The application outlines studies to examine the molecular basis of visual signalling in cone photoreceptors of the mammalian retina. Cones signal using a cGMP cascade pathway similar to that of rod photoreceptors; they differ from rods in their sensitivity, rate and amplitude of signaling, and adaptation properties. In lieu of biochemical methods, the identity of the components will be established through molecular cloning; their functional properties will be examined following in vitro expression. In previous research periods, we have identified and characterized the cDNAs and genes encoding the components of visual signaling in rods.
Our specific aims i n this term are to use the set of rod genes as probes to identify and characterize the homologous components that contribute to signaling in the red/green cones and blue cones. Potential candidates for cone cDNAs will be identified through the technique of homology cloning. Their specific cone-type identity will be determined by in situ hybridization studies. To examine the structure and function of cone signaling proteins, the components will be expressed in eucaryotic host cells. Comparative functional studies for rod, red/green cone and blue cone components will be carried out for the opsins by examination of the bleaching profile and activation of G-proteins. The differences in functioning of cGMP phosphodiesterases in rod and cones will be studied in systems reconstituted with opsins and G-proteins. The potential differences in rod and cone cGMP-dependent channels and Na+/Ca2+ exchangers will be examined biochemically in lipid vesicle flux assays. We seek to provide basic information about our most intensively used and least understood photoreceptors. The identification of genes expressed in cone cell types and the characterization of their gene products is a first step towards defining how cones function. The studies will provide tools to investigate the underlying basis of macular degeneration and retinal degenerations that affect cone cells. The characterization of gene structure will enable ourselves and other investigators to undertake studies of the molecular basis of development of rods and cones and ultimately to understand defects in development.
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