A HIGH-AFFINITY SITE FOR ACETYLCHOLINE OCCURS CLOSE TO THE ALPHA-GAMMA-SUBUNIT INTERFACE OF TORPEDO NICOTINIC ACETYLCHOLINE-RECEPTOR

Affinity labeling techniques have been used to investigate the location of high-affinity binding sites for cholinergic agonists on the Torpedo acetylcholine receptor and the extent of overlap of these sites with those for long alpha-neurotoxins. Following reduction of the receptor by dithiothreitol, reaction with [H-3] bromoacetylcholine leads to covalent incorporation into each of the two alpha subunits. A thigh concentrations of [H-3]bromoacetylcholine (240 muM) and with prolonged incubation times (1-2 h), this labeling was not inhibited by either alpha-bungarotoxin or alpha-najatoxin. Following maximum labeling by [H-3] bromoacetylcholine, no residual high binding sites for [I-125]-alpha-bungarotoxin could be detected in the membrane-bound receptor, but 50% of the original sites were recovered by receptor solubilization. Since it has previously been reported that one of the two sites for alpha-bungarotoxin in the membrane-bound receptor is readily reversible but is converted to a high-affinity state by solubilization [Conti-Tronconi, B. M., Tang, F., Walgrave, S., & Gallagher, W. (1990) Biochemistry 29, 1046-1054], these results demonstrate that the covalently bound agonist inhibits the binding of alpha-bungarotoxin only to its higher affinity site in the membrane. When [H-3]bromoacetylcholine labeling was carried out after reduction of the receptor by sodium borohydride rather than dithiothreitol, both alpha and gamma subunits of the receptor were labeled. Labeling of both subunits was completely inhibited if the receptor was first reduced with dithiothreitol and the alpha subunit sites were previously covalently labeled by unlabeled bromoacetylcholine. These results provide evidence that a high-affinity site for the agonist is located at, or close to, the alpha-gamma subunit interface and that disulfide bonds on each of the alpha and the gamma subunit are located at positions approximately equidistant from the quaternary ammonium binding site.