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Interactions of Orthosteric and Allosteric Ligands with [³H]Dimethyl-W84 at the Common Allosteric Site of Muscarinic M₂ Receptors

Department of Pharmacology and Toxicology, Institute of Pharmacy, University of Bonn, Bonn, Germany (C.T., O.W., U.V., K.M.); Division of Physical Biochemistry, National Institute for Medical Research, Mill Hill, London, United Kingdom (A.M., N.J.M.B.); and MRC Technology, Mill Hill, London, United Kingdom (S.L.)

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.

Address correspondence to: Dr. Christian Tränkle, Department of Pharmacology and Toxicology, Institute of Pharmacy, University of Bonn, Gerhard-Domagk-Strasse 3, D-53121 Bonn, Germany. E-mail: traenkle{at}uni-bonn.de

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