Site-directed mutagenesis reveals two epitopes involved in the subtype selectivity of the allosteric interactions of gallamine at muscarinic acetylcholine receptors.

Gallamine allosterically modulates the binding of classical muscarinic ligands with a potency order of M(2) > M(1),M(4) > M(3), M(5). We have suggested previously that the M(2)/M(5) and M(2)/M(3) selectivities are attributable to an epitope in the sixth transmembrane region or third outer loop (o3) region of the receptor. In this study, analysis of numerous point mutations in this region of the M(5) receptor found that a mutation of V --> N resulted in an increased affinity toward gallamine, suggesting that the asparagine residue at M(2)(419) is responsible for gallamine's M(2)/M(5) selectivity. Mutations in the other subtypes indicated that the acidic residues found at this position in M(1) and M(4) are associated with slightly higher affinity toward gallamine, whereas the valine and lysine residues of M(5) and M(3), respectively, are associated with significantly lower affinity. In the o2 region, replacement of an acidic sequence of M(2) (EDGE) by the corresponding neutral sequence of M(1) (LAGQ) reduced the affinity toward gallamine, as reported previously by others; the converse substitution of the acidic sequence into M(1) significantly increased affinity for gallamine. Substitution of the M(1) sequence into this region of M(5) markedly reduced affinity toward gallamine, whereas substitution into M(4) had no effect. All of the above mutations are consistent with gallamine binding with a similar orientation at each subtype, such that it interacts with acidic residues in the o2 region of M(3) and M(5) and with acidic residues in the o3 region of M(1) and M(4); gallamine appears to interact with both regions of the M(2) subtype.