MODULATION OF THE CGMP-GATED ION-CHANNEL IN FROG RODS BY CALMODULIN AND AN ENDOGENOUS INHIBITORY FACTOR

1. Outer segment patches excised in the light were used to investigate the effects of exogenous calmodulin and an endogenous inhibitory fact or on the cGMP-gated channel of frog rods. 2. Calmodulin shifted to th e right the dose-response relation for activation of the channels by 8 -Br-cGMP, but did not change the maximum current or the form of the re lation. Reversal of this effect by removal of calmodulin was accelerat ed by brief exposure to saturating [8-Br-cGMP]. Inhibition by calmodul in required calcium and gave as much as a 5-fold decrease in current f or an [8-Br-cGMP] functionally comparable to the presumed physiologica l [cGMP]. 3. Exposure to low [Ca2+](i) (tens of nanomolar) appeared to irreversibly remove or inactivate an endogenous channel inhibitory fa ctor from the patches, increasing the current at low [8-Br-cGMP]. Like calmodulin, this factor slowed the voltage-dependent channel-gating k inetics and did not change the maximum current. However, unlike calmod ulin, the endogenous factor remained stably associated with the patche s at high [Ca2+](i) (1 mu M), even with exposure to saturating [8-Br-c GMP]. 4. After the low-Ca2+ treatment increased the current, calmoduli n reduced the current to about the same level as it had before the low -Ca2+ treatment, giving a larger fractional suppression. Furthermore, patches with high initial. sensitivity to 8-Br-cGMP had small low-Ca2 effects and large calmodulin effects, while the reverse was true for patches with low initial agonist sensitivity. 5. Application of trypsi n to the intracellular surface of the patch prevented the responses to calmodulin and to low [Ca2+](i), suggesting involvement of a cytoplas mic portion of the channel. However, trypsin also reduced the total ag onist-induced patch current. 6. Our results are consistent with a mode l in which calmodulin and an endogenous calcium-binding protein compet e for the same site, inhibiting channel opening or cGMP binding. The t ight association of the endogenous factor with the channel even at rel atively low [Ca2+](i) suggests that in the transducing; rod it may inh ibit the channels most of the time in darkness and in dim light, preve nting any potential inhibitory effects of calmodulin. The endogenous f actor would be expected to leave the channel only in bright or prolong ed light,when the [Ca2+](i) is thought to be very low.