Ligand-induced changes in membrane-bound acetylcholine receptor observed by ethidium fluorescence. 2. Stopped-flow studies with agonists and antagonists

Abstract

The kinetics of cholinergic ligand binding to membrane-bound acetylcholine receptor from Torpedo californica [electroplax] were followed in a stopped-flow photometer, by using the fluorescent probe ethidium. The overall reaction amplitude, as a function of ligand concentration, can be fit to the law of mass action for both agonists and antagonists. All agonists show at least biphasic kinetics, and the concentration dependence of the kinetic parameters is fit by a common mechanism involving sequential binding of ligands with increasingly lower affinity. The receptor-ligand precomplexes isomerize to different noninterconvertible final complexes depending on the number of ligands bound. The kinetics observed with antagonists cannot be fit to a common model. These kinetics are always much slower than those observed with agonists, and the relaxation rates depend only weakly on antagonist concentration.