Ligand-induced changes in membrane-bound acetylcholine receptor observed by ethidium fluorescence. 3. Stopped-flow studies with histrionicotoxin

Abstract

Rapid kinetic studies of histrionicotoxin interactions with [Torpedo californica] membrane-bound acetylcholine receptor showed a conformational change in the receptor-histrionicotoxin complex as reflected by a decrease in fluorescence intensity of the extrinsic probe ethidium. The simplest kinetic mechanism consistent with the observed data is one in which a rapid preequilibrium exists between receptor and toxin (K = 3.33 .mu.M), followed by a slow conformational change (k1 .simeq. 2 .times. 10-2 s-1 and k-1 .simeq. 1.5 .times. 10-3 s-1). The overall equilibrium constant (Kov) determined from a fit of the amplitude dependence on toxin concentration had a value of 0.25 .mu.M. The data preclude kinetic mechanisms where histrionicotoxin acts as an effector, shifting equilibria between preexisting, discrete and slowly interconverting receptor forms.