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Equilibrium binding of acetylcholine to the membrane-bound acetylcholine receptor.

We have studied the binding of acetylcholine to membrane-bound acetylcholine receptor from Torpedo marmorata employing a highly accurate airfuge assay procedure. At equilibrium the receptor displays two classes of acetylcholine binding sites; these interact with only weak positive cooperativity. As a further difference to binding data deduced from electrophysiological dose/response curves, the equilibrium constants for the two classes of sites (Kd1 = 25 nM, Kd2 = 8 nM) are orders of magnitude lower than the concentration required for half-maximal response. Both the weaker-than-expected cooperativity of sites and the high binding affinities are likely to be due to desensitisation of the receptor during the period of incubation. The positively cooperative interaction of acetylcholine binding sites is only observed with membrane preparations obtained in the presence of appropriate chelating, sulfhydryl-blocking and active-serine-blocking agents. Aged membrane preparations loose the ability of site interactions while only small changes in the total number of binding sites are observed. In the absence of divalent ions, the affinity of binding of acetylcholine to the receptor is reduced. To assess the significance of the binding data obtained, several alternative reaction schemes for non-random binding to two sites at the receptor are considered. In addition, the effects of possible sources of experimental error on the shape of Scatchard plots are analysed.