We propose to investigate the molecular function and regulation of a key molecule in visual transduction, the cyclic-GMP-activated channel of retinal rods. This channel generates the electrical response to light. cGMP keeps channels open in darkness; light closes channels by activating an enzyme cascade that lowers the concentration of cGMP. Some of the channel's basic functions have been described, but the detailed mechanisms that underlie its behavior and whether it is regulated by cellular agents other than cGMP are largely unknown. Using a combination of electrophysiological and biochemical methods, we will approach the following questions: 1. What is the chemical nature of the cGMP binding sites on the channel? How far apart are the binding sites? How does the binding of multiple-cGMP's to a single channel unit cause it to open? 2. How do open states of different conductance arise, and what is their functional significance? What are the opening and closing kinetics of each state, and how do these rates depend on voltage? How do the different open states interact with divalent cations? 3. Is the number of channels capable of responding to cGMP actively regulated by covalent modification or tightly bound factors? Are the affinity of the channel binding sites for cGMP and the affinity of the pore for divalent cations also regulated? If modifications occur, what signals trigger them, how do they affect the overall response properties of the rod, and what are the molecular mechanisms? Answers to these questions will provide additional insight into how retinal rods transduce light into a neural signal, and how this type of channel may operate in other cells that use a similar molecular strategy. The experiments should also lead to a greater understanding of visual defects that occur in retinal disease.
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