Studies on the electrogenic action of acetylcholine with Torpedo marmorata electric organ : II. The permeability response of the receptor-rich membrane fragments to cholinergic agonists in vitro

Abstract

Purified receptor-rich membrane fragments (spec. act. 1000 ± 400 nmol 3H-labelled α-toxin sites/g protein) from Torpedo marmorata make closed vesicles which retain 22Na + in free and membrane-bound compartments. Acetylcholine (after blocking acetylcholinesterase with 0.01 m m-Tetram † † Abbreviations used: Tetram, O,O′-diethyl S-(β-diethylamino)ethyl phosphorothiolate; SKF-525A, diethylamino ester of diphenylpropylacetic acid; methyldilvasene, 2-methyl-4-dimethylaminomethyl-1,3-dioxolane methiodide; EGTA, ethyleneglycol bis(β-aminoethylether)- N,N′-tetraacetic acid. ), carbamylcholine, phenyltrimethylammonium, suberyldicholine and methyldilvasene enhance the release of the free 22Na + from these microsacs and the effect of carbamylcholine is blocked by Naja nigricollis α-toxin, d-tubocurarine, flaxedil and hexamethonium. Decamethonium, an agonist on T. marmorata electroplaque, acts as a competitive antagonist on this in vitro system. Two local anaesthetics, dimethisoquin and SKF-525A block the response in vitro. Apparent dissociation constants for cholinergic ligands are determined from the dose-response curves. The values of the constants are, for most of them, significantly larger than those measured in vivo with the electroplaque by following steady-state depolarization in the presence of agonist added in the bath (on the other hand, they coincide rather well with the equilibrium dissociation constants given by direct binding studies with the cholate-solubilized receptor). The real dissociation constants measured with the membrane-bound receptor are close to the apparent ones for all the antagonists tested. However, the agonists bind at concentrations two orders of magnitude smaller than those necessary to trigger the response.