Kinetic mechanism of acetylcholine receptor-controlled ion flux: Flow quench kinetic measurements of acetylcholine-induced flux in membrane vesicles

Abstract

The dependence of acetylcholine receptor-controlled transmembrane ion flux on acetylcholine concentration was measured in the msec time region using membrane vesicles and a quench flow technique. Four measurements were made: (1) transmembrane ion flux, (2) rate of inactivation of the receptor, (3) rate of recovery, and (4) ion flux mediated by “inactivated” receptor. A minimum model which relates the ligand binding and ion translocation processes, which has been previously proposed to account for carbamylcholine-induced ion flux, can also account for acetylcholine-induced flux. The integrated rate equation, based on the model, predicts the time dependence of the ion flux over the 160-fold concentration range of acetylcholine investigated. The receptor-controlled ion flux exhibits simple kinetics, and this has permitted the use of simple analytical expressions for the dependence on acetylcholine concentration of the various constants of the minimum mechanism. The evaluation of the constants, and methods for the separation of vesicles which contain functional receptors from those which do not, have led to the determination of the specific reaction rate, J , of the acetylcholine receptor-controlled translocation of inorganic ions. J = 3 × 10 7 M −1 sec −1. The value for J allows one to calculate the number of ions translocated per receptor per msec, ∼6 × 10 3. A value of 1 × 10 7 ions translocated per receptor site per unit time has also been determined by analysis of acetylcholine-induced noise in cells. Therefore it becomes possible to integrate the results obtained in two types of measurement of receptor function: chemical kinetics, which establishes the relationship between the ligand binding and ion translocation processes, and noise analysis, which measures elementary steps in the formation of receptor-formed ion channels.