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The Torpedo nicotinic acetylcholine receptor is a heteropentamer (α2βγδ) in which structurally homologous subunits assemble to form a central ion pore. Viewed from the synaptic cleft, the likely arrangement of these subunits is α–γ–α–δ–β lying in an anticlockwise orientation. High affinity binding sites for agonists and competitive antagonists have been localized to the α–γ and α–δ subunit interfaces. We investigated the involvement of amino acids lying at an adjacent interface (γ–α) in receptor properties. Recombinant Torpedo receptors, expressed in Xenopus oocytes, were used to investigate the consequences of mutating αArg55 and γGlu93, residues that are conserved in most species of the peripheral nicotinic receptors. Based on homology modeling, these residues are predicted to lie in close proximity to one another and it has been suggested that they may form a salt bridge in the receptor's three-dimensional structure (Sine et al. 2002 J Biol Chem277, 29 210–29 223). Although substitution of αR55 by phenylalanine or tryptophan resulted in approximately a six-fold increase in the EC50 value for acetylcholine activation, the charge reversal mutation (αR55E) had no significant effect. In contrast, the replacement of γE93 by an arginine conferred an eight-fold increase in the potency for acetylcholine-induced receptor activation. In the receptor carrying the double mutations, αR55E-γE93R or αR55F-γE93R, the potency for acetylcholine activation was partially restored to that of the wild-type. The results suggest that, although individually these residues influence receptor activation, direct interactions between them are unlikely to play a major role in the stabilization of different conformational states of the receptor.