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 [ 3 H]NMS (5 mM Na,K,P i buffer, pH 7.4, 23°C 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 NMSfree 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 ϳ10-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.Muscarinic acetylcholine receptors are members of the rhodopsin-like family of G protein-coupled receptors, which share general structural motifs, including seven hydrophobic transmembrane helices connected by intracellular and extracellular loops, an extracellular amino terminus, and a cytoplasmic carboxyl terminus. Molecular cloning studies revealed the existence of five (M 1 -M 5 ) muscarinic acetylcholine receptors (Bonner et al., 1987), all of which are susceptible to allosteric modulation (Ellis et al., 1991). The orthosteric acetylcholine binding site seems to be lined by the transmembrane helices (Wess, 1993) and seems to be highly conserved among the five subtypes (Hulme et al., 1990). The allosteric binding site is located at the entrance of the ligand binding pocket (Ellis et al., 1993;Leppik et al., 1994) and is likely to be less well conserved than the orthosteric ligand binding site, thus potentially allowing the design of ligands with greater subtype selectivity (Tuček and Proška, 1995). The family of the muscarinic acetylcholine receptors has been widely studied as a model system for the interaction of allosteric modulators with G protein-coupled receptors (Christopoulos and Kenakin, 2002).It is striking that muscarinic allosteric ligands exhibit generally the highest affinity to the M 2 receptor subtype (Lee and El-Fakahany, 1991;Ellis et al., 1991;Ellis and Seidenberg, 2000). A number of studies aimed to identify receptor This article is dedicated to the late Dr. Stanislav Tuček, Academy of Scien...

No abstract