Synergy Between Pairs of Competitive Antagonists at Adult Human Muscle Acetylcholine Receptors

Abstract

Background Synergistic neuromuscular blocking effects have been observed clinically with certain pairs of nicotinic acetylcholine receptor (nAChR) competitive antagonists. The mechanism for synergy has not been elucidated. We tested the hypothesis that synergy arises from a differential selectivity of antagonists for the two ligand binding sites on adult human nAChR. Methods We expressed nAChR in BOSC23 cells. We applied ACh with or without antagonists to outside-out patches and measured macroscopic currents at room temperature. We determined the IC90 for (+)-tubocurarine, metocurine, pancuronium, vecuronium, cisatracurium, rocuronium, and atracurium. For 15 combinations of two antagonists, we determined the IC90 for one antagonist in the presence of the IC70 of a second antagonist. We constructed isobolograms for 90% inhibition. For single antagonists, we measured inhibition of receptors containing mutations in the ε- and δ-subunits to determine site selectivity. Results Two pairs of antagonists, metocurine+cisatracurium and cisatracurium+atracurium exhibited additive inhibition. Ten combinations, including (+)-tubocurarine+pancuronium and pancuronium+vecuronium, were highly synergistic such that the combination was 2−3 times more effective than expected for additivity. Three combinations were 1.5−1.6 times more effective than expected for additivity. Inhibition by (+)-tubocurarine and metocurine was sensitive to mutations in the ε-subunit only. Vecuronium was affected by the δ-subunit mutation only. Inhibition by other antagonists was decreased by mutations in either subunit. Conclusions Many combinations of antagonists exhibited synergistic effects on adult human nAChR. Synergy was observed with structurally similar as well as dissimilar antagonists. The degree of synergy did not always correlate well with site specificity assayed with mutants. In some, but not all cases, the synergy at the receptor level correlated with clinical determinations of synergy. We conclude that the synergistic actions of muscle relaxants can be partially explained by direct interactions with adult human nAChR.

Site Selectivity of Competitive Antagonists for the Mouse Adult Muscle Nicotinic Acetylcholine Receptor

“…In our previous paper (Liu and Dilger, 2008), we measured inhibition of adult human nAChR in the presence of one or two antagonists. An analysis of this data in terms of Equation 3 is presented in Table 4.…”

“…This approach circumvents some the problems that can arise from using mutagenesis to determine site selectivity (Dilger et al, 2007; Liu and Dilger, 2008) and may have more general applicability to other ligand-gated ion channels.…”

“… Previously published one- and two- antagonist inhibition data for adult human nAChR (Liu and Dilger, 2008) were fitted to Equation 3 using the same approach that was used for adult mouse nAChR (Table 3). Because the Hill slope of the rocuronium (alone) data on adult human nAChR was small (0.67), the fits to Equation 3 did not converge.…”

“…This provides a more sensitive test for synergistic effects (Nigrovic and Amann, 2004; Waud and Waud, 1985). Previously, we used this approach in a study of adult human nAChR (Liu and Dilger, 2008) and found evidence for synergy; in some cases, synergy at the receptor level correlated with the clinical phenomenon of muscle relaxant synergy in which pairs of antagonist are more effective at inducing paralysis than expected from their individual potencies (Lebowitz et al, 1980; Waud and Waud, 1984). …”

Three Arginines in the GABAAReceptor Binding Pocket Have Distinct Roles in the Formation and Stability of Agonist- versus Antagonist-Bound Complexes

“…Interestingly, competitive antagonists for the homologous nACh receptor known to bind preferentially to one or the other nonidentical binding site illustrate that binding of a single antagonist molecule is sufficient to prevent channel opening in a cysteine-loop receptor (Wenningmann and Dilger, 2001;Dilger et al, 2007). Furthermore, pairs of nACh receptor antagonists often exhibit cooperative effects, possibly because antagonist binding at one site allosterically influenced antagonist binding at the other site (Liu and Dilger, 2008). We conclude that SR-95531 either preferentially binds to only one of the two GABA binding sites or allosterically inhibits itself from binding to both sites simultaneously.…”