Allosteric interactions between the membrane-bound acetylcholine receptor and chemical mediators: equilibrium measurements.

An approach to equilibrium dialysis measurements has been developed which enables one to study the interaction of chemical mediators with the membrane-bound acetylcholine receptor and to gain information of a type previously obtainable only with soluble proteins. Equilibrium dialysis experiments conducted at pH 7.0,4 degrees C, and mu = 0.18 M, with electroplax membrane preparations from Electrophorus electricus revealed apparently homogeneous binding isotherms for decamethonium with dissociation constants in the range of 0.2-0.4 muM. The following new information has been obtained. (1) The activators of neural transmission, decamethonium and carbamylcholine, occupy overlapping binding sites. (2) These activators and the inhibitors, alpha-bungarotoxin and d-tubocurarine, compete for only one-half of the sites available to them even through the stoichiometry of these is 1:1 as measured with decamethonium (a reversibly binding activator) and alpha-bungarotoxin (an irreversible specific inhibitor). Different receptor molecules, preexisting nonequivalent binding sites, or an allosteric mechanism involving ligand-induced conformational changes are often considered to account for such observations.