INTERACTION OF CALMODULIN WITH THE CYCLIC GMP-GATED CHANNEL OF ROD PHOTORECEPTOR CELLS - MODULATION OF ACTIVITY, AFFINITY PURIFICATION, ANDLOCALIZATION

The cGMP-gated cation channel of rod photoreceptor cells plays a centr al role in the phototransduction process by controlling the influx of cations into the rod outer segment in response to changes in cGMP leve ls. Previous studies have shown that the cGMP-gated channel in native rod outer segment membrane vesicles is modulated by calmodulin in a ca lcium-dependent manner. In this study we report that the immunoaffinit y-purified channel consisting of the 63-kDa alpha-subunit and a 240-kD a protein is also modulated by calmodulin when reconstituted into lipi d vesicles. In the absence of calmodulin, the purified channel had an apparent k(m) of 33 mu M and a Hill coefficient of 33 for cGMP-depende nt efflux of Ca2+ from reconstituted lipid vesicles. In the presence o f calmodulin, the K-m increased to 44 mu M without affecting the Hill coefficient or maximum velocity of ion efflux. CalModulin modulation o f the channel is inhibited by the calmodulin antagonist, mastoparan. I n the absence of mastoparan, the half-maximum inhibition of channel ac tivity (IC50) occurred at 1.85 +/- 0.25 nM calmodulin at a cGMP concen tration of 12.5 mu M in the presence of mastoparan, the IC50 value inc reased to 20.3 +/- 3.8 mu M calmodulin. Based on the strong, selective interaction of calmodulin with the channel, an efficient, general met hod has been developed to isolate functionally active cGMP-gated chann els from mammalian and amphibian photoreceptor membranes. Calmodulin e xtraction studies, Western blotting, and channel activity measurements indicate that endogenous rod outer segment calmodulin modulates the a ctivity of the channel through its binding to the 240-kDa protein. Fro m these studies we conclude that the 240-kDa protein of the cGMP-gated channel is a major calmodulin target protein of rod outer segment mem branes.