Interactions of orthosteric and allosteric ligands with [3H]dimethyl-W84 at the common allosteric site of muscarinic M2 receptors

An optimized assay for the binding of 3H]dimethyl-W84 to its allosteric site on M2 muscarinic receptors has been used to directly measure the affinities of allosteric ligands. Their potencies agree with those deduced indirectly by their modulation of the equilibrium binding and kinetics of 3H]N-methylscopolamine (3H]NMS) binding to the orthosteric site. The affinities and cooperativities of orthosteric antagonists with 3H]dimethyl-W84 have also been quantitated. These affinities agree with those measured directly in a competition assay using 3H]NMS. All these data are compatible with the predictions of the allosteric ternary complex model. The association and dissociation kinetics of 3H]dimethyl-W84 are rapid but the estimate of its association rate constant is nevertheless comparable with that found for the orthosteric radioligand, 3H]NMS. This is unexpected, given that the allosteric site to which 3H]dimethyl-W84 binds is thought to be located on the external face of the receptor and above the 3H]NMS binding site that is buried within the transmembrane helices. The atypical allosteric ligands tacrine and 4,4'-bis-(2,6-dichloro-benzyloxy-imino)-methyl]-1,1'-propane-1,3-diyl-bis-pyridinium dibromide (Duo3) inhibit 3H]dimethyl-W84 binding with the same potencies and comparably steep slope factors as found for inhibition of 3H]NMS binding. Tacrine and Duo3 decrease 3H]dimethyl-W84 affinity, not the number of binding sites. It is suggested that these atypical ligands either bind to the two known spatially separated allosteric sites on muscarinic receptors with positive cooperativity or their binding to the common allosteric site modulates receptor-receptor interactions such that homotropic positive cooperativity within a dimer or higher oligomer is generated.