FUNCTIONAL-EFFECTS ON THE ACETYLCHOLINE-RECEPTOR OF MULTIPLE MUTATIONS OF GAMMA-ASP174 AND DELTA-ASP180

Residues gamma Asp174 and delta Asp180, previously implicated in the b inding of acetylcholine (ACh) by the mouse muscle ACh receptor, were e ach mutated to nine other residues, Asn, Glu, Thr, Ala, Cys, I-Iis, Va l, Tyr, and Lys, The effects of the mutations on ACh-induced current w as determined on surface receptors containing wild-type alpha and beta subunits and mutant gamma and delta subunits. The mutations increased the concentration of ACh eliciting half-maximal current (EC50) by fac tors 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 area s of the mutant and wild-type side chains and the difference in side-c hain charges Indicated that, per binding site, Delta Delta G(0) for ac tivation was a sum of 10 cal mol(-1) Angstrom(-2) Of change in side-ch ain 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 4 2 mV. The effects on the concentration of ACh (IC50,ACh) and of d-tubo curarine (IC50,dTC) causing half-maximal retardation of cx-bungarotoxi n binding were determined on complexes containing wild-type a and beta subunits and either mutant gamma or mutant delta 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 electrost atic potential at the binding site. The effects on IC50,dTC were in al l 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 lowe r d-tubocurarine affinity of the alpha-delta binding site compared to the alpha-gamma binding site, mutations of delta Asp180 had a greater effect on IC50,ACh than did the same mutations of gamma Asp174, and vi ce versa for effects on IC50,dTC. Consequently, all mutations decrease d the asymmetry in the binding properties of the two types of sites.