TY - JOUR T1 - Molecular Mechanisms of Methoctramine Binding and Selectivity at Muscarinic Acetylcholine Receptors JF - Molecular Pharmacology JO - Mol Pharmacol SP - 180 LP - 192 DO - 10.1124/mol.114.093310 VL - 86 IS - 2 AU - Jan Jakubík AU - Pavel Zimčík AU - Alena Randáková AU - Květoslava Fuksová AU - Esam E. El-Fakahany AU - Vladimír Doležal Y1 - 2014/08/01 UR - http://molpharm.aspetjournals.org/content/86/2/180.abstract N2 - Methoctramine (N,N'-bis[6-[[(2-methoxyphenyl)-methyl]hexyl]-1,8-octane] diamine) is an M2-selective competitive antagonist of muscarinic acetylcholine receptors and exhibits allosteric properties at high concentrations. To reveal the molecular mechanisms of methoctramine binding and selectivity we took advantage of reciprocal mutations of the M2 and M3 receptors in the second and third extracellular loops that are involved in the binding of allosteric ligands. To this end we performed measurements of kinetics of the radiolabeled antagonists N-methylscopolamine (NMS) in the presence of methoctramine and its precursors, fluorescence energy transfer between green fluorescent protein–fused receptors and an Alexa-555–conjugated precursor of methoctramine, and simulation of molecular dynamics of methoctramine association with the receptor. We confirm the hypothesis that methoctramine high-affinity binding to the M2 receptors involves simultaneous interaction with both the orthosteric binding site and the allosteric binding site located between the second and third extracellular loops. Methoctramine can bind solely with low affinity to the allosteric binding site on the extracellular domain of NMS-occupied M2 receptors by interacting primarily with glutamate 175 in the second extracellular loop. In this mode, methoctramine physically prevents dissociation of NMS from the orthosteric binding site. Our results also demonstrate that lysine 523 in the third extracellular loop of the M3 receptors forms a hydrogen bond with glutamate 219 of the second extracellular loop that hinders methoctramine binding to the allosteric site at this receptor subtype. Impaired interaction with the allosteric binding site manifests as low-affinity binding of methoctramine at the M3 receptor. ER -