Calcium is an important messenger in the outer segment of vertebrate photoreceptors. In darkness, it enters through light-dependent channels at a rapid rate, but it is equally rapidly removed, so that its concentration is regulated to within a narrow range. Exposure to light causes a transient decrease in calcium that serves as an important signal during light adaptation. Calcium may also be critically important in retinal degeneration. Increasing evidence indicates that a rise or fall of outer segment calcium, if sufficiently large and prolonged, can trigger programmed cell death. The goal of this application is to understand how calcium is regulated in the photoreceptor, and to determine the relationship between calcium concentration and the rate of photoreceptor degeneration in animal models of retinal dystrophy. We shall study movements of calcium between rod outer segment disks and cytoplasm, using two fluorescent dyes to label each compartment independently. These measurements should enable us to estimate the rate of calcium exchange, to show whether release or uptake of calcium from disks is sufficient to affect the cytoplasmic concentration. We shall in addition use fluorescent dyes and suction-electrode recording on rods in animals with targeted modifications or deletions of outer segment proteins to address the following questions: Is the continuous activation of transduction by opsin and a concomitant decrease in calcium the cause of degeneration during prolonged vitamin A deprivation? Why do some mutations of outer segment proteins cause benign stationary night blindness, but others the death of photoreceptors and blindness? How large a change in calcium is required to trigger photoreceptor death? Can the death of the rods and cones be rescued by manipulating the calcium level? Does an increase in outer segment calcium kill the photoreceptor by diffusing into the inner segment, and in particular into the mitochondria? It is our hope that our experiments will provide a more complete understanding of the role of calcium in photoreceptor homeostasis and disease.
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