Diacylglycerol analogs inhibit the rod cGMP-gated channel by a phosphorylation-independent mechanism.
The electrical response to light in retinal rods is mediated by cyclic nucleotide-gated, nonselective cation channels in the outer segment plasma membrane. Although cGMP appears to be the primary light-regulated 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 nucleoside triphosphates, indicating that a phosphorylation reaction was not involved. DAG was more effective at low than at high [cGMP]: with 50 microM 8-Br-cGMP, the DAG analog 1,2-dioctanoyl-sn-glycerol (1,2-DiC8) reduced the current with an IC50 of approximately 22 microM (Hill coefficient, 0.8), whereas with 1.2 microM 8-Br-cGMP, only approximately 1 microM 1,2-DiC8 was required to halve the current. DAG reduced the apparent affinity of the channels for cGMP: 4 microM 1,2-DiC8 produced a threefold increase in the K1/2 for channel activation by 8-Br-cGMP, as well as a threefold reduction in the maximum current, without changing the apparent stoichiometry or cooperativity of cGMP binding. Inhibition by 1,2-DiC8 was not relieved by supersaturating concentrations of 8-Br-cGMP, suggesting 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-glycerol (1,3-DiC8)--suppressed the current with the same potency as 1,2-DiC8, whereas an ethylene glycol of identical chain length (DiC8-EG) was much less effective. Our results suggest that DAG allosterically interferes with channel opening, and raise the question of whether DAG is involved in visual transduction.
Department of Physiology, Brown University, Providence, Rhode Island 02912, USA.
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Gordon
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Downing-Park
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Tam
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Zimmerman
D000557:Ambystoma; D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D006152:Cyclic GMP / Q000378:metabolism; D054815:Cyclic Nucleotide-Gated Cation Channels; D004075:Diglycerides / Q000737:chemistry / Q000494:pharmacology*; D004594:Electrophysiology; D066298:In Vitro Techniques; D015640:Ion Channel Gating / Q000187:drug effects; D007473:Ion Channels / Q000737:chemistry / Q000187:drug effects* / Q000528:radiation effects; D007700:Kinetics; D008027:Light; D010766:Phosphorylation; D011894:Rana pipiens; D012374:Rod Cell Outer Segment / Q000737:chemistry / Q000187:drug effects* / Q000528:radiation effects; D015290:Second Messenger Systems
D054815:Cyclic Nucleotide-Gated Cation Channels; D004075:Diglycerides; D007473:Ion Channels; C044295:1,2-dioctanoylglycerol; D006152:Cyclic GMP