Cyclic GMP-gated channels of bovine rod photoreceptors: Affinity, density and stoichiometry of Ca2+-calmodulin binding sites

1. Ca2+-loaded vesicles of bovine rod outer segment (ROS) membranes were used to examine the influence of Ca2+-calmodulin (Ca2+-CaM) on the activity of cGMP-gated channels. 2. In vesicles prepared from ROS membranes which were washed at zero free Ca2+, Ca2+-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 mu M in the CaM-free state, and of 55.6 +/- 5.2 mu M in the Ca2+-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 mu M Ca2+, the dose-response curve was identical to the Ca2+-CaM-bound state. 4. From the slope of the linear regression of EC(50)(CaM) plotted vs. the rhodopsin concentration, the molar ratio of rhodopsin to externally accessible Ca2+-CaM binding sites of fused ROS membranes was determined to be 1439 +/- 109. Therefore, there are about 720 molecules of rhodopsin per Ca2+-CaM binding site present in ROS. 5. From the slope of the linear regression of EC(50)(CaM) plotted vs. the rhodopsin concentration, the molar ratio of rhodopsin to externally accessible Ca2+-CaM binding sites of fused ROS membranes was determined to be 1439 +/- 109. Therefore, there are about 720 molecules of rhodopsin per Ca2+-CaM binding site present in ROS. 6. Based on these data, a density of 560 Ca2+-CaM binding sites mu m(-2) is estimated for the plasma membrane of bovine ROS, suggesting that there are two Ca2+-CaM binding sites per channel. 7. The Ca2+-CaM effect did not become noticeable until the ROS membranes were hypotonically washed at free [Ca2+] below 100 nM, suggesting that an endogenous Ca2+-binding protein was washed off in the absence of Ca2+. 8. If the endogenous Ca2+-binding protein of bovine ROS membranes was washed off at zero Ca2+ and then Ca2+-CaM added, Ca2+-CaM could only be washed off again at free [Ca2+] below 100 nM. 9. These findings strongly suggest that the endogenous Ca2+-binding protein of the bovine cGMP-gated channel is CaM.