Abstract

Gallamine allosterically modulates the binding of classical muscarinic ligands with a potency order of M₂ > M₁,M₄ > M₃,M₅. We have suggested previously that the M₂/M₅ and M₂/M₃ 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 M₅ receptor found that a mutation of V → N resulted in an increased affinity toward gallamine, suggesting that the asparagine residue at M₂ ⁴¹⁹is responsible for gallamine's M₂/M₅selectivity. Mutations in the other subtypes indicated that the acidic residues found at this position in M₁ and M₄are associated with slightly higher affinity toward gallamine, whereas the valine and lysine residues of M₅ and M₃, respectively, are associated with significantly lower affinity. In the o2 region, replacement of an acidic sequence of M₂(EDGE) by the corresponding neutral sequence of M₁(LAGQ) reduced the affinity toward gallamine, as reported previously by others; the converse substitution of the acidic sequence into M₁ significantly increased affinity for gallamine. Substitution of the M₁ sequence into this region of M₅ markedly reduced affinity toward gallamine, whereas substitution into M₄ 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 M₃ and M₅ and with acidic residues in the o3 region of M₁ and M₄; gallamine appears to interact with both regions of the M₂subtype.