Acetylcholine receptor in planar lipid bilayers: Characterization of the Channel Properties of the Purified Nicotinic Acetylcholine Receptorfrom Torpedo californica Reconstituted in Planar Lipid Bilayers

Abstract

The properties of the channel of the purified acetylcholine receptor (AChR) were investigated after reconstitution in planar lipid bilayers. The time course of the agonist-induced conductance exhibits a transient peak that relaxes to a steady state value. The macroscopic steady state membrane conductance increases with agonist concentration, reaching saturation at 10-5 M for carbamylcholine (CCh). The agonist-induced membrane conductance was inhibited by d-tubocurarine (50% inhibition, IC50, at ~10-6 M) and heitamethonium (IC50 ~10-5 M). The single channel conductance, γ, is ohmic and independent of the agonist. At 0.3 M monovalent salt concentrations, γ = 28 pS for Na+, 30 pS for Rb+, 38 pS for Cs+, and 50 pS for NH4+. The distribution of channel open times was fit by a sum of two exponentials, reflecting the existence of two distinct open states. τo1, and τo2, the fast and slow components of the distribution of open times, are independent of the agonist concentration: for CCh this was verified in the range of 10-6 M ≤ C ≤ 10-3 M. τo1, and τo2 are approximately three times longer for suberyldicholine (SubCh) than for CCh. τo1, and τo2 are moderately voltage dependent, increasing as the applied voltage in the compartment containing agonist is made more positive with respect to the other. At desensitizing concentrations of agonist, the AChR channel openings occurred in a characteristic pattern of sudden paroxysms of channel activity followed by quiescent periods. A local anesthetic derivative of lidocaine (QX- 222) reduced both τo1, and τo2. This effect was dependent on both the concentration of QX-222 and the applied voltage. Thus, the AChR purified from Torpedo electric organ and reconstituted in planar lipid bilayers exhibits ion conduction and kinetic and pharmacological properties similar to AChR in intact muscle postsynaptic membranes. © 1984, Rockefeller University Press., All rights reserved.