Specific reaction rate of acetylcholine receptor-controlled ion translocation: a comparison of measurements with membrane vesicles and with muscle cells.

Abstract

The specific reaction rate (J) of the acetylcholine receptor-controlled ion translocation has been determined. In eel Ringer's solution (pH 7.0) at 10C, j = 3 x 107 M'1 sec-1. j is an intrinsic constant that is characteristic of the receptor and independent of other properties of a receptor-containing cell that also determine the rates of ion translocation. Membrane vesicles (prepared , which allows one to measure elementary steps in the formation of ion channels through the cell membrane.The initial rate of acetylcholine receptor-controlled movement of ions across the membrane of a particular cell determines the initial change in transmembrane potential (1, 2) and therefore also determines whether or not the electrical signal generated by a nerve cell will be propagated (3) or, in muscle cells, whether a contraction will occur. This initial rate is characterized by the rate coefficient, JA The value of JA is determined not only by the intrinsic properties of the receptor but also by certain properties of the cell, such as the number of receptor sites and the internal volume, and also the internal and external concentrations of freely mobile inorganic ions. We have, therefore, determined the specific reaction rate, J, which is characteristic of the receptor-controlled ion translocation and independent of these other properties of nerve and muscle cells. The rate of receptor-controlled ion translocation over a wide range of both carbamoylcholine and acetylcholine concentrations can be measured (12-15). The interpretation of all these measurements has led to the formulation of a minimum mechanism (Fig. 1) that relates the formation of ligand-receptor complexes to ion translocation (13,14). The integrated rate equation (Eq. 1) based on this mechanism predicts the rate of receptor-controlled flux over the concentration ranges of carbamoylcholine (13,14) and acetylcholine that we have investigated (15).JA is the observed rate coefficient associated with the initial fast influx before the onset of inactivation (desensitization) of the receptor. J. is the observed rate coefficient associated with the slow phase that occurs after the inactivation process has gone to completion, and a is the rate coefficient for the inactivation process: JA = Jm ([AL2]

Acetylcholine-induced cation translocation across cell membranes and inactivation of the acetylcholine receptor: chemical kinetic measurements in the millisecond time region.

“…vesicles), and the time resolution of the flow method that we use is sufficient to make measurements in the physiologically relevant time region. The number of ions translocated per receptor per msec (6 x 103), which we determined by chemical kinetic measurements using eel vesicles (7,8), is in agreement with results obtained in electrical noise measurements using muscle cells (14,15).…”

“…1. The linearity of this plot, demonstrated previously (7,8), confirms the square dependence of-ion flux on acetylcholine concentration, showing that two ligand molecules bound to the receptor are required for channel opening and that the process is cooperative.…”

“…vesicles), and the time resolution of the flow method that we use is sufficient to make measurements in the physiologically relevant time region. The number of ions translocated per receptor per msec (6 x 103), which we determined by chemical kinetic measurements using eel vesicles (7,8), is in agreement with results obtained in electrical noise measurements using muscle cells (14,15).Originally we used carbamoylcholine (4-6, 10), because it is a stable analogue of acetylcholine, and measured the effect of carbamoylcholine concentration on (i) the ion flux rate corresponding to the active state of the receptor, (ii) the inactivated receptor (16), and (iii) the rates ofreceptor inactivation and reactivation (4-6, 10). The quench-flow measurements, over a 200-fold range ofcarbamoylcholine concentration that included saturation of the active state, supported a minimum reaction scheme as shown in Fig.…”

“…Although the rate constants for inactivation are comparable for the two ligands, the equilibrium constants are more in favor of inactivation for acetylcholine. The rate constant for ion translocation, Im, pertains to the open-channel form of the receptor and is independent of ligand (7,8).…”

Effect of a benzodiazepine (chlordiazepoxide) on a GABAA receptor from rat brain Requirement of only one bound GABA molecule for channel opening

“…2), which is a measure of the concentration of open channels per unit internal volume (inside the vesicles) [22,23], has been measured for the faster desensitizing receptor in the presence and absence of the benzodiazepine, CDPX. This receptor gives the major fraction (approx.…”