Fellowship for Stuart Licht: Energy Landscapes for Acetylcholine Recptor Gating

The goal of this proposal is to investigate the nature of the transition state of the allosteric conformational change that controls the gating reaction of the acetylcholine receptor, an ion channel that opens (i.e., becomes ion-permeable) in response to the neurotransmitter acetylcholine. The transition state for the gating reaction may be a unique state that represents a saddle point on a free energy surface describing the reaction, like many transition states of reactions of small organic molecules. Alternatively, the gating reaction may proceed through a transition state ensemble composed of many closely spaced microstates, similar to the transition regions of some protein folding reactions. Linear free energy relationship analysis of multiple reaction coordinates will allow these possibilities to be distinguished. Measurement of single-channel currents via patch-clamp electrophysiology will allow determination of microscopic rate constants and equilibrium constants for gating. Site- directed mutagenesis, variation of ligand occupancy, and control of transmembrane voltage will be used to perturb the gating equilibrium for the linear free energy relationship analysis.