Abstract

Three azido derivatives of ethidium bromide, a potent noncompetitive antagonist of the nicotinic acetylcholine receptor from Torpedo californica, were synthesized, namely 8-azido-ethidium chloride, 3-azido-ethidium chloride, and 3,8-diazido-ethidium chloride. These derivatives were tested for their ability to interact with the noncompetitive antagonist binding site and the acetylcholine binding sites on the acetylcholine receptor. The derivatives bound to the noncompetitive antagonist site with 2-5-fold lower affinity than did ethidium bromide, as determined by competitive inhibition of [3H]phencyclidine binding, indicating a moderate effect of the azide groups upon binding. Inhibition of [3H]-acetylcholine binding by ethidium and its azide derivatives indicated differential binding to the two agonist sites, with high affinity binding to the same site that exhibits high affinity for d-tubocurarine. Photoaffinity labeling by these derivatives revealed reaction with the alpha and gamma subunits that was specific for the acetylcholine binding sites. Inhibition of labeling by d-tubocurarine showed reaction with alpha subunits at both of the acetylcholine binding sites, whereas reaction with the gamma subunit was consistent with reaction only at the site with high affinity for d-tubocurarine. There was no corresponding reaction with the delta subunit, which forms part of the second acetylcholine binding site, despite reaction with the apposing alpha subunit. The azides, therefore, display preferential reaction with the gamma subunit. The selectivity of the reaction must reflect structural differences between the two sites, and subsequent determination of the labeled site(s) should reveal the nature of the differences.