DIACYLGLYCEROL ANALOGS INHIBIT THE ROD CGMP-GATED CHANNEL BY AT PHOSPHORYLATION-INDEPENDENT MECHANISM

The electrical response to light in retinal rods is mediated by cyclic nucleotide-gated, nonselective cation channels in the outer segment p lasma membrane. Although cGMP appears to be the primary light-regulate d second messenger, cellular levels of other substances, including Ca2 + and phosphatidylinositol-4,5-bisphosphate, are also sensitive to the level of illumination. We now show that diacylglycerol (DAG) analogs reversibly suppress the cGMP-activated conductance in excised patches from frog rod outer segments. This suppression did not require nucleos ide triphosphates, indicating that a phosphorylation reaction was not involved. DAG was more effective at low than at high [cGMP]: with 50 m u M 8-Br-cGMP, the DAG analog 1,2-dioctanoyl-sn-glycerol (1,2-DiC8) re duced the current with an IC50 of similar to 22 mu M (Hill coefficient , 0.8), whereas with 1.2 mu M 8-Br-cGMP, only similar to 1 mu M 1,2-Di C8 was required to halve the current. DAG reduced the apparent affinit y of the channels for cGMP: 4 mu M 1,2-DiC8 produced a threefold incre ase in the K-1/2 for channel activation by 8-Br-cGMP, as well as a thr eefold reduction in the maximum current, without changing the apparent stoichiometry or cooperativity of cGMP binding. Inhibition by 1,2-DiC 8 was not relieved by supersaturating concentrations of 8-Br-cGMP, sug gesting that DAG did not act by competitive inhibition of cGMP binding , Furthermore, DAG did not seem to significantly reduce single-channel conductance. A DAG analog similar to 1,2-DiC8-1,3-dioctanoyl-sn-glyce rol (1,3-DiC8)-suppressed the current with the same potency as 1,2-DiC 8, whereas an ethylene glycol of identical chain length (DiC8-EG) was much less effective. Our results suggest that DAG allosterically inter feres with channel opening, and raise the question of whether DAG is i nvolved in visual transduction.