title: 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.
abstract: 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 degrees 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 approximately 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.
authors.affiliation: Department of Pharmacology and Toxicology, Institute of Pharmacy, University of Bonn, 53121 Bonn, Germany.
authors.forename: Uta
authors.initials: U
authors.lastname: Voigtländer
authors.forename: Kirstin
authors.initials: K
authors.lastname: Jöhren
authors.forename: Marion
authors.initials: M
authors.lastname: Mohr
authors.forename: Alexandra
authors.initials: A
authors.lastname: Raasch
authors.forename: Christian
authors.initials: C
authors.lastname: Tränkle
authors.forename: Stefan
authors.initials: S
authors.lastname: Buller
authors.forename: John
authors.initials: J
authors.lastname: Ellis
authors.forename: Hans-Dieter
authors.initials: HD
authors.lastname: Höltje
authors.forename: Klaus
authors.initials: K
authors.lastname: Mohr
mesh_terms: D000494:Allosteric Regulation / Q000502:physiology*; D000495:Allosteric Site; D000596:Amino Acids; D000818:Animals; D019556:COS Cells; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D019832:N-Methylscopolamine / Q000494:pharmacology*; D010276:Parasympatholytics / Q000494:pharmacology; D043585:Receptor, Muscarinic M2; D011976:Receptors, Muscarinic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D008024:Ligands; D010276:Parasympatholytics; D043585:Receptor, Muscarinic M2; D011976:Receptors, Muscarinic; D019832:N-Methylscopolamine