During the last funding period we combined transgenic mouse technology with electrophysiological, biochemical, and morphological analyses in a multidisciplinary approach to study rhodopsin deactivation in vivo. We obtained detailed information on mechanisms of rhodopsin deactivation. In addition, we observed the cellular consequence of defective rhodopsin shut off. In this proposal I will use a similar approach to (1) study cone opsin deactivation and (2) investigate pathogenic mechanisms of light-dependent photoreceptor cell death. Due to the paucity of cone cells in the retina of most experimental animal models, little is known about phototransduction mechanisms underlying the cone response. In the first aim I propose to use transgenic techniques to reconstitute the S-opsin deactivation components in rod cells of transgenic mice. In our studies, we will measure and compare rates of thermal isomerization, efficiency of transducin activation, time constant of MII decay and time constant of pigment regeneration between rhodopsin and S-opsin to see whether these parameters contribute to differences in behavior between rod and cone responses. We will also test the hypothesis that multiple phosphorylations of S-opsin, followed by cone arrestin binding, are steps necessary for its rapid and timely deactivation. I anticipate that a detailed and systematic comparison between rod and cone deactivation pathways in an environment that approximates the native state will provide a good start to understanding phototransduction mechanism in cones. In the second aim, I propose to study the mechanisms underlying light-induced photoreceptor cell death. First, I will test the hypothesis that accumulation of rhodopsin/arrestin complex, a pathogenic mechanism in Drosophila, is conserved in vertebrates. Second, I propose to use a combination of expression arrays and proteomics analyses to compare and contrast expression changes in different mouse models of light damage due to constitutive signaling
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