Functional acetylcholine receptor from Torpedo marmorata in planar membranes.

Abstract

Planar bilayer membranes containing functional acetylcholine receptor were formed from vesicles of T. marmorata electric organ without extracting the acetylcholine receptor from its native environment. Native vesicles were transformed into monolayers which were apposed into planar bilayers. In the absence of agonists the membrane conductance was similar to lipid bilayers. Addition of carbamoylcholine or succinylcholine caused increased membrane conductance. Conductance was competitively inhibited by d-tubocurarine and suppressed by .alpha.-bungarotoxin. The amplitude of the conductance response was proportional to the number of .alpha.-bungarotoxin binding sites in the bilayers. Asymmetric membranes were formed with the ligand binding sites on only 1 membrane surface. Desensitization of acetylcholine receptor was evident from the equilibrium and kinetics of the carbamoylcholine-activated conductance. Carbamoylcholine-induced membrane permeability was about 7 times higher for K+ and Na+ ions than for Cl-. At low levels of conductance, single-channel fluctuations of 20-25 pS [picroSiemens] in conductance and 1.3 ms lifetime were resolved in physiological saline containing carbamoylcholine. The ratio of observed channels to .alpha.-bungarotoxin sites showed that a significant fraction of acetylcholine receptor in the membrane was functional. The quantitative aspects of the cation channel, the desensitization, and ligand binding properties were in close agreement with established values. This transformation of natural acetylcholine receptor vesicles to planar bilayers conserves the essential properties of the in vivo receptor.