We will investigate the mechanisms that regulate the sensitivity of vertebrate retinal rods and cones during and following bright light. To do this, we will make electrophysiological recordings of membrane current microspectrophotometric measurements of visual pigment, and microfluorometric measurements of retinal and retinol in solitary rods and cones in darkness before and following bleaching and following exogenous incorporation of retinal and its analogs. The following hypotheses will be tested: (1) The reduction of all-trans retinal to all- trans retinol plays an important rote in regulation of response kinetics and sensitivity in vertebrate photoreceptors. We will perform microfluorometric experiments on solitary salamander rods and cones as well as mouse rods to determine and compare the kinetics, spatial distribution and metabolic dependence of reduction of retinal to retinol following bleaching and treatment with retinal analogs. We shall determine the kinetics of removal of retinal from bleached cells by extracellular agents (e.g. IRBP). We will make simultaneous determinations of the physiological state of cells under these same conditions using extracellular current recordings to determine the effect that reduction of bleached photopigment has on sensitivity recovery. Finally, we shall make simultaneous fluorometric and electrophysiological measurements to determine if retinal reduction occurs before, at the same time as, or after retinal is released from the opsin binding site. (1) The state of occupation of the non-covalent binding site in opsin plays a crucial role in determining the adaptational state of photoreceptors. We shall measure PDE and guanylyl cyclase velocities in salamander photoreceptors that have been bleached and treated with analogs of retinal to determine the essential structural features of the opsin/retinal interaction required for pigment regeneration and recovery of sensitivity during dark-adaptation.
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