Acetylcholine receptor: evidence for a voltage-dependent regulatory site for acetylcholine. Chemical kinetic measurements in membrane vesicles using a voltage clamp

Abstract

Acetylcholine receptor mediated ion translocation in membrane vesicles prepared from the Electrophorus electricus electroplax was investigated under voltage clamp conditions by using a quench-flow technique that allows the translocation to be measured in the millisecond to second time region. Two rate coefficients were measured over a 500-fold concentration range of acetylcholine, at a transmembrane voltage, V_m, of −45 mV, at pH 7.0, 1°C. J_A is the rate coefficient for ion translocation by the active state of the receptor in the absence of inactivation (desensitization), and α is the rate coefficient for the inactivation of the receptor by acetylcholine. (1) The values of J_A and α increase with increasing acetylcholine concentration up to 300 µM. At higher concentrations, α concentration-dependent decrease in the ion flux rate was observed without a concomitant change in the inactivation rate. This inhibitory effect has not been reported previously and was not observed with acetylcholine or carbamoylcholine in the absence of a transmembrane voltage. (2) The value of the maximum influx rate coefficient, 26 s^−l, is approximately twice that observed at 0 mV [J_A(max)=15 s^−l]. This is consistent with previous interpretations that related J_A(max) values to the channel-opening equilibrium constant, l/φ, and with the relation of l/φ to the mean lifetime of the open receptor channel in muscle cells, which is dependent on V_m. (3) The maximum observed inactivation rate coefficient [α(max)=8.5 s^−l] is somewhat larger than that observed at 0 mV [α(max)=5 s^−l]. The consequence of the newly discovered voltage-dependent regulatory site, characterized by a dissociation constant K_R=800 µM (V_m=−45 mV), is that the ability of the receptor to translocate ions, and therefore to initiate the transfer of signals between cells, depends significantly and unsuspectedly on the resting transmembrane voltage of the cell. The regulatory site for acetylcholine can be fitted into a general model, proposed in this paper, which also explains activation, inactivation, and voltage-dependent modulation of the receptor function.