Modulation of phencyclidine-sensitive ethidium binding to the Torpedo acetylcholine receptor: interaction of noncompetitive inhibitors with carbamylcholine and cobra alpha-toxin

CF Valenzuela, JA Kerr, P Duvvuri and DA Johnson

Division of Biomedical Sciences, University of California, Riverside 92521-0121.

Ethidium is one of two fluorescent ligands known to bind to the noncompetitive inhibitor (NCI) site in the central ion channel of the Torpedo acetylcholine receptor with a micromolar dissociation constant. To further characterize heterotropic allosteric regulation of ligand binding in general, and of ethidium binding in particular, to the Torpedo receptor, we measured the effects of three liquid anesthetics (diethyl ether, halothane, and butanol), two barbiturates (secobarbital and thiamylal), and urethane. The phencyclidine-sensitive chromatic shift and the quantum yield increase associated with ethidium binding to the channel NCI site were used as indicators of ethidium binding. In the absence of other ligands, halothane, diethyl ether, and butanol increased the affinity of ethidium toward the channel NCI site to the same extent as carbamylcholine (400-600-fold), whereas the barbiturates and urethane were without effect. Cobra alpha-toxin blocked anesthetic- induced ethidium binding, confirming that cobra alpha-toxin stabilizes the AcChR in the resting-like state. In the presence of carbamylcholine, when ethidium was bound to the channel NCI site, several ligand-dependent effects were observed. 1) Without affecting further the affinity of ethidium for the NCI binding site, diethyl ether and halothane increased and butanol had no effect on the fluorescence emission of channel-bound ethidium. This indicated that there is little relation between the affinity and the quantum yield of the channel-bound ethidium. 2) Addition of secobarbital and thiamylal had no effect, beyond the effect of carbamylcholine, on ethidium binding to the channel NCI site, indicating that the barbiturates did not bind to the channel NCI site. 3) Urethane inhibited carbamylcholine- induced ethidium binding to the channel NCI binding site, suggesting direct interaction of urethane with the channel NCI binding site, at least when the receptor is in a desensitized state. The results confirm the conformational sensitivity of ethidium binding to the channel NCI binding site and demonstrate at least three different modes of action of anesthetics to inhibit the Torpedo receptor noncompetitively.

Volume 41, Issue 2, pp. 331-336, 02/01/1992
Copyright © 1992 by American Society for Pharmacology and Experimental Therapeutics