Modified cyclic nucleotide gated ion channels
View Patent ↗The present invention provides modified cyclic nucleotide gated (CNG) channels. In particularly preferred embodiments, the modified CNG channels exhibit increased sensitivity and specificity for cAMP, as compared to wild-type CNG channels. In additional embodiments, regulation by Ca 2+ -calmodulin has been removed in the modified CNG channels. Convenient optical methods for detecting changes in cAMP, taking advantage of the Ca 2+ permeability of the channel are also provided by the present invention. In addition, electrophysiological methods are further provided.
1. 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) an isolated eukaryotic cell expressing a modified mammalian olfactory cyclic nucleotide-gated ion channel alpha subunit, wherein said channel comprises a mutation, wherein said mutation comprises a substitution at a residue corresponding to position 583 of SEQ ID NO:8, and wherein said channel has increased cAMP sensitivity, and decreased cGMP sensitivity as compared to a wild type channel, 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.
2. The method of claim 1 , wherein said expressing of said modified olfactory cyclic nucleotide-gated ion channel is accomplished by infection of said eukaryotic cell with a recombinant adenovirus comprising a polynucleotide encoding said modified mammalian olfactory cyclic nucleotide-gated ion channel alpha subunit.
3. The method of claim 1 , 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.
4. The method of claim 3 , wherein said measuring intracellular calcium concentration comprises monitoring calcium flux with a fluorescent calcium indicator.
5. The method of claim 4 , wherein said fluorescent calcium indicator is selected from the group consisting of fura-2, indo-1, quin-2, fluo-3 and rhod-2.
6. The method of claim 1 , 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.
7. The method of claim 6 , wherein said measuring electric current across the plasma membrane comprises a perforated patch-clamp technique.
8. The method of claim 6 , wherein said measuring electric current across the plasma membrane comprises a whole-cell patch-clamp technique.
9. The method of claim 1 , wherein said channel alpha subunit comprises a glutamic acid (E) to methionine (M) substitution at a residue corresponding to position 583 of SEQ ID NO:8.
10. The method of claim 9 , wherein said channel alpha subunit further comprises a cysteine (C) to tryptophan (W) substitution at a residue corresponding to position 460 of SEQ ID NO:8.
11. The method of claim 10 , wherein said channel alpha subunit further comprises a 61-90 deletion at residues corresponding to positions 61-90 of SEQ ID NO:8.
12. The method of claim 1 , 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.
13. The method of claim 1 , wherein said modified mammalian olfactory cyclic nucleotide-gated ion channel alpha subunit is expressed from a recombinant adenovirus expression vector.
14. The method of claim 1 , wherein said eukaryotic cell is selected from the group consisting of a human embryonic kidney-293 cell and a rat GH4C1 pituitary cell.
15. The method of claim 1 , wherein said substitution at a residue corresponding to position 583 of SEQ ID NO:8 is selected from the group consisting of E583M, E583V, E583L and E583I.
16. The method of claim 1 , wherein said increased cAMP sensitivity and decreased cGMP sensitivity is determined by measuring cAMP-induced and cGMP-induced current, and wherein said cGMP-induced current is 40% or less than said cAMP-induced current.
17. The method of claim 1 , wherein said increased cAMP sensitivity comprises a K 1/2 at least ten-fold lower than that observed for a a wild type channel.
18. The method of claim 1 , wherein said decreased cGMP sensitivity comprises a K 1/2 at least ten-fold higher than that observed for a a wild type channel.
19. The method of claim 1 , wherein said mutation further comprises a substitution at a residue corresponding to position 460 of SEQ ID NO:8.
20. The method of claim 19 , wherein said substitution at a residue corresponding to position 460 of SEQ ID NO:8 is selected from the group consisting of C460W, C460F and C460Y.
21. The method of claim 19 , wherein said mutation further comprises a 61-90 deletion at residues corresponding to positions 61-90 of SEQ ID NO:8.