Multiple Activity Patterns of Acetylcholine

The nicotinic acetylcholine receptor channel (AChR) is an allosteric membrane protein that mediates neuromuscular transmission in vertebrates. Experiments are proposed regarding the kinetic properties of wild type and mutant mouse recombinant AChR, probed using single-channel electrophysiology. Three areas that are of fundamental importance with regard to understanding synaptic receptor channels are addressed: agonist binding, channel gating, and desensitization. One goal of this proposal is to understand the mechanisms of molecular recognition at the transmitter binding sites. Experiments are proposed to study the nature of the agonist-protein interactions with both high and low affinity conformations of the AChR. The second objective is to use linear free energy relationships to obtain a snapshot of the gating transition state. These experiments will report the order of events during the closed-open allosteric transition. The third aim is to understand the molecular basis of desensitization. Experiments are proposed to make high-resolution measurements of desensitization recovery rate constants in both wild type and mutant AChR. The results of the experiments will provide a deep, mechanistic understanding of AChR both at normal synapses, and at synapses that have been compromised by neuromuscular disease.