Localization of azidophencyclidine-binding site on the nicotinic acetylcholine receptor α-subunit

Nicotinic acetylcholine receptors in receptor-rich membranes from Torpedo californica and from T. marmorata electric tissue were photolabeled with the non-competitive inhibitor [ 3H]azidophencyclidine. The receptor subunits were separated on SDS-polyacrylamide gels and the α-subunits recovered from the gel, were subjected to Staphylococcus aureus V8 protease cleavage. The proteolytic fragments were resolved by SDS-polyacrylamide gel electrophoresis and were identified on protein blots by 125I-labeled α-bungarotoxin binding and by staining with concanavalin A. The site of specific azidophencyclidine labeling has been localized to the V8–18 kDa fragment which binds toxin. Labeling of the V8–18 kDa fragment was observed in the absence and in the presence of carbamylcholine. This was found for both the species of Torpedo used here.