CHARACTERIZATION OF GUANYLATE-CYCLASE ACTIVITY IN SINGLE RETINAL ROD OUTER SEGMENTS

cGMP mediates vertebrate phototransduction by directly gating cationic channels on the plasma membrane of the photoreceptor outer segment. This second messenger is produced by a guanylate cyclase and hydrolyzed by a light-activated cGMP-phosphodiesterase. Both of these enzyme activities are Ca2+ sensitive, the guanylate cyclase activity being inhibited and the light-activated phosphodiesterase being enhanced by Ca2+. Changes in these activities due to a light-induced decrease in intracellular Ca2+ are involved in the adaptation of photoreceptors to background light. Mie describe here experiments to characterize the guanylate cyclase activity and its modulation by Ca2+ using a truncated rod outer segment preparation, in order to evaluate the enzyme's role in light adaptation. The outer segment of a tiger salamander rod was drawn into a suction pipette to allow recording of membrane current, and the remainder of the cell was sheared off with a probe to allow internal dialysis. The cGMP-gated channels on the surface membrane were used to monitor conversion of GTP, supplied from the bath, into cGMP by the guanylate cyclase in the outer segment. At nominal 0 Ca2+, the cyclase activity had a K-m, of 250 mu M MgGTP and a V-max of 25 mu M cGMP s(-1) in the presence of 1.6 mM free Mg2+ in the presence of 0.5 mM free Mg2+, the K-m was 310 mu M MgGTP and the V-max was 17 mu M cGMP s(-1). The stimulation by Mg2+ had an EC(50) of 0.2 mM Mg2+ for MgGTP at 0.5 mM. Ca2+ inhibited the cyclase activity. In a K+ intracellular solution, with 0.5 mM free Mg2+ and 2.0 mM GTP, the cyclase activity was 13 mu M cGMP s(-1) at nominal 0 Ca2+; Ca2+ decreased this activity with a IC50 Of similar to 90 nM and a Hill coefficient of similar to 2.0.