The long term goal of this research program is to characterize the molecular structure and properties of rod and cone plasma membrane proteins and to define their functional role in phototransduction, structural organization of the outer segment organelle and retinal degenerative diseases, including Retinitis Pigmentosa.
The specific aims for this grant period are: 1) to define the structural, functional and antigenic domains of the cGMP-gated channel as a means to test a new, proposed model for the molecular organization of this channel in rod and cone photoreceptor membranes; this study will involve i. the generation and characterization of site-directed monoclonal and polyclonal antibodies for mapping the topographical organization of the channel within the membrane by biochemical and immunocytochemical techniques; ii. the development of site-specific mutations in the putative pore region of the channel for analysis of its ion translocation properties in Xenopus oocytes and COS-1 cell expression systems; and iii. a comparison of the molecular properties of rod and cone cGMP-gated channel proteins. 2) to study the molecular properties of the 240 kD channel-associated protein and its interaction with calmodulin as a means to define its role in the modulation of channel activity during photoexcitation and recovery and as a possible cytoskeletal protein to maintain the spatial organization of the channel in rod outer segment plasma membranes. this study will involve cloning and sequencing the cDNA for this 240 kD protein for comparison with protein sequences in the Data Base and for identification of consensus sequences for calmodulin binding, phosphorylation, prenylation, etc.; the binding and modulation of the channel-240 kD complex by Ca2+-calmodulin will also be studied to define its role in phototransduction mechanisms. 3) to identify and characterize the molecular properties of other ROS plasma membrane proteins including the Na/Ca-K exchanger and several proteins to which monoclonal antibodies have been generated. A variety of biochemical, immunochemical, cell biology and molecular biology techniques will be used in these studies. The results of this study should provide new insight into the molecular structure and function of important membrane proteins of photoreceptor cells and serve as a basis for defining their role in photoreceptor degenerative diseases.
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