Acetylcholine receptor: characterization of the voltage-dependent regulatory (inhibitory) site for acetylcholine in membrane vesicles from Torpedo californica electroplax

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Abstract
Evidence for a voltage-dependent regulatory (inhibitory) site on the nicotinic acetylcholine receptor to which acetylcholine binds was obtained in membrane vesicles prepared from the Torpedo californica electric organ. Two rate coefficients, JA and .alpha., which pertain to the receptor-controlled ion flux, were measured. A 1000-fold concentration range of acetylcholine was used in a transmembrane voltage (Vm) range from 0 to -48 mV under a voltage-clamped condition at pH 7.4, 1.degree. C. The following observations were made. (i) At low acetylcholine concentrations, the value of JA, the rate coefficient for ion translocation by the active (nondesensitized) state of the receptor, increased with increasing concentration. (ii) JA decreased at high acetylcholine concentrations. (iii) In contrast, .alpha., the rate coefficient for receptor desensitization, did not show such a decrease. (iv) When the transmembrane potential of the vesicle membrane was changed to more negative values, the value of KR (the dissociation constant for binding of acetylcholine to the regulatory site) decreased by a factor of .apprx. 9 for a 25 mV change in Vm, while K1 (the dissociation constant for binding of acetylcholine to the receptor site that controls channel opening) did not show such a change and has a value of 80 .mu.M. When Vm is -48 mV, KR has a value of 8 .mu.M. (v) The effect of a transmembrane voltage on the regulatory site was reversible and occurred within the time resolution (5 ms) of the quench-flow technique used in the measurements. These results can be explained by a simple model in which the function of the receptor is regulated by binding of acetylcholine to a voltage-dependent regulatory site, a site that is distinct from the sites responsible for receptor activation and desensitization, and from the inhibitory site for cationic noncompetitive inhibitors such as the local anesthetic procaine. The biological significance of the voltage-dependent regulatory site on the acetylcholine receptor, specific for acetylcholine, is discussed.

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