Claims
- 1. A method for screening for a candidate bioactive agent capable of binding to TRPM4b, said method comprising:
a) contacting a TRPM4b protein or an amino-terminal fragment thereof with said candidate agent; and b) determining the binding of said candidate agent to said TRPM4b protein or amino-terminal fragment thereof.
- 2. The method of claim 1 wherein a library of two or more of said candidate agents are contacted with said TRPM4b protein or fragment thereof.
- 3. The method of claim 2 wherein one of said candidate agents is Ca2+.
- 4. The method of claim 1 wherein said TRPM4b protein comprises amino acids from 1 through about 1214 of SEQ ID NO:2.
- 5. The method of claim 1 wherein said TRPM4b protein is encoded by a nucleic acid comprising sequences from 1 through about 4061 of SEQ ID NO:1.
- 6. A method for screening a candidate bioactive agent comprising
a) contacting a TRPM4b channel with the candidate agent, and b) detecting whether said agent modulates the monovalent cationic permeability of said TRPM4b channel.
- 7. The method of claim 6 wherein said modulating activity opens said TRPM4b channel.
- 8. The method of claim 7 wherein said candidate agent is Ca2+.
- 9. The method of claim 6 wherein said modulating activity closes said TRPM4b channel.
- 10. The method of claim 6 wherein said monovalent cation is selected from the group consisting of Na+, K+, and Cs+.
- 11. A method for screening for a candidate bioactive agent capable of modulating monovalent cation permeability of a TRPM4b channel, said method comprising:
a) providing a recombinant cell comprising a recombinant nucleic acid comprising nucleic acid encoding TRPM4b and an inducible promoter operably linked thereto which is capable of expressing said TRPM4b, and further comprising a monovalent cation indicator; b) inducing said recombinant cell to express said TRPM4b; c) contacting said recombinant cell with a monovalent cation and said candidate agent; and d) detecting the intracellular levels of said monovalent cation with said indicator.
- 12. The method of claim 11 wherein said contacting is of said candidate agent followed by said monovalent cation.
- 13. The method of claim 11 wherein said monovalent cation is selected from the group consisting of Na+, K+, and Cs+.
- 14. The method of claim 11 wherein the modulating activity increases said monovalent cation permeability of said TRPM4b channel.
- 15. The method of claim 14 wherein said candidate agent is Ca+.
- 16. The method of claim 11 wherein the modulating activity decreases said monovalent cation permeability of said TRPM4b channel.
- 17. The method of claims 13 or 16 wherein said modulating activity alters the membrane potential of said recombinant cell.
- 18. The method of claim 11 wherein said indicator comprises a fluorescent molecule.
- 19. The method of claim 18 wherein said fluorescent molecule comprises fura-2.
- 20. A method for measuring monovalent cation permeability of a TRPM4b channel, said method comprising:
a) providing a recombinant cell wherein said cell comprises a recombinant nucleic acid which expresses TRPM4b and further comprises a monovalent cation indicator; b) contacting said recombinant cell with a monovalent cation which selectively interacts with said indicator to generate a signal; and c) measuring the intracellular levels of said monovalent cation by detecting said indicator signal.
- 21. The method of claim 20 wherein said indicator comprises a fluorescent molecule.
- 22. The method of claim 20 wherein said fluorescent molecule comprises fura-2.
- 23. The method of claim 20 wherein said monovalent cation is selected from the group consisting of Na+, K+, and Cs+.
- 24. The method of claim 20 further comprising contacting said recombinant cell with a candidate bioactive agent.
- 25. The method of claim 24 wherein said modulating activity increases said monovalent cation permeability of said TRPM4b channel.
- 26. The method of claim 25 wherein the candidate agent is Ca2+.
- 27. The method of claim 24 wherein said modulating activity decreases said monovalent cation permeability of said TRPM4b channel.
- 28. The method of claims 25 or 27 wherein said modulating activity alters the membrane potential of said recombinant cell.
- 29. The method of claim 24 wherein said measuring further comprises comparing said intracellular monovalent cation levels to intracellular monovalent cation levels in a cell which does not express recombinant TRPM4b.
- 30. The method of claim 24 wherein said measuring further comprises comparing said intracellular monovalent cation levels to intracellular monovalent cation levels in a cell which does not express recombinant TRPM4b but which is in contact with said candidate agent.
- 31. A method for screening for a candidate bioactive agent capable of modulating expression of a TRPM4b protein or fragment thereof comprising:
a) providing a recombinant cell capable of expressing a recombinant nucleic acid encoding a TRPM4b protein; b) contacting said cell with said candidate agent; and c) determining the effect of said candidate agent on the expression of said recombinant nucleic acid.
- 32. The method of claim 31 wherein said determining is a phenotype of said cell.
- 33. The method of claim 31 wherein the determining comprises determining the level of expression of TRPM4b in the presence of said candidate agent and comparing said level of expression to endogenous TRPM4b levels.
- 34. The method of claims 1, 6, 11, 19 and 31, wherein said candidate agent comprises a small molecule, protein, polypeptide or nucleic acid.
CROSS-REFERENCE
[0001] This application claims priority to (1) U.S. provisional application Serial No. 60/351,938, filed Jan. 25, 2002, entitled “LTRPC4 is a CA2+-Activated Non-Selective Cation Channel Mediating Cell Membrane Depolarization” and (2) U.S. provisional application Serial No. [unknown], filed May 2, 2002, entitled “Methods of Screening for TRPM4b Modulators.”
Provisional Applications (2)
|
Number |
Date |
Country |
|
60351938 |
Jan 2002 |
US |
|
60377937 |
May 2002 |
US |