Functional Effects on the Acetylcholine Receptor of Multiple Mutations of γAsp174 and δAsp180

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Abstract
Residues γAsp174 and δAsp180, previously implicated in the binding of acetylcholine (ACh) by the mouse muscle ACh receptor, were each mutated to nine other residues, Asn, Glu, Thr, Ala, Cys, His, Val, Tyr, and Lys. The effects of the mutations on ACh-induced current was determined on surface receptors containing wild-type α and β subunits and mutant γ and δ subunits. The mutations increased the concentration of ACh eliciting half-maximal current (EC50) by factors from 22 for the Glu mutant to 660 for the Lys mutant. Analysis of the effects in terms of the difference in the accessible surface areas of the mutant and wild-type side chains and the difference in side-chain charges indicated that, per binding site, ΔΔG0 for activation was a sum of 10 cal mol-1 Å-2 of change in side-chain accessible surface area and of 0.95 kcal mol-1 positive step-1 in side-chain charge, equivalent to 1 mol of charge falling through 42 mV. The effects on the concentration of ACh (IC50,ACh) and of d-tubocurarine (IC50,dTC) causing half-maximal retardation of α-bungarotoxin binding were determined on complexes containing wild-type α and β subunits and either mutant γ or mutant δ subunit. The effects on IC50,ACh correlated well with the effects on EC50, with a similar magnitude for the influence of side-chain charge on the free energy of binding (in this case to the desensitized state) and on the electrostatic potential at the binding site. The effects on IC50,dTC were in all cases less than the effects on IC50,ACh, and the two sets of effects were poorly correlated. In line with the higher ACh affinity and lower d-tubocurarine affinity of the α-δ binding site compared to the α-γ binding site, mutations of δAsp180 had a greater effect on IC50,ACh than did the same mutations of γAsp174, and vice versa for effects on IC50,dTC. Consequently, all mutations decreased the asymmetry in the binding properties of the two types of sites.