Photoaffinity Labeling of Torpedo Acetylcholine Receptor at Multiple Sites

The acetylcholine receptor from Torpedo californica electroplax was labeled with the photoaffinity reagent bis(3-azidopyridinium)decane perchlorate. All four receptor subunits (α, β, γ, and δ) were specifically labeled. In the presence of cholinergic agonists the γ-, β-, and δ-subunit labeling was decreased significantly, whereas labeling of the α subunit was minimally affected. Full occupancy of the two high-affinity sites involving the α subunits in the vicinity of α-Cys-192-Cys-193 by covalent reaction with bromoacetylcholine also caused a large decrease of γ-subunit labeling by the photoaffinity reagent and lesser but significant decreases in β- and δ-subunit labeling. No decrease in labeling of the α subunit was seen. Labeling of the α subunit could, however, be inhibited by high concentrations of the agonist carbamoylcholine. We conclude that the binding sites of high-affinity reside at interfaces of the α subunit and other subunits and that the α subunit also contributes to formation of a low-affinity site(s) for cholinergic compounds.