Interaction of a fluorescent agonist with the membrane-bound acetylcholine receptor from Torpedo marmorata in the millisecond time range: Resolution of an "intermediate" conformational transition and evidence for positive cooperative effects

Stopped-flow measurements of the rapid kinetics of interaction of the fluorescent agonist Dns-C₆-Cho with the membrane-bound acetylcholine receptor (AChR) were extended to the 0.1 to 1 mM range of agonist concentration. In this domain of concentrations, a fluorescent intensity increase is observed in the milliseconds time range. The signal follows a monoexponential time course and is abolished upon preincubation of the membrane fragments with saturating concentrations of Naja nigricollis α-toxin. A plot of the rate constant of the signal as a fonction of agonist concentration deviates from linearity: k_app first increases in a sigmoid manner (n_H = 1.86) for concentrations up to 300 μM, and then decreases approx. two-fold for concentrations up to 1 mM. A minimal scheme involving a conformational transition between discrete low affinity states of the receptor, with two agonist binding sites per molecule is proposed; the relevance of these states and transition to the physiological mechanism of activation and to the fast and slow desensitisation processes is discussed.