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.