Intracellular biochemical manipulation of phototransduction in detached rod outer segments.
Recent progress in understanding phototransduction has come primarily from studies on cell-free systems. To investigate the transduction process under physiological conditions, a fully functional preparation of retinal rod outer segments without attached inner segments was developed that allows electrical recording of light-sensitive current during intracellular dialysis with defined solutions. No light-sensitive current is recorded from detached outer segments dialyzed with nucleotide-free solutions, whereas cells detached from the retina into Ringer's solution containing 3-isobutyl-1-methyl-xanthine (a phosphodiesterase inhibitor) develop a light-sensitive inward dark current. This indicates that there is a basal level of cGMP-specific phosphodiesterase activity in the dark. Detached outer segments dialyzed with greater than or equal to 20 microM cGMP rapidly develop a light-suppressible current. A current of similar magnitude is generated more slowly during dialysis with a 50-fold greater concentration of GTP. Apparently, cGMP can be synthesized from GTP by guanylate cyclase in the outer segment. Cells dialyzed with cGMP alone show a reduced light sensitivity that is restored to normal by addition of 20 microM GTP. This action of GTP is antagonized by guanosine 5'-[beta-thio]diphosphate. These findings are in good agreement with biochemical evidence indicating that a GTP-binding protein (transducin) plays a pivotal role in the generation of responses to light. The recovery of photocurrent following a brief flash is delayed or abolished by dialysis with solutions that lack ATP or contain guanosine 5'-[gamma-thio]triphosphate, a nonhydrolyzable GTP analog. These results support the view that both GTP hydrolysis by activated transducin and ATP-dependent phosphorylation of a rhodopsin photoproduct are necessary for termination of the transduction process.
Sather
W A
WA
University of Washington, Department of Physiology and Biophysics, Seattle, WA 98195.
Detwiler
P B
PB
D000255:Adenosine Triphosphate / Q000502:physiology; D000818:Animals; D002118:Calcium / Q000502:physiology; D006152:Cyclic GMP / Q000502:physiology; D004553:Electric Conductivity; D006150:Guanine Nucleotides / Q000502:physiology; D006160:Guanosine Triphosphate / Q000502:physiology; D066298:In Vitro Techniques; D007700:Kinetics; D008027:Light; D008116:Lizards; D008565:Membrane Proteins / Q000502:physiology*; D010786:Photoreceptor Cells / Q000502:physiology*; D012168:Retinal Pigments / Q000502:physiology*; D012243:Rhodopsin / Q000502:physiology*; D012374:Rod Cell Outer Segment / Q000502:physiology*; D015293:Transducin; D014785:Vision, Ocular
D006150:Guanine Nucleotides; D008565:Membrane Proteins; D012168:Retinal Pigments; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D012243:Rhodopsin; D015293:Transducin; D006152:Cyclic GMP; D002118:Calcium