title: Mutation of a single residue in the S2-S3 loop of CNG channels alters the gating properties and sensitivity to inhibitors.
abstract: We previously found that native cyclic nucleotide-gated (CNG) cation channels from amphibian rod cells are directly and reversibly inhibited by analogues of diacylglycerol (DAG), but little is known about the mechanism of this inhibition. We recently determined that, at saturating cGMP concentrations, DAG completely inhibits cloned bovine rod (Brod) CNG channels while only partially inhibiting cloned rat olfactory (Rolf) channels (Crary, J.I., D.M. Dean, W. Nguitragool, P.T. Kurshan, and A.L. Zimmerman. 2000. J. Gen. Phys. 116:755-768; in this issue). Here, we report that a point mutation at position 204 in the S2-S3 loop of Rolf and a mouse CNG channel (Molf) found in olfactory epithelium and heart, increased DAG sensitivity to that of the Brod channel. Mutation of this residue from the wild-type glycine to a glutamate (Molf G204E) or aspartate (Molf G204D) gave dramatic increases in DAG sensitivity without changing the apparent cGMP or cAMP affinities or efficacies. However, unlike the wild-type olfactory channels, these mutants demonstrated voltage-dependent gating with obvious activation and deactivation kinetics. Interestingly, the mutants were also more sensitive to inhibition by the local anesthetic, tetracaine. Replacement of the position 204 glycine with a tryptophan residue (Rolf G204W) not only gave voltage-dependent gating and an increased sensitivity to DAG and tetracaine, but also showed reduced apparent agonist affinity and cAMP efficacy. Sequence comparisons show that the glycine at position 204 in the S2-S3 loop is highly conserved, and our findings indicate that its alteration can have critical consequences for channel gating and inhibition.
authors: Department of Molecular Pharmacology, Physiology and Biotechnology, Brown University, Providence, Rhode Island 02912, USA.
authors: J I
authors: JI
authors: Crary
authors: D M
authors: DM
authors: Dean
authors: F
authors: F
authors: Maroof
authors: A L
authors: AL
authors: Zimmerman
mesh_terms: D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D002417:Cattle; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000494:pharmacology; D054815:Cyclic Nucleotide-Gated Cation Channels; D004075:Diglycerides / Q000494:pharmacology; D004351:Drug Resistance / Q000235:genetics; D004594:Electrophysiology; D015640:Ion Channel Gating / Q000502:physiology*; D007473:Ion Channels / Q000819:agonists / Q000037:antagonists & inhibitors* / Q000235:genetics*; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation / Q000502:physiology*; D009865:Oocytes; D051381:Rats; D012016:Reference Values; D013748:Tetracaine / Q000494:pharmacology; D013997:Time Factors; D014982:Xenopus laevis
chemical_list: D054815:Cyclic Nucleotide-Gated Cation Channels; D004075:Diglycerides; D007473:Ion Channels; D013748:Tetracaine; D000242:Cyclic AMP