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

• Published in: Molecular pharmacology Vol. 64; no. 1; p. 21
• Main Authors: Voigtländer, Uta, Jöhren, Kirstin, Mohr, Marion, Raasch, Alexandra, Tränkle, Christian, Buller, Stefan, Ellis, John, Höltje, Hans-Dieter, Mohr, Klaus
• Format: Journal Article
• Language: English
• Published: United States American Society for Pharmacology and Experimental Therapeutics 01.07.2003
• Subjects: Allosteric Regulation - physiology; Allosteric Site; Amino Acids; Animals; COS Cells; Humans; Ligands; Models, Molecular; Mutagenesis, Site-Directed; N-Methylscopolamine - pharmacology; Parasympatholytics - pharmacology; Receptor, Muscarinic M2; Receptors, Muscarinic - chemistry; Receptors, Muscarinic - genetics; Receptors, Muscarinic - metabolism
• ISSN: 0026-895X; 1521-0111
• DOI: 10.1124/mol.64.1.21

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 wild-type 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). 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 ∼10-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.