Effects of thio-group modifications on the ion permeability control and ligand binding properties of Torpedo californica acetylcholine receptor.

Chemical modification of membrane-bound Torpedo californica acetylcholine receptor by the disulfide reducing agent dithiothreitol has two major effects on receptor function: (1) it shifts the dose-response curve for agonist-induced increases in 22Na+ permeability to 10-fold higher concentrations, and (2) it decreases the binding affinity of the receptor for the same agonist about 6-fold. In the experiments reported here, the agonist used was carbamoylcholine. Despite the quantitative changes in agonist binding and flux response, dithiothreitol-treated membranes display all other functional properties expected of a receptor. The flux response is blocked by preincubation of the membranes with carbamoylcholine, a phenomenon known as desensitization. In parallel, the receptor undergoes a carbamoylcholine-induced shift from a low-affinity to a high-affinity binding state for the same agonist. All of the effects of dithiothreitol are reversed by the oxidizing agent 5,5'-dithiobis(2-nitrobenzoic acid). Alkylation of the membranes with N-ethylmaleimide after dithiothreitol reduction results in complete inhibition of the flux response, and the effect is not reoxidation treatment. The N-ethylmaleimide also shifts the receptor into a very low-affinity binding state for carbamylcholine that is shifted to only a slightly higher affinity by preincubation with carbamoylcholine. Prior to reduction, N-ethylmaleimide has no effect on receptor binding or flux properties. Detailed binding studies on affinity-alkylated receptor membranes indicate that the alpha-neurotoxin binding site not occupied by the affinity label displays all the same properties as unlabeled membranes, including the dithiothreitol and N-ethylmaleimide effects. The results are discussed in the context of several hypotheses previously proposed to account for the diverse effects of thio-group modifications on the acetylcholine receptor.