to be an allele of stargazer (Sweet et al. (1991) Mouse Genome 89, 552). Waggler mice are severely ataxic but head-toss less frequently than stargazer and have a more pronounced side-to-side head motion. The fine-mapping of the stargazer mutation on mouse chromosome 15 and the construction of a 1.3 Mb physical contig across the critical genetic interval was described in an earlier study (Letts et al. (1997) Genomics 43, 62-68). The present invention describes the first member of a multi-gene family that encodes a neuronal specific voltage-gated calcium channel .gamma. subunit disrupted in stargazer and waggler mice. SUMMARY OF THE INVENTION In one aspect, the present invention relates to a mammalian nucleic acid sequences encoding a neuronal-specific subunit of a voltage-gated calcium channel. Specifically disclosed are .gamma..sub.2, .gamma..sub.3 and .gamma..sub.4 subunits. In other aspects, the invention relates to expression vectors which encode neuronal-specific subunits, as well as cells containing such vectors. In other aspects, the invention relates to antigenic fusion proteins comprising at least a portion of a mammalian neuronal-specific subunit of a voltage-gated calcium channel. Such fusion proteins are useful, for example, in the production of antibodies specifically reactive with the subunits of the invention. Such antibodies are also encompassed within the scope of the invention. In another embodiment, the invention relates to a method for screening for compounds which modulate the activity of neuronal voltage-gated calcium channels. The method involves providing a cell transformed with a DNA expression vector comprising a mammalian cDNA sequence encoding a neuronal-specific .gamma. subunit of a voltage-gated calcium channel, the cell comprising additional calcium channel subunits necessary and sufficient for assembly of a functional voltage-gated calcium channel. The cell is contacted with a test compound and agonistic or antagonistic action of the test compound on the reconstituted calcium channels is determined. Also encompassed within the scope of the invention are diagnostic methods based on the experiments described in the Exemplification section set forth below. These include, for example, a method of diagnosing Lambert-Eaton Syndrome and a method for diagnosing a defect in a .gamma. subunit gene of a patient with a neuronal disease such as epilepsy. Therapeutic methods based on the disclosure are also described. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagrammatic representation which shows the stargazer locus on mouse chromasome 15 and an ETn insertion that is genetically and physically linked to the stg locus. a) Restriction map of the 12 kb HindIII fragment from BAC28, showing the locations of exon 2, cDNA selection products 416, 211, 417, 113, 401, 144, 198 and the ETn insertion. Abbreviations: B, BamHI; E, EcoRI; H, HindIII; S, SacI. Several additional BamHI sites are not shown. The locations of oligonucleotide primers used in genomic DNA PCR reactions are shown here as black arrowheads (left, ETn-OR; right, JS167) and white arrowheads (left, 109F; right, E/Ht7). Below the restriction map are the exons from cDNA clone c2 shown relative to the mouse BAC and PAC contig of Letts et al. (1997) Genomics 43, 62-68. Coding regions are shown in black, untranslated regions in white. Splice site sequences are Exon 1: (donor only) CCTGCTGCCTCGAAG ' gtatttgatttccaa SEQ ID NO: 1; Exon 2: (acceptor) tgcattatcttgcag ' GGAACTTCAAAGGTC SEQ ID NO: 2, (donor) CAGAGTATTTCCTCC ' gtgagtcacacacgg SEQ ID NO: 3; Exon 3: (acceptor) ctctatgttccgcag ' GGGCCGTGAGGGCCT SEQ ID NO: 4, (donor) TCTTCGTGTCTGCAG ' gtaaggcaggggtgt SEQ ID NO: 5; Exon 4: (acceptor only ) cctcttctcctccag ' GTCTTAGTAATATCA SEQ ID NO: 6. FIG. 2 is a diagrammatic representation of the cDNA sequence SEQ ID NO: 7 and conceptual translation of mouse Cacng2 SEQ ID NO: 8. FIGS. 3A-3B are a diagrammatic representation comparing the .gamma..sub.2 subunit protein to the .gamma. subunit of voltage-gated skeletal muscle calcium channels. FIG. 3a) The predicted open reading frame of cDNA clone c2 SEQ ID NO: 8 is shown on the top line and is aligned with that of the previously identified rat calcium channel .gamma. subunit Genbank accession number CAA70602 on the bottom line. Underlined sequences in each are putative transmembrane regions. The predicted N-glycosylation sites are shown in double underlining, and stippled underlining at the C-terminus indicates the peptide used for antibody generation. FIG. 3b) Secondary structure prediction plot. Positive scores show residues that are likely to be in the membrane, and negative scores show those that are not. FIG. 4 is a diagrammatic representation of the brain RT-PCR analysis performed, spanning Cacng2 exons in stargazer and waggler mutants. Lanes A, S, M, W and C correspond to A/J, B6C3Fe-stg/stg, MRL/MpJ, B6-stg.sup.wag /stg.sup.wag and no DNA (water) control, respectively, and lane X is the size marker (HaeIII restriction-digested PhiX DNA). FIGS. 5A-5D is a diagrammatic representation of patch clamp data on the functional effect of the wild-type stargazer protein on neuronal calcium channel activity. FIG. 5a) Average whole-cell calcium current in BHK cells. Each bar represents mean .+-.SE peak calcium current amplitude, and the number of recorded cells is indicated in parentheses. FIG. 5b) Average normalized current-voltage relationship for control and transfected BHK cells. Symbols represent mean .+-.SE of six to fourteen cells. FIG. 5c) Representative superimposed current traces illustrating voltage-dependent inactivation of the channels at steady state from single control and transfected BHK cells. To facilitate comparison of records, currents have been scaled to similar size and only the first 10 ms are displayed. FIG. 5d) Average steady-state inactivation curves for control and transfected BHK cells. FIG. 6 is a diagrammatic representation of the cDNA sequence SEQ ID NO:9 and conceptual translation SEQ ID NO: 10 of mouse Cacng3. FIG. 7 is a diagrammatic representation of the cDNA sequence SEQ ID NO: 11 and conceptual translation SEQ ID NO: 12 of mouse Cacng4. FIG. 8 is a diagrammatic representation of the protein alignments of neuronal calcium channel .gamma. subunits, mCacng2pep SEQ ID NO: 8, mCacng3pep SEQ ID NO: 10, and mCacng4pep SEQ ID NO: 12. |
| PATENT EXAMPLES | This data is not available for free |
| PATENT PHOTOCOPY | Available on request |
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