Cyclic GMP‐gated channels of bovine rod photoreceptors: affinity, density and stoichiometry of Ca(2+)‐calmodulin binding sites.

1. Ca(2+)‐loaded vesicles of bovine rod outer segment (ROS) membranes were used to examine the influence of Ca(2+)‐calmodulin (Ca(2+)‐CaM) on the activity of cGMP‐gated channels. 2. In vesicles prepared from ROS membranes which were washed at zero free Ca2+, Ca(2+)‐CaM reduced the Ca2+ flux to maximally 40%. The dose‐response curve for activation of the cGMP‐gated channel had a half‐maximal value of 36.8 +/‐ 2 microM in the CaM‐free state, and of 55.6 +/‐ 5.2 microM in the Ca(2+)‐CaM‐bound state. In both cases the Hill coefficients were 2.2 +/‐ 0.2. 3. In vesicles prepared from ROS membranes which were washed at 100 microM Ca2+, the dose‐response curve was identical to the Ca(2+)‐CaM‐bound state. 4. Titration of the Ca(2+)‐CaM‐dependent decrease of the channel activity upon addition of 40 microM cGMP yielded half‐maximal Ca(2+)‐CaM concentrations (EC50CaM) which were linearly correlated with the concentration of membrane vesicles. Extrapolation of EC50CaM to infinite dilution of vesicles yielded a Ca(2+)‐CaM affinity constant for the cGMP‐gated channel of 1.01 +/‐ 0.20 nM. Hill analysis of the Ca(2+)‐CaM titrations resulted in a Hill coefficient of 1.36 +/‐ 0.15. 5. From the slope of the linear regression of EC50CaM plotted vs. the rhodopsin concentration, the molar ratio of rhodopsin to externally accessible Ca(2+)‐CaM binding sites of fused ROS membranes was determined to be 1439 +/‐ 109. Therefore, there are about 720 molecules of rhodopsin per Ca(2+)‐CaM binding site present in ROS. 6. Based on these data, a density of 560 Ca(2+)‐CaM binding sites micron‐2 is estimated for the plasma membrane of bovine ROS, suggesting that there are two Ca(2+)‐CaM binding sites per channel. 7. The Ca(2+)‐CaM effect did not become noticeable until the ROS membranes were hypotonically washed at free [Ca2+] below 100 nM, suggesting that an endogenous Ca(2+)‐binding protein was washed off in the absence of Ca2+. 8. If the endogenous Ca(2+)‐binding protein of bovine ROS membranes was washed off at zero Ca2+ and then Ca(2+)‐CaM added, Ca(2+)‐CaM could only be washed off again at free [Ca2+] below 100 nM. 9. These findings strongly suggest that the endogenous Ca(2+)‐binding protein of the bovine cGMP‐gated channel is CaM.