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Allosteric Site on Muscarinic Acetylcholine Receptors: Identification of Two Amino Acids in the Muscarinic M₂ Receptor That Account Entirely for the M₂/M₅ Subtype Selectivities of Some Structurally Diverse Allosteric Ligands in N-Methylscopolamine-Occupied Receptors

Department of Pharmacology and Toxicology, Institute of Pharmacy, University of Bonn, Bonn, Germany (U.V., M.M., A.R., C.T., S.B., K.M.); Institute of Pharmaceutical Chemistry, University of Düsseldorf, Düsseldorf, Germany (K.J., H.-D. H.); and Departments of Psychiatry and Pharmacology, Penn State College of Medicine, Hershey, Pennsylvania, USA (S.B., J.E.).

Two epitopes have been identified recently to be responsible for the high-affinity binding of alkane-bisammonium and caracurine V type allosteric ligands to N-methylscopolamine (NMS)-occupied M₂ muscarinic acetylcholine receptors, relative to M₅ receptors: the amino acid M₂-Thr⁴²³ at the top of transmembrane region (TM) 7 and an epitope comprising the second extracellular loop (o2) of the M₂ receptor including the flanking regions of TM4 and TM5. We aimed to find out whether a single amino acid could account for the contribution of this epitope to binding affinity. Allosteric interactions were investigated in wild-type and mutant receptors in which the orthosteric binding site was occupied by [³H]NMS (5 mM Na,K,P_i buffer, pH 7.4, 23°C). Using M₂/M₅ chimeric and point-mutated receptors, the relevant epitope was narrowed down to M₂-Tyr¹⁷⁷. A double point-mutated M₂ receptor in which both M₂-Tyr¹⁷⁷ and M₂-Thr⁴²³ were replaced by the corresponding amino acids of M₅ revealed that these two amino acids account entirely for the (approximately 100-fold) M₂/M₅ selectivity of the alkane-bisammonium and the caracurine V type allosteric ligands. At NMS-free M₂ receptors, the caracurine V derivative also displayed approximately 100-fold M₂/M₅ selectivity, but the double point mutation reduced the M₂ affinity by only 10-fold; thus, additional epitopes may influence selectivity for the free receptors. A three-dimensional model of the M₂ receptor was used to simulate allosteric agent docking to NMS-occupied receptors. M₂-Tyr¹⁷⁷ and M₂-Thr⁴²³ seem to be located near the junction of the allosteric and the orthosteric areas of the M₂ receptor ligand binding cavity.

Address correspondence to: Dr. Klaus Mohr, Department of Pharmacology and Toxicology, Institute of Pharmacy, University of Bonn, 53121 Bonn, Germany. E-mail: k.mohr{at}uni-bonn.de

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