Acetylcholine receptor: evidence for a regulatory binding site in investigations of suberyldicholine-induced transmembrane ion flux in Electrophorus electricus membrane vesicles

Abstract

Suberyldicholine-induced ion translocation in the millisecond time region in acetylcholine receptor rich membrane vesicles prepared from the electric organ of Electrophorus electricus was investigated in eel Ringer's solution, pH 7.0, 1°C. A quench-flow technique with a time resolution of 5 ms was used to measure the transmembrane flux of a radioactive tracer ion (⁸⁶Rb⁺). J_A, the rate coefficient for ion flux mediated by the active form of the receptor, and α, the rate coefficient for the inactivation of the ion flux, increase with increasing suberyldicholine concentrations and reach a plateau value at about 15 µM. At higher suberyldicholine concentrations (>50 µM), a concentration-dependent decrease in the ion flux rate was observed without a corresponding decrease in the rate of receptor inactivation. This regulatory effect was not observed with acetylcholine or carbamoylcholine. The minimal kinetic scheme previously presented for acetylcholine and carbamoylcholine, modified by the inclusion of an additional regulatory ligand-binding site for suberyldicholine and characterized by a single dissociation constant, K_R, is consistent with the results obtained over a 10000-fold concentration range of this ligand. Rate and equilibrium constants pertaining to this scheme were elucidated. Suberyldicholine binds to the regulatory site (K_R=500 µM) approximately 100-fold less well than to its activating sites, and the binding to the regulatory site has no effect on the inactivation (desensitization) rate coefficient α[α(max)=5.7 s^−l], which is comparable to that observed with acetylcholine. The maximum influx rate coefficient [J_A(max)=18.5 s^−l is approximately twice that obtained when carbamoylcholine is the activating ligand and somewhat higher than when acetylcholine is used. Previous interpretations of the specific reaction rate, J̅, for the receptor-controlled ion translocation process required that while the maximum observed flux rates can depend on the activating ligand used, J̅ is the same for all activating ligands. Here we find that J̅ for suberyldicholine (J̅=3×l0⁷ M^−l s^−l) has the same value as was found earlier for carbamoylcholine and acetylcholine. This is the first report of chemical kinetic investigations of the inhibitory effect exerted by suberyldicholine at high concentrations.