Claims
- 1. An isolated nucleic acid encoding a modified olfactory cyclic nucleotide-gated ion channel, wherein said channel comprises mutations which together impart increased cAMP sensitivity, decreased cGMP sensitivity, decreased nitric oxide sensitivity and decreased calcium-calmodulin sensitivity.
- 2. An isolated nucleic acid encoding a modified olfactory cyclic nucleotide-gated ion channel, wherein said channel comprises at least one mutation selected from the group consisting of a C460W mutation and a E583M mutation.
- 3. The isolated nucleic acid of claim 2, wherein said channel comprises a E583M mutation; a C460W mutation and a E583M mutation; or a 61-90 deletion, a C460W mutation, and a E583M mutation.
- 4. The isolated nucleic acid of claim 2, wherein said channel comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7.
- 5. An isolated polypeptide encoded by the nucleic acid of claim 2.
- 6. An expression vector comprising the nucleic acid of claim 2.
- 7. The expression vector of claim 6, wherein said vector is a recombinant adenovirus vector.
- 8. A host cell comprising the expression vector of claim 6, wherein said host cell is selected from the group consisting of a prokaryotic cell and a eukaryotic cell.
- 9. The host cell of claim 8, wherein said eukaryotic cell is selected from the group consisting of a human embryonic kidney-293 cell, a GH4C1 pituitary cell, and a rat C6-2B glioma cell.
- 10. A method for determining local intracellular cAMP concentration within a eukaryotic cell, comprising the steps of:
a) providing:
i) at least one eukaryotic cell, and ii) a nucleic acid encoding a modified olfactory cyclic nucleotide-gated ion channel, wherein said channel comprises at least one mutation selected from the group consisting of a C460W mutation and a E583M mutation; and b) contacting said eukaryotic cell with said nucleic acid under conditions suitable for expressing said modified olfactory cyclic nucleotide-gated ion channel in said eukaryotic cell; c) measuring intracellular calcium concentration in said eukaryotic cell; and d) determining local intracellular cAMP concentration based upon said intracellular calcium concentration.
- 11. The method of claim 10, wherein said contacting step comprises infecting said eukaryotic cell with a recombinant adenovirus comprising said nucleic acid.
- 12. The method of claim 10, wherein said measuring step comprises contacting said eukaryotic cell with a stimulus.
- 13. The method of claim 12, wherein said stimulus is selected from the group consisting of an adenylate cyclase activator, a G-protein activator, and a phosphodiesterase inhibitor.
- 14. The method of claim 10, wherein said measuring step comprises monitoring calcium flux with a fluorescent calcium indicator.
- 15. The method of claim 14, wherein said fluorescent calcium indicator is selected from the group consisting of fura-2, indo-1, quin-2, fluo-3 and rhod-2.
- 16. A method for determining local intracellular cAMP concentration within a eukaryotic cell, comprising the steps of:
a) providing:
i) at least one eukaryotic cell, and ii) a nucleic acid encoding a modified olfactory cyclic nucleotide-gated ion channel, wherein said channel comprises at least one mutation selected from the group consisting of a C460W mutation and a E583M mutation; and b) contacting said eukaryotic cell with said nucleic acid under conditions suitable for expressing said modified olfactory cyclic nucleotide-gated ion channel in said eukaryotic cell; c) measuring the electric current across the plasma membrane of said eukaryotic cell; and d) determining local intracellular cAMP concentration based upon said electric current.
- 17. The method of claim 16, wherein said contacting step comprises infecting said eukaryotic cell with a recombinant adenovirus comprising said nucleic acid.
- 18. The method of claim 16, wherein said measuring step comprises contacting said eukaryotic cell with a stimulus.
- 19. The method of claim 18, wherein said stimulus is selected from the group consisting of an adenylate cyclase activator, a G-protein activator, and a phosphodiesterase inhibitor.
- 20. The method of claim 16, wherein said measuring step comprises monitoring said electric current with a patch-clamp technique selected from the group consisting of perforated patch-clamp technique and a whole-cell patch-clamp technique.
- 21. The method of claim 16, wherein said determining step comprises calibrating the electric current with respect to cAMP concentration by obtaining a cAMP-dose response curve for said modified cyclic nucleotide-gated ion channel with an inside-outside patch clamp technique.
- 22. A method for determining whether a candidate compound is capable of modulating local intracellular cAMP concentration within a eukaryotic cell, comprising the steps of:
a) providing:
i) at least one eukaryotic cell expressing a modified olfactory cyclic nucleotide-gated ion channel, wherein said channel comprises at least one mutation selected from the group consisting of a C460W mutation and a E583M mutation, and ii) a drug candidate; and b) determining local intracellular cAMP concentration within said eukaryotic cell in the presence and absence of said drug candidate.
- 23. The method of claim 22, wherein said expressing of said modified olfactory cyclic nucleotide-gated ion channel is accomplished by infection of said eukaryotic cell with a recombinant adenovirus.
- 24. The method of claim 22, wherein said determining of said local intracellular cAMP concentration is accomplished by measuring intracellular calcium concentration in said eukaryotic cell in the presence and absence of said drug candidate.
- 25. The method of claim 22, wherein said determining of said local intracellular cAMP concentration is accomplished by measuring the electric current across the plasma membrane of said eukaryotic cell in the presence and absence of said drug candidate.
Parent Case Info
[0001] This application claims benefit of provisional patent application U.S. Ser. No. 60/332,494, filed on Nov. 16, 2001. This invention was made with government support under grants GM32438, NS28389, HL58344, DC00385, EY09275, from the National Institutes Health. The United States Government has certain rights in the invention.
Provisional Applications (1)
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Number |
Date |
Country |
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60332494 |
Nov 2001 |
US |