Excised patches of plasma membrane from vertebrate rod outer segments retain a functional phototransduction enzymatic cascade.
Ion channels in excised patches of plasma membrane are generally considered to be isolated from any intracellular regulation mechanisms. For example, in excised patches of vertebrate rod outer segment plasma membrane, the cGMP-activated cation channels have traditionally been studied in room light because the enzyme cascade linking photon absorption to channel closure was assumed to be inoperative. To investigate the possibility that, in fact, such excised patches retain a functional phototransduction enzymatic cascade, this same preparation was studied in darkness. Patches excised in the dark were found to retain the light sensitivity of their cGMP-induced conductance and the ability to synthesize cGMP. In the presence of guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]), a nonhydrolyzable GTP analog, light suppresses the cGMP-induced conductance irreversibly. Furthermore, inhibitors of phosphodiesterase activity reduce light sensitivity, whereas activated phosphodiesterase or activated transducin does not directly affect the channels. These results (i) establish that excised patches from rod outer segment retain functional phototransduction enzymes, (ii) support the classical view that channel opening is modulated by phosphodiesterase-mediated cGMP hydrolysis, and, most surprisingly, (iii) demonstrate that diffusion in excised patches is so restricted that local enzymes can induce variations in the concentration of small molecules. The indication that excised patches are not as simple as usually surmised opens the possibility of using them to study other intracellular transduction mechanisms.
Department of Physiology and Biophysics, School of Medicine, University of Washington, Seattle 98195.
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D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000502:physiology; D000818:Animals; D002462:Cell Membrane / Q000502:physiology; D006152:Cyclic GMP / Q000502:physiology; D004553:Electric Conductivity; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology / Q000502:physiology; D006162:Guanylate Cyclase / Q000502:physiology; D066298:In Vitro Techniques; D008027:Light; D008116:Lizards; D010786:Photoreceptor Cells / Q000502:physiology*; D015398:Signal Transduction; D013873:Thionucleotides / Q000494:pharmacology; D014785:Vision, Ocular
D013873:Thionucleotides; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D006162:Guanylate Cyclase; D006152:Cyclic GMP; D015056:1-Methyl-3-isobutylxanthine