Gating by cyclic GMP and voltage in the a subunit of the cyclic GMP-gated channel from rod photoreceptors

Gating by cGMP and voltage of the alpha subunit of the cGMP-gated channel f rom rod photoreceptor was examined with a patch-clamp technique. The channe ls were expressed in Xenopus oocytes. At low [cGMP] (<20 mu M), the current displayed strong outward rectification. At low and high (700 mu M) [cGMP], the channel activity was dominated by only one conductance level. Therefor e, the outward rectification at low [cGMP] results solely from an increase in the open probability, P-o. Kinetic analysis of single-channel openings r evealed two exponential distributions. At low [cGMP], the larger P-o at pos itive voltages with respect to negative voltages is caused by an increased frequency of openings in both components of the open-time distribution. In macroscopic currents, ;depolarizing voltage steps, starting from -100 mV, g enerated a time-dependent current that increased with the step size (activa tion). At low [cGMP] (20 mu M), the degree of activation was large and the time course was slow, whereas at saturating [cGMP] (7 mM) the respective ch anges were small and fast. The dose-response relation at -100 mV was shifte d to the right and saturated at significantly lower P-o values with respect to that at +100 mV (0.77 vs. 0.90). P-o was determined as function of the [cGMP] (at +100 and -100 mV) and voltage (at 20, 70, and 700 mu M, and 7 mM cGMP). Both relations could be fitted with an allosteric state model consi sting of four independent cGMP-binding reactions cold one voltage-dependent allosteric opening reaction. At saturating [cGMP] (7 mM), the activation t ime course was monoexponential, which allowed us to deter-mine the individu al rate constants for the allosteric reaction. For the rapid rate constants of cGMP binding and unbinding, lower limits are determined. It is conclude d that an allosteric model consisting of four independent cGMP-binding: rea ctions and one voltage-dependent allosteric reaction, describes the cGMP- a nd voltage-dependent gating of cGMP-gated channels adequately.