Thiol-group modification of Torpedo californica acetylcholine receptor: subunit localization and effects on function

The effects of thiol-group modifications on acetylcholine receptor (ACHR) function were measured with purified ACHR reconstituted into asolectin vesicles. N-Phenylmaleimide (NPM) was used to modify sulfhydryl groups on ACHR in the absence of any prior reduction of dithiothreitol, so that only the functional relevance of free sulfhydryls was examined. Modification by NPM led to the inhibition of ion-channel activity without a detectable effect on ligand binding. The ion flux inhibition by NPM primarily affected channel activation, since the initial rates of activation were decreased over a wide range of carbamylcholine concentrations. The [3H]NPM subunit labeling pattern of ACHR (a multisubunit membrane protein with alpha 2 beta gamma delta stoichiometry) revealed that there was preferential labeling of the gamma subunit. At high NPM concentrations, the number of sulfhydryl groups on the gamma subunit that could be modified with NPM was approximately two. Detergent was required during labeling for functionally relevant thiol-group modifications, and most of the label was protected from protease digestion in the reconstituted membranes. These results are consistent with the presence of the NPM modification in a bilayer and/or cytoplasmic domain.