Photoreceptors have several important physiological functions. They detect light but they also adapt to constant illumination and darkness. Phosphorylation of rhodopsin is a critical biochemical reaction that modulates the phototransduction cascade. In this proposal we outline methods to precisely characterize the contributions of rhodopsin phosphorylation to light and dark adaptation and to the kinetics of the photoresponse. Rhodopsin phosphorylation will be measured both in isolated intact mouse retinas and in vivo using high-resolution rapid quench and mass spectrometry methodology. The time course of rhodopsin phosphorylation will be established under a variety of physiologically important conditions. Physiological responses of rod photoreceptors will be established by quantitative electroretinography under conditions identical to those used for phosphorylation measurements. A variety of mouse strains will be examined in which genes encoding proteins that influence rhodopsin phosphorylation and regeneration are mutated. Retinoid metabolism will be monitored and it will also be compared with the kinetics of physiological responses to light and darkness. These studies will precisely define the contributions of phosphorylation and visual cycle reactions to light and dark adaptation and photoresponse kinetics in mammalian retinas.
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