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CELLULAR AND MOLECULAR

Application of a Kinetic Model to the Apparently Complex Behavior of Negative and Positive Allosteric Modulators of Muscarinic Acetylcholine Receptors

Department of Pharmacology (V.A., L.T.M., P.M.S., A.C.) and Howard Florey Institute of Experimental Physiology and Medicine (V.A., P.M.S., A.C.), University of Melbourne, Parkville, Victoria, Australia

The binding of allosteric modulators to G protein-coupled receptors (GPCRs) is often described by an equilibrium allosteric ternary complex model (ATCM). This study evaluated the effects of three modulators on the binding of [³H]N-methylscopolamine ([³H]NMS) to the human M₂ muscarinic acetylcholine receptor (mAChR). The binding of each modulator was more complex than predicted by the ATCM; the inhibitors heptane-1,7-bis-(dimethyl-3-phthalimidopropyl)-ammonium bromide and gallamine yielded biphasic curves that were described empirically by a two-site binding model, whereas the enhancer alcuronium yielded a bell-shaped curve. Radioligand dissociation assays revealed that the modulators retarded [³H]NMS kinetics such that the system never attained equilibrium. Subsequent application of a kinetic ATCM accommodated and quantified all experimental observations. Our findings confirm and extend previous studies on the use of a kinetic ATCM for mAChR allosteric enhancers, but also highlight how complex curves displayed by allosteric inhibitors can be misinterpreted in terms of multisite orthosteric binding. It is possible that similar behavior of other allosteric modulators at GPCRs may reflect nonequilibrium binding artifacts rather than deviation from an ATCM.

Address correspondence to: Dr. Arthur Christopoulos, National Health and Medical Research Council Senior Research Fellow, Department of Pharmacology, University of Melbourne, Grattan St., Parkville, 3010, VIC, Australia. E-mail: arthurc1{at}unimelb.edu.au

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