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 M-2 muscarinic acetylcholine receptors, relative to M-5 receptors: the amino acid M-2-Thr(423) at the top of transmembrane region (TM) 7 and an epitope comprising the second extracellular loop (o2) of the M-2 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 [H-3] NMS (5 mM Na, K, P-i buffer, pH 7.4, 23 degreesC). Using M-2/M-5 chimeric and point-mutated receptors, the relevant epitope was narrowed down to M-2-Tyr(177). A double point-mutated M-2 receptor in which both M-2-Tyr(177) and M-2-Thr(423) were replaced by the corresponding amino acids of M-5 revealed that these two amino acids account entirely for the ( approximately 100-fold) M-2/M-5 selectivity of the alkane-bisammonium and the caracurine V type allosteric ligands. At NMS-free M-2 receptors, the caracurine V derivative also displayed approximately 100-fold M-2/M-5 selectivity, but the double point mutation reduced the M-2 affinity by only similar to10-fold; thus, additional epitopes may influence selectivity for the free receptors. A three-dimensional model of the M-2 receptor was used to simulate allosteric agent docking to NMS-occupied receptors. M-2-Tyr(177) and M-2-Thr(423) seem to be located near the junction of the allosteric and the orthosteric areas of the M-2 receptor ligand binding cavity.