Abstract
The interaction of a cholinergic agonist, suberyldicholine, with the membrane-bound acetylcholine receptor from Torpedo marmorata was studied in vitro by a combination of kinetic and steady-state techniques. Exposure of the receptor-rich membranes to the agonist resulted in a time and concentration dependent, saturable and reversible series of changes in receptor properties. The apparent affinity of the receptor for the ligand increased about 50-fold in the course of this exposure, as measured indirectly by the inhibition of α-toxin association kinetics. The modifications induced by suberyldicholine in the receptor could be studied in a more direct manner by following the kinetics of the (intrinsic) protein fluorescence quenching. An initial step occurring in the time scale of milliseconds could be observed with stopped-flow fluorimetry, its characteristics being consistent with a binding process of the agonist to a “low affinity” form of the receptor ( K app ~ 1 μM) with on and off-rates of 9.8 × 10 6 m −1 s −1 and 10 s −1, respectively. This step was followed by a larger fluorescence change taking place in a time range of seconds (forward rate 1.2 to 1.5 s −1; backward rate 0.06 to 0.1 s −1). The final, “high-affinity” state of the complex, which differed in its fluorescence properties from both the free (initial) and liganded-forms of the receptor, is tentatively attributed to the “desensitised” conformation postulated from in vivo studies (Katz & Thesleff, 1957). The rate of formation of this complex elicited by suberyldicholine was higher than those of acetyl- or carbamoylcholine. The extent of the total fluorescence quenching was, on the other hand, approximately the same for the three agonists, suggesting that the final conformation does not depend on the nature of the ligand. The specific agonist-induced quenching probably operates via a “static” mechanism, since the lifetime of the excited state did not change in the presence of the ligand. The accessibility of the intrinsic fluorophores to acrylamide or to nitroxide spin labels, as sensed by quenching of fluorescence from the bulk and lipid phases, respectively, was also affected by suberyldicholine. Both fluorescence and toxin-inhibition studies have been analysed in terms of a reaction mechanism derived from in vivo observations, involving interconvertible conformational states of the acetylcholine receptor in its membrane environment, differing in affinity for the agonist, and in their intrinsic fluorescence properties.
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