Recoverin and rhodopsin kinase activity in detergent-resistant membrane rafts from rod outer segments

Cholesterol-rich membranes or detergent-resistant membranes (DRMs) have recently been isolated from bovine rod outer segments and were shown to contain several signaling proteins such as, for example, transducin and its effector, cGMP-phosphodiesterase PDE6. Here we report the presence of rhodopsin kinase and recoverin in DRMs that were isolated in either light or dark conditions at high and low Ca(2+) concentrations. Inhibition of rhodopsin kinase activity by recoverin was more effective in DRMs than in the initial rod outer segment membranes. Furthermore, the Ca(2+) sensitivity of rhodopsin kinase inhibition in DRMs was shifted to lower free Ca(2+) concentration in comparison with the initial rod outer segment membranes (IC(50) = 0.76 muM in DRMs and 1.91 muM in rod outer segments). We relate this effect to the high cholesterol content of DRMs because manipulating the cholesterol content of rod outer segment membranes by methyl-beta-cyclodextrin yielded a similar shift of the Ca(2+)-dependent dose-response curve of rhodopsin kinase inhibition. Furthermore, a high cholesterol content in the membranes also increased the ratio of the membrane-bound form of recoverin to its cytoplasmic free form. These data suggest that the Ca(2+)-dependent feedback loop that involves recoverin is spatially heterogeneous in the rod cell.