Covalent labeling of the acetylcholine receptor from Torpedo electric tissue with the channel blocker [3H]triphenylmethylphosphonium by ultraviolet irradiation

Abstract

The lipophilic cation [3H]triphenylmethylphosphonium, frequently used as a voltage sensor in membrane systems, binds reversibly to a site different from the acetylcholine [Ach] binding site, according to different pH dependences of the binding of these 2 ligands. [3H]triphenylmethylphosphonium, previously identified as a channel blocker, can be covalently incorporated into Ach receptor-rich membranes from Torpedo electric tissue by UV irradiation of the receptor-ligand complex. In the absence of effector, predominantly the .alpha.-polypeptide chains (MW 40,000) of the receptor protein are labeled by the radioactive ligand. The agonist carbamoylcholine strongly stimulates the labeling, but it directs the label predominantly to the .delta.- and .beta.-polypeptide chains. The antagonist D-tubocurarine and the virtually irreversible competitive antagonist .alpha.-bungarotoxin have qualitatively the same effect as the agonist carbamoylcholine. Significant differences were obtained with receptor-rich membranes prepared from Torpedo marmorata and T. californica. No agonist- or antagonist-stimulated reaction was observed with the latter. The results are interpreted as an indication of a rearrangement of the receptor''s quaternary structure caused by cholinergic effector binding preceding discrimination between agonists and antagonists.