The muscle acetylcholine receptor (AChR) is an ion channel that mediates transmission at the nerve-muscle synapse. After binding two transmitter molecules, the AChR switches rapidly (and with high probability) from a closed-channel (C) to an open-channel (O) conformation. With prolonged exposure to agonist, AChRs also adopt inactivated (desensitized) conformations. We seek to understand the dynamics of the molecular events that constitute the binding, gating and desensitization reactions. Results to date suggest that the allosteric gating conformational change is asynchronous, with residues in the extracellular domain of the protein moving in advance of those in the membrane domain during the C-to-O isomerization. Could it be visualized, we hypothesize that this conformational change would appear as a staggering sequence of back and forth motions of a few rigid body domains rather than as a smooth transition between the C and O conformations. Perturbations to the protein (for example mutations that cause the disease slow-channel congenital myasthenic syndromes) alter gating, and, hence, synaptic function, by changing the propagation of this Brownian conformational 'wave'. We will use single-molecule electrophysiology and kinetic (phi-value) analysis to probe the properties of the brief intermediates that constitute the transition state of the gating reaction.
Our specific aims are to i) extend the map of phi-values, ii) measure the degree of synchrony between the five AChR subunits, iii) explore the discreteness of the rigid body gating domains, iv) quantify the temperature dependence of the channel-opening speed limit, and v) extend our theoretical analyses of the transition state. We also propose to use similar approaches to study the intermediate states of the transmitter binding and desensitization reactions. The results will illuminate the dynamic machinery of the AChR, and will provide fundamental insight into the mechanisms by which drugs, toxins, cellular perturbations and disease-causing mutations modify ion channel function. They will also serve as the basis for rational protein engineering. ? ? ?
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