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

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 wildtype 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.