ANTAGONISTS OF CYCLIC NUCLEOTIDE-GATED CHANNELS AND MOLECULAR MAPPINGOF THEIR SITE OF ACTION

Activation of photoreceptor and olfactory cyclic nucleotide-gated (CNG ) channels involves distinct ligand-binding and channel-gating reactio ns. To dissociate binding from gating, we identified the first competi tive antagonists of CNG channels: specific phosphorothioate derivative s of cAMP and cGMP. We also identified membrane-permeant forms of thes e molecules that are antagonists and that will be useful for elucidati ng physiological roles for CNG channels in intact cells. The photorece ptor and olfactory CNG channels determine which of the phosphorothioat e derivatives are agonists and which are antagonists based on differen t structural features of the ligand. The photoreceptor channel uses th e nature of the purine ring (adenine vs guanine), whereas the olfactor y channel uses the isomeric position of the thiophosphate S atom (Rp v s Sp). Interestingly, the same ligand, Rp-cGMPS, has opposite effects on the two channels, activating the photoreceptor channel and antagoni zing the olfactory channel. Because Rp-cGMPS binds to both channels bu t activates only one, the channels must differ in a protein region tha t couples binding to gating. Chimeric photoreceptor and olfactory CNG channels reveal that the cytoplasmic C-terminal domain determines whet her bound ligand activates the channel successfully. Hence, the C term inus contains not only the cyclic nucleotide-binding site, but also a region that couples ligand binding to channel gating.