This project will focus on mechanisms which regulate the sensitivity of vertebrate retinal rods and cones. Electrophysiological recordings of membrane current, microspectrophotometric measurements of visual pigment and fluorometric determinations of cytosolic calcium ([Ca2+]i) will be obtained from solitary rods and cones in which retinal and its analogs have been incorporated in darkness and following bright light (bleaching) to determine how cytosolic messenger substances are regulated during dark-adaptation. Three general questions will be addressed: (1) what the nature of the opsin-retinoid interaction is during dark- and bleaching-adaptation: the minimum structural interactions of opsin and retinal required for recovery of sensitivity during dark-adaptation will be determined; PDE and guanylyl cyclase velocities will be estimated in cells which have been bleached and treated with 11-cis retinal and analogs; and 13-demethyl retinal and 10-demethyl retinal will be incorporated into bleached rods and cones to test the importance of torsion in the C-11:C-12 bond in retinal during dark-adaptation; (2) what the involvement of [Ca2+]i is in light adaptation and pigment regeneration: changes in cytosolic calcium and membrane current will be measured in solitary dark-adapted and bleach-adapted rods and cones in the presence and absence of retinal and its analogs in order to determine the relationship between recovery of sensitivity during dark-adaptation and levels of [Ca2+]i; 9-demethyl retinal will be incorporated into bleached photoreceptors in order to determine whether an early phase of adaptation is due to rhodopsin turn-off since 9-demethyl rhodopsin is thought to exhibit an abnormally prolonged activated lifetime; whether or not this prolonged lifetime is modified by changes in [Ca2+]i will be determined; and (3) whether all-trans retinal occupancy of the opsin binding site is required for opsin adaptation. This latter question will be addressed by measuring sensitivity and estimating PDE and cyclase velocities in bleached rods and cones in the presence and absence of agents which are known to leach all-trans retinal/retinol from the intact photoreceptors.
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