Abstract

Acetylcholine receptors in the innervated membrane of an electroplax cell from the electric eel Electrophorus electricus may be affinity-labeled, after disulfide reduction, by a variety of sulfhydryl alkylating and acylating agents. The conductance state of a cell after this receptor modification remains under receptor control but is extremely sensitive both to the structure of the affinity reagent and, frequently, to the concentration of the reagent during modification. When the affinity reagents 3-(α-bromomethyl)-3'-[α-(trimethylammonium)methyl]azobenzene bromide or 4-(4'-nitrophenoxycarbonyl)-phenyltrimethylammonium iodide are used in low concentration to label the receptor in situ, subsequent sequential applications of disulfides and dithiothreitol give rise to cycles of repolarization and depolarization of the innervated membrane. Since other mild oxidizing agents cannot substitute for disulfides in this activation cycle, it is concluded that the activation cycle arises from the reversible formation of a mixed disulfide on the receptor. This mixed disulfide probably involves the remaining sulfhydryl group near the acetylcholine binding site, which is formed by the initial disulfide reduction but not labeled by the affinity reagents. The membrane responses to these chemical manipulations of the receptor in situ suggest useful criteria for evaluating the similarity of an isolated receptor after reconstitution into black lipid membranes to the receptor in its native state.