PT - JOURNAL ARTICLE AU - Uta Voigtländer AU - Kirstin Jöhren AU - Marion Mohr AU - Alexandra Raasch AU - Christian Tränkle AU - Stefan Buller AU - John Ellis AU - Hans-Dieter Höltje AU - Klaus Mohr TI - Allosteric Site on Muscarinic Acetylcholine Receptors: Identification of Two Amino Acids in the Muscarinic M<sub>2</sub> Receptor That Account Entirely for the M<sub>2</sub>/M<sub>5</sub> Subtype Selectivities of Some Structurally Diverse Allosteric Ligands in <em>N</em>-Methylscopolamine-Occupied Receptors AID - 10.1124/mol.64.1.21 DP - 2003 Jul 01 TA - Molecular Pharmacology PG - 21--31 VI - 64 IP - 1 4099 - http://molpharm.aspetjournals.org/content/64/1/21.short 4100 - http://molpharm.aspetjournals.org/content/64/1/21.full SO - Mol Pharmacol2003 Jul 01; 64 AB - 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,Pi 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.