We will investigate the mechanisms that regulate recovery of sensitivity and responsiveness of cones and rods during and following exposure to bright light which allow these cells to respond to light over an immense intensity range. To do this we will make electrophysiological recordings of receptor current together with microspectrophotometric measurements of visual pigment and microfluorometric measurements of retinal to retinol reduction in solitary rods and cones. Measurements will be made in darkness before and following bleaching and following exogenous incorporation of retinal and its analogs. The following hypotheses will be tested: (1) Critical non-covalent interactions between prosthetic groups on retinal and opsin in different cone types regulate the decay of light-activated cone opsin (Meta II) by different mechanisms. These mechanisms assist to affect the rapid recovery of dark current and flash responses that occur during and following bright light exposure. We shall correlate effects on Meta II decay, retinal production, and receptor response recovery following bleaching and subsequent incorporation of a variety of native and analog retinoids into the visual pigment of intact cells to determine specific sites of interaction that on retinal that regulate response recovery. (2) Non-covalent occupancy of the cone binding site during pigment regeneration assists in the very rapid recovery of sensitivity that occurs during dark-adaptation. Such occupancy has the opposite effects in rods. We shall determine the time course of sensitivity recovery relative to that of pigment regeneration in cones and rods that have been exposed to retinoids that either simply occupy the opsin binding site or bind to opsin and subsequently form a visual pigment. We will use different retinoid analogs to determine the essential features that a retinoid must have to accelerate/impede sensitivity recovery during dark adaptation in these cells. (3) The reduction of all-trans retinal to all-trans retinol by NADPH, and subsequent retinol release from opsin limit the regeneration of visual pigment and recovery of visual sensitivity in vertebrate rods. We shall measure the recovery of sensitivity locally within outer segments of bleached rods before and following dialysis with solutions containing NADPH or its metabolic precursors (glucose-6 phosphate, ATP, etc.) to determine whether the reduction of retinal to retinol must occur to allow recovery of dark current and flash sensitivity, and to see if this response recovery can be limited by the supply of NADPH in the OS. Lay Summary: Recovery of sensitivity and responsiveness of rods and cones during and following bright light exposure is important for our ability to sense changes in our light environment. We propose physiological and biochemical experiments designed to increase our understanding of the cellular mechanisms that underlie this ability. Our ultimate goal is to understand retinal function under normal and disease related conditions.
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