Interaction of calmodulin with the cyclic GMP-gated channel of rod photoreceptor cells. Modulation of activity, affinity purification, and localization.

The cGMP-gated cation channel of rod photoreceptor cells plays a central role in the phototransduction process by controlling the influx of cations into the rod outer segment in response to changes in cGMP levels. Previous studies have shown that the cGMP-gated channel in native rod outer segment membrane vesicles is modulated by calmodulin in a calcium-dependent manner. In this study we report that the immunoaffinity-purified channel consisting of the 63-kDa alpha-subunit and a 240-kDa protein is also modulated by calmodulin when reconstituted into lipid vesicles. In the absence of calmodulin, the purified channel had an apparent Km of 33 microM and a Hill coefficient of 3.3 for cGMP-dependent efflux of Ca2+ from reconstituted lipid vesicles. In the presence of calmodulin, the Km increased to 44 microM without affecting the Hill coefficient or maximum velocity of ion efflux. Calmodulin modulation of the channel is inhibited by the calmodulin antagonist, mastoparan. In the absence of mastoparan, the half-maximum inhibition of channel activity (IC50) occurred at 1.85 +/- 0.25 nM calmodulin at a cGMP concentration of 12.5 microM; in the presence of mastoparan, the IC50 value increased to 20.3 +/- 3.8 nM calmodulin. Based on the strong, selective interaction of calmodulin with the channel, an efficient, general method has been developed to isolate functionally active cGMP-gated channels from mammalian and amphibian photoreceptor membranes. Calmodulin extraction studies, Western blotting, and channel activity measurements indicate that endogenous rod outer segment calmodulin modulates the activity of the channel through its binding to the 240-kDa protein. From these studies we conclude that the 240-kDa protein of the cGMP-gated channel is a major calmodulin target protein of rod outer segment membranes.