The rod cells of vertebrate retinas are responsible for vision at low levels of light. The visual pigment, rhodopsin, functions in the primary event in visual transduction. Absorption of light by rhodopsin leads to a hyperpolarization of the plasma membrane of the rod outer segment. Rhodopsin is found in the disk membranes of the rod outer segment. These disks are stacked and appear to differ in cholesterol content, depending upon their location in the stack. This may be related to aging of the disks. This proposal will separates disks based upon their cholesterol content and document the differences in protein and lipid composition of the separated fractions. The consequences of these compositional differences to disk membrane function will then be studied in disk membranes whose composition has been appropriately altered, mimic the above mentioned compositional differences, or in membranes in which rhodopsin has been reconstituted and whose composition has been determined by the reconstitution. The effects of cholesterol and phospholipid compositional differences on activation of PDEase, photolysis of rhodopsin and regenerability will be determined. Furthermore, since disks lipids participate in fusion events, preferentially at the end of the stack near the inner segment, the influence of the compositional differences on the ability to promote membrane fusion will be examined. And since the disk membrane lipids have been shown to be inherently unstable in the normal lamellar configuration required for disk membrane integrity (which may play a role in retinal degeneration), the effect of the compositional differences on the bilayer stability of the disk lipids will be determined. Finally, because cholesterol content is already known to be one of the compositional variables, the behavior of cholesterol in disk membranes under the influence of the defined compositional differences will be examined. This project will continue to examine an interesting co- reconstitution of a calcium pump protein with rhodopsin to delineate rhodopsin function under the influence of a naturally produced calcium gradient such as is expected in the disk membrane.