Site selectivity of competitive antagonists for the mouse adult muscle nicotinic acetylcholine receptor

The muscle-type nicotinic acetylcholine receptor has two non-identical binding sites for ligands. The selectivity of acetylcholine and the competitive antagonists (+)-tubocurarine and me-tocurine for adult mouse receptors is known. Here, we examine the site selectivity for four other competitive antagonists: cisa-tracurium, pancuronium, vecuronium, and rocuronium. We rapidly applied acetylcholine to outside-out patches from trans-fected BOSC23 cells and measured macroscopic currents. We have reported the IC ₅₀ of the antagonists individually in prior publications. Here, we determined inhibition by pairs of competitive antagonists. At least one antagonist was present at a concentration producing >67% receptor inhibition. Metocurine shifted the apparent IC ₅₀ of (+)-tubocurarine in quantitative agreement with complete competitive antagonism. The same was observed for pancuronium competing with vecuronium. However, pancuronium and vecuronium each shifted the apparent IC ₅₀ of (+)-tubocurarine less than expected for complete competition but more than expected for independent binding. The situation was similar for cisatracurium and (+)-tubocurarine or metocurine. Cisatracurium did not shift the apparent IC ₅₀ of pancuronium or vecuronium, indicating independent binding of these two pairs. The data were fit to a two-site, two-antagonist model to determine the antagonist binding constants for each site, L _αε and L _αδ. We found L _αε/L _αδ = 0.22 (range, 0.14-0.34), 20 (9-29), 21 (4-36), and 1.5 (0.3-2.9) for cisatracurium, pan-curonium, vecuronium, and rocuronium, respectively. The wide range of L _αε/L _αδ for some antagonists may reflect experimental uncertainties in the low affinity site, relatively poor selectivity (rocuronium), or possibly that the binding of an antagonist at one site affects the affinity of the second site.