The goal of this rese77arch project is to elucidate how cembranoids interact with and inhibit noncompetitively the nicotinic acetylcholine receptor (AChR). Based on previous studies in the applicant and ~ collaborator laboratories, the working hypothesis is that there is one nonluminal cembranoid site on the AChR which, when occupied, produces inhibition of current by stabilizing the resting conformation of the AChR. To test this hypothesis the project will use two exciting new probes of this site, a photoreactive cembranoid derivative developed at the applicant institution and a set of RNA aptamers developed at the collaborating institution. The applicant institution will study the binding of radiolabelled probes to and the functional effects of unlabelled probes on weld-type and mutant AChR. This will be the first time that both radioligand binding and electrophysiology will be used simultaneously on the same cells to study how a noncompetitive inhibitor interacts with a heterologously-expressed muscle-type AchR. The collaborating institution will refine the RNA aptamers ~y identifying the shortest sequences which still bind to the AChR with high-affinity in a cembranoid-displaceable manner and will study in detail the effects of the RNA aptamers and cembranoids on native cell-expressed muscle-type AChR using rapid kinetics methods developed at the collaborating institution. These studies will elucidate the mechanism of how cembranoids, which have substantial pharmacological potential,~block the muscle-type AChR and will lead to a better understanding of the structure and function of this important prototype of all ligand-gated ion channels.
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