Nucleic acids encoding modified olfactory 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. An isolated nucleic acid encoding 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.
2. The nucleic acid 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.
3. The nucleic acid of claim 1 , wherein said cGMP-induced current is 40% or less than said cAMP-induced current.
4. The nucleic acid of claim 1 , wherein said increased cAMP sensitivity comprises a K 1/2 at least ten-fold lower than that observed for a nucleic acid encoding a wild type channel.
5. The nucleic acid of claim 1 , wherein said decreased cGMP sensitivity comprises a K 1/2 at least ten-fold higher than that observed for a nucleic acid encoding a wild type channel.
6. The nucleic acid of claim 1 , wherein said mutation further comprises a substitution at a residue corresponding to position 460 of SEQ ID NO:8.
7. The nucleic acid of claim 6 , 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.
8. The nucleic acid of claim 6 , wherein said mutation further comprises a 61–90 deletion at residues corresponding to positions 61–90 of SEQ ID NO:8.
9. An isolated nucleic acid encoding a modified rat olfactory cyclic nucleotide-gated ion channel alpha subunit, 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 isolated nucleic acid 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 isolated nucleic acid 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 isolated nucleic acid of claim 9 , 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. An expression vector comprising the nucleic acid of claim 9 .
14. The expression vector of claim 13 , wherein said vector is a recombinant adenovirus vector.
15. An isolated host cell comprising the expression vector of claim 13 , wherein said host cell is selected from the group consisting of a prokaryotic cell and a eukaryotic cell.
16. The host cell of claim 15 , wherein said eukaryotic cell is selected from the group consisting of a human embryonic kidney-293 cell and a rat GH4C1 pituitary cell.
17. An isolated polypeptide of 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.