RT Journal Article
SR Electronic
T1 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
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 21
OP 31
DO 10.1124/mol.64.1.21
VO 64
IS 1
A1 Uta Voigtländer
A1 Kirstin Jöhren
A1 Marion Mohr
A1 Alexandra Raasch
A1 Christian Tränkle
A1 Stefan Buller
A1 John Ellis
A1 Hans-Dieter Höltje
A1 Klaus Mohr
YR 2003
UL http://molpharm.aspetjournals.org/content/64/1/21.abstract
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.