Claims
- 1. An isolated cell that recombinantly expresses an N-type calcium channel comprising a CaV2.2 subunit that comprises exon e37a (CaV2.2e[37a]).
- 2. The isolated cell of claim 1, wherein the CaV2.2e[37a] subunit has a human sequence.
- 3. The isolated cell of claim 1, wherein the CaV2.2e[37a] subunit has a mouse sequence.
- 4. The isolated cell of claim 1, wherein the CaV2.2e[37a] subunit has a rat sequence.
- 5-6. (Canceled)
- 7. An isolated neuron that expresses an N-type calcium channel comprising a CaV2.2 subunit that comprises exon e37a (CaV2.2e[37a]).
- 8. The isolated neuron of claim 7, wherein the neuron further expresses a marker of nociceptive neurons.
- 9. The isolated neuron of claim 8, wherein the marker of nociceptive neurons is NaV1.8.
- 10. The isolated neuron of claim 8, wherein the marker of nociceptive neurons is vanilloid receptor VR1.
- 11. The isolated neuron of claim 8, wherein the neuron expresses both NaV1.8 and vanilloid receptor VR1.
- 12. A method for identifying lead compounds for a pharmacological agent useful in the treatment of disease associated with increased or decreased voltage regulated calcium influx mediated by a N-type calcium channel containing a CaV2.2e[37a] subunit comprising
providing a cell or other membrane-encapsulated space comprising a CaV2.2e[37a] polypeptide; contacting the cell or other membrane-encapsulated space with a candidate pharmacological agent under conditions which, in the absence of the candidate pharmacological agent, cause a first amount of voltage regulated calcium influx into the cell or other membrane-encapsulated space; and determining a test amount of voltage regulated calcium influx as a measure of the effect of the lead compounds for a pharmacological agent on the voltage regulated calcium influx mediated by a N-type calcium channel containing a CaV2.2e[37a] subunit, wherein a test amount of voltage regulated calcium influx which is less than the first amount indicates that the candidate pharmacological agent is a lead compound for a pharmacological agent which reduces voltage regulated calcium influx and wherein a test amount of voltage regulated calcium influx which is greater than the first amount indicates that the candidate pharmacological agent is a lead compound for a pharmacological agent which increases voltage regulated calcium influx.
- 13. The method of claim 12, further comprising the step of loading the cell or other membrane-encapsulated space with a calcium-sensitive compound which is detectable in the presence of calcium, wherein the calcium-sensitive compound is detected as a measure of the voltage regulated calcium influx.
- 14. The method of claim 12, wherein the pharmacological agent that specifically reduces voltage regulated calcium influx mediated by a N-type calcium channel containing a CaV2.2e[37a] subunit is an agent that reduces N-type calcium channel current densities in nociceptive neurons.
- 15. The method of claim 14, wherein the pharmacological agent that specifically reduces voltage regulated calcium influx mediated by a N-type calcium channel containing a CaV2.2e[37a] subunit is useful as an analgesic agent.
- 16. A method for identifying compounds which selectively or preferentially bind a N-type calcium channel containing a CaV2.2e[37a] subunit comprising,
providing a first cell or membrane encapsulated space which expresses a N-type calcium channel that contains a CaV2.2e[37a] subunit, providing a second cell or membrane encapsulated space which expresses a N-type calcium channel that does not contain a CaV2.2e[37a] subunit, wherein the second cell or membrane encapsulated space is identical to the first cell except for the N-type calcium channel expressed, contacting the first cell or membrane encapsulated space and the second cell or membrane encapsulated space with a compound, and determining the binding of the compound to the first cell or membrane encapsulated space and the second cell or membrane encapsulated space, wherein a compound which binds the first cell or membrane encapsulated space but does not bind the second cell or membrane encapsulated space is a compound which selectively binds the N-type calcium channel that contains a CaV2.2e[37a] subunit, and wherein a compound which binds the first cell or membrane encapsulated space in an amount greater than the compound binds the second cell or membrane encapsulated space is a compound which preferentially binds the N-type calcium channel that contains a CaV2.2e[37a] subunit.
- 17. The method of claim 16, wherein the N-type calcium channel that does not contain a CaV2.2e[37a] subunit is a N-type calcium channel that contains a CaV2.2e[37b] subunit.
- 18. A method for identifying compounds which selectively or preferentially bind to a CaV2.2e[37a] isoform comprising,
providing a CaV2.2e[37a] isoform polypeptide or nucleic acid, providing a CaV2.2e[37b] isoform polypeptide or nucleic acid, contacting the CaV2.2e[37a] isoform polypeptide or nucleic acid and the CaV2.2e[37b] subunit isoform polypeptide or nucleic acid with a compound, and determining the binding of the compound to the CaV2.2e[37a] isoform polypeptide or nucleic acid and the CaV2.2e[37b] isoform polypeptide or nucleic acid, wherein a compound which binds the CaV2.2e[37a] isoform polypeptide or nucleic acid but does not bind the human N-type calcium channel CaV2.2e[37b] isoform polypeptide or nucleic acid is a compound which selectively binds the CaV2.2e[37a] isoform, and wherein a compound which binds the CaV2.2e[37a] isoform polypeptide or nucleic acid in an amount greater than the compound binds the CaV2.2e[37b] isoform polypeptide or nucleic acid is a compound which preferentially binds the CaV2.2e[37a] isoform.
- 19-23. (Canceled)
- 24. A method for preparing an analgesic agent, comprising
identifying an agent that selectively or preferentially reduces calcium channel current densities in nociceptive neurons mediated by N-type calcium channels containing a CaV2.2e[37a] subunit, and formulating the agent for administration to a subject in need of such treatment.
- 25. (Canceled)
- 26. A double stranded RNA molecule specific for CaV2.2e[37a] RNA.
- 27. The double stranded RNA molecule of claim 26, wherein the molecule is 21-23 nucleotides in length.
- 28. The double stranded RNA molecule of claim 26, wherein the molecule has a 3′ overhang.
- 29. The double stranded RNA molecule of claim 28, wherein the 3′ overhang is 2 nucleotides in length.
- 30. The double stranded RNA molecule of claim 26, wherein the molecule is a single molecule that comprises a hairpin structure.
- 31. A method for inhibiting calcium influx in a neuronal cell mediated by a N-type calcium channel containing a CaV2.2e[37a] subunit comprising
contacting the neuronal cell with an amount of a CaV2.2e[37a] inhibitor effective to inhibit calcium influx in the mammalian cell.
- 32. The method of claim 31, wherein the inhibitor is selected from the group consisting of an antibody which selectively binds the CaV2.2e[37a] polypeptide, an antisense nucleic acid that reduces expression of a CaV2.2e[37a] polypeptide, a siRNA that reduces expression of a CaV2.2e[37a] polypeptide.
- 33. A method for treating a subject afflicted by pain mediated by a N-type calcium channel containing a CaV2.2e[37a] subunit comprising
administering to a subject in need of such treatment an inhibitor of the CaV2.2e[37a] polypeptide in an amount effective to inhibit voltage regulated calcium influx and thereby to reduce the pain.
- 34. The method of claim 33, wherein the inhibitor is selected from the group consisting of an antibody which selectively binds the CaV2.2e[37a] polypeptide, an antisense nucleic acid that reduces expression of a CaV2.2e[37a] polypeptide, a siRNA that reduces expression of a CaV2.2e[37a] polypeptide.
- 35. The method of claim 33, wherein the inhibitor is administered prophylactically to a subject at risk of being afflicted with pain.
- 36. The method of claim 33, wherein the pain is neuropathic pain.
RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C. § 19(e) of U.S. provisional application serial No. 60/443,474, filed Jan. 29, 2003, the disclosure of which is incorporated by reference herein.
GOVERNMENT SUPPORT
[0002] This work was funded in part by the National Institutes of Health under grant numbers NS29967 and NS43082. The government may have certain rights in this invention.
Provisional Applications (1)
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Number |
Date |
Country |
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60443474 |
Jan 2003 |
US |