Abstract

The purpose of this study was to explore the role of acidic amino acids in the allosteric behavior of gallamine at the m2 receptor. This was achieved by first mutating the acidic residues to neutral residues by site-directed mutagenesis. Both the parent and mutated receptors were expressed in mouse fibroblast A9L cells and characterized pharmacologically. The two main methods used were (i) Schild analysis of equilibrium binding data and (ii) study of the effect of gallamine on the dissociation kinetics of N-methylscopolamine. The Schild analysis gave an estimate of the affinity of gallamine for the allosteric site (KdA) and also a measure of the level of cooperativity (alpha) between the allosteric and primary binding sites. For the receptors studied, a good agreement was found between the alpha KdA values calculated from the Schild analysis and the IC50 values for the effect of gallamine on the N-methylscopolamine off-rate. One mutated receptor, in which the acidic EDGE (Glu-Asp-Gly-Glu) sequence of the putative third outer domain was changed to the neutral LAGQ (Leu-Ala-Gly-Gin) sequence, displayed an 8-fold reduction in affinity for gallamine at the allosteric site, in comparison with the parent receptor. The level of cooperatively between the allosteric and primary binding sites in this mutant was 46% of that of the parent receptor. A second mutated receptor, in which Asp-97 (near the top of putative transmembrane domain 3) was changed to asparagine, was found to have a level of cooperativity between sites 58% of that of the parent but was found not to be affected with respect to the affinity of gallamine for the allosteric site. When all of the acidic groups on the outer side were changed to neutral residues, there was still only an 8.6-fold reduction in gallamine affinity for the allosteric site, but the level of cooperativity was reduced to 19% of that found in the parent receptor. The results suggest that the allosteric site for gallamine binding in the m2 receptor residues at or near the putative third outer domain and that both the EDGE motif and Asp-97 play an essential role in the interaction between the two sites. However, none of the acidic amino acids mutated were found to be critical for binding at the allosteric site.