Energy Changes at the Acetylcholine Receptor Gate Region

Abstract

Abstract The neuromuscular acetylcholine receptor (AChR) alternatively switches between an inactive (closed) and active (open) conformation. during the gating isomerization (R↔R∗) process. The ‘gate’ region is formed by the equatorial residues (9’-16’). We and others have speculated that channel gating involves a wetting/de-wetting of this region of the pore, based on measurements of gating equilibrium changes caused by mutations, phi value analysis and molecular dynamics simulations. We are studying the coupling energies between 9’-13’ residues in different subunits, by estimating the effect of mutations, separately vs. in combination, on the unliganded gating equilibrium constant E0. The end states and conformational pathway for AChR gating is fundamentally the same with and without agonists, and E0 can be estimated without obfuscation by channel block. We characterized two background mutants (αA96N/W149S and αA96H/Y96H) that greatly increase spontaneous gating and exhibit simple two-state kinetics (E0 = 0.076 and 0.031). These E0 values predict an E0wt value that is similiar to previous estimates (∼6.5x10−7). With these spontaneously-opening backgrounds, the individual free energy changes for βV13A and δV13'L (−4.1 kcal/mol and −1.9 kcal/mol) are similar to previous estimates obtained by using diliganded AChRs. Initial results suggest that these 13’ mutants are only weakly coupled (∼-1.2 kcal/mol). We expect these studies will help unravel the structural changes in protein and water that occur at the AChR gate region.