PHOTOAFFINITY-LABELING OF TORPEDO ACETYLCHOLINE-RECEPTOR AT MULTIPLE SITES

The acetylcholine receptor from Torpedo californica electroplax was la beled with the photoaffinity reagent bis(3-azidopyridinium)decane perc hlorate. All four receptor subunits (alpha, beta, gamma, and delta) we re specifically labeled. In the presence of cholinergic agonists the g amma-, beta-, and delta-subunit labeling was decreased significantly, whereas labeling of the a subunit was minimally affected. Full occupan cy of the two high-affinity sites involving the alpha subunits in the vicinity of alpha-Cys-192-Cys-193 by covalent reaction with bromoacety lcholine also caused a large decrease of gamma-subunit labeling by the photoaffinity reagent and lesser but significant decreases in beta- a nd delta-subunit labeling. No decrease in labeling of the alpha subuni t was seen. Labeling of the alpha subunit could, however, be inhibited by high concentrations of the agonist carbamoylcholine. We conclude t hat the binding sites of high-affinity reside at interfaces of the alp ha subunit and other subunits and that the a subunit also contributes to formation of a low-affinity site(s) for cholinergic compounds.