SITE-SELECTIVE PHOTOAFFINITY-LABELING OF THE TORPEDO-CALIFORNICA NICOTINIC ACETYLCHOLINE-RECEPTOR BY AZIDE DERIVATIVES OF ETHIDIUM-BROMIDE

Three azido derivatives of ethidium bromide, a potent noncompetitive a ntagonist of the nicotinic acetylcholine receptor from Torpedo califor nica, were synthesized, namely 8-azido-ethidium chloride, 3-azido-ethi dium chloride, and 3,8-diazido-ethidium chloride. These derivatives we re tested for their ability to interact with the noncompetitive antago nist binding site and the acetylcholine binding sites on the acetylcho line receptor. The derivatives bound to the noncompetitive antagonist site with 2-5-fold lower affinity than did ethidium bromide, as determ ined by competitive inhibition of [H-3]phencyclidine binding, indicati ng a moderate effect of the azide groups upon binding. Inhibition of [ H-3]-acetylcholine binding by ethidium and its azide derivatives indic ated differential binding to the two agonist sites, with high affinity binding to the same site that exhibits high affinity for d-tubocurain e. Photoaffinity labeling by these derivatives revealed reaction with the alpha and gamma subunits that was specific for the acetylcholine b inding sites. Inhibition of labeling by d-tubocurarine showed reaction with alpha subunits at both of the acetylcholine binding sites, where as reaction with the gamma subunit was consistent with reaction only a t the site with high affinity for d-tubocuraine. There was no correspo nding reaction with the delta subunit, which forms part of the second acetylcholine binding site, despite reaction with the apposing alpha s ubunit. 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 th e labeled site(s) should reveal the nature of the differences.