Light-induced currents in Xenopus oocytes expressing bovine rhodopsin.
1. We have investigated the functioning of bovine rod opsin, which is efficiently synthesized from RNA made by in vitro transcription, following injection into Xenopus oocytes. We found that oocytes expressing the gene for opsin exhibit light-dependent ionic currents only after pigment generation by incubation with 11-cis-retinal. These currents are similar to the endogenous muscarinic acetylcholine (ACh) response of oocytes, but their amplitude is substantially smaller. 2. In order to optimize the conditions for obtaining light-induced currents in RNA-injected oocytes, the native ACh response was examined under several conditions. It was found that elevated external calcium markedly enhances the muscarinic response and that these currents have a non-linear dependence on membrane voltage, increasing substantially with depolarization. 3. Using the optimal conditions for evoking the largest ACh responses, (28 mM [Ca2+]o, 0 mV, omission of serum and Hepes from the media), the light-evoked currents obtained in RNA-injected oocytes were remarkably enhanced, and responses to multiple light stimuli could be obtained. 4. The light response appeared to desensitize, even after long periods of recovery and pigment regeneration. By contrast, the ACh responses continued to appear normal. These results suggest that desensitization of photoresponses expressed in Xenopus oocytes involve changes at early stages of the pathway, resulting in a reduced ability of rhodopsin to couple to the endogenous signalling system.
Knox
B E
BE
Department of Chemistry, Massachusetts Institute of Technology, Cambridge 02139.
Khorana
H G
HG
Nasi
E
E
D000109:Acetylcholine / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D005260:Female; D007473:Ion Channels / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects*; D008027:Light; D008564:Membrane Potentials; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects*; D012243:Rhodopsin / Q000096:biosynthesis / Q000235:genetics / Q000528:radiation effects*; D015398:Signal Transduction; D014158:Transcription, Genetic; D014981:Xenopus
D007473:Ion Channels; D012243:Rhodopsin; D000109:Acetylcholine; D002118:Calcium