We propose to investigate the molecular function and pharmacology of a key molecule in visual transduction, the cyclic nucleotide- gated (CNG) channel of retinal rods. This channel generates the electrical response to light. A thorough understanding of the rod CNG channel should provide important insights into how it participates in phototransduction. We also plan to use this knowledge to develop potent and specific pharmacological agents to dissect the physiological roles of CNG channels in different cells. These agents may also prove useful in treating some forms of retinal degeneration. The following questions will be addressed: 1. How do the two subunits of native rod CNG channels participate in channel activation and modulation? How are subconductance states generated? The rod channel is a tetramer consisting of two different subunits: alpha and beta. There is little known about the roles that these subunits play in channel activation and modulation. Using a photoaffinity analog of cGMP developed in this lab, we will covalently tether cGMP moieties to the channel to determine the functional consequences of binding to each subunit. 2. Where is the gate in relation to the ion binding sites in the pore of rod CNG channels? Does the substitution of a K+ channel pore region substantially change the position and function of the gate? How is ion selectivity affected? The recent high-resolution structure of a bacterial K+ channel, and the study of ion permeation mechanisms in Ca2+ channels provide templates for understanding permeation and how the """"""""gate"""""""" controls the flow of ions in CNG channels. We plan to study how the structures of ion binding sites change during gating. 3. Can highly potent and specific blockers of CNG channels be developed by joining a nonspecific pore blocker like tetracaine with cGMP, using polymers of different lengths? We have developed a new method of ligand and drug design for multi- site proteins, in which two ligands are joined together by variable length polymers until a compound is found that spans two binding sites. We plan to produce heterodimeric channel blockers in which a pore blocker is linked to cGMP to increase the overall affinity and specificity.
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