Permeability control by cholinergic receptors in Torpedo postsynaptic membranes: agonist dose-response relations measured at second and millisecond times

Abstract

A quantitative analysis of nicotinic acetylcholine receptor function in Torpedo postsynaptic membranes is presented. 22Na+ efflux induced by carbamylcholine (Carb) and the partial agonist phenyltrimethylammonium (PTA) is assessed by determining dose-response relations using 3 approaches: a filtration assay measuring responses on the 10-s time scale, the same filtration assay after blocking different fractions of the receptor sites with .alpha.-bungarotoxin (.alpha.-BgTx), and a rapid-mix quenched-flow technique which permits measurement of the initial rate of 22Na+ efflux on the ms time scale. The concentrations of agonist producing half-maximal responses in these 3 assays at 4.degree. C are 13, 150 and 600 .mu.M, respectively, for Carb and 50, 50 and 200 .mu.M, respectively, for PTA. The rate constants for 22Na+ efflux are 1.3 .times. 10-4/s in the absence of agonist and 65/s and 0.8/s in the presence of maximal concentrations of Carb and PTA, respectively, representing a stimulation of 5 .times. 105 by Carb. The Hill coefficient for the Carb response, expressed as rate constants for 22Na+ efflux, is 1.97 .+-. 0.06 for Carb concentrations between 3 .mu.M and 1 mM. The inhibition of the agonist-stimulated 22Na+ efflux by a .alpha.-BgTx is compatible with 2 .alpha.-BgTx (and acetylcholine) sites per functional unit. Inhibition of Carb responses (slow assay) by d-tubocurarine appears competitive with a KI .apprx. 0.5 .mu.M, while responses to PTA are inhibited noncompetitively with KI = 0.3 .mu.M. This paradox is due to the presence of spare receptors and to complexities in the binding of dTC to the nicotinic acetylcholine receptor. Determination of responses without the complication of spare receptors allows a meaningful comparison to direct measurements of agonist and antagonist binding in the same system. A model is proposed to account for both binding and response.