Methoctramine high-affinity binding to the M2 receptors involves simultaneous interaction with both the orthosteric and the allosteric binding sites. 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.
Jakubík, J., Zimčík, P., Randáková, A., Fuksová, K., El-Fakahany, E.E. & Doležal, V. Molecular mechanisms of methoctramine binding and selectivity at muscarinic acetylcholine receptors. Mol Pharmacol 86, 180-92 (2014).
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.
Simulation of molecular dynamic of methoctramine association with M2 receptors.
Three stages of molecular dynamics are displayed: Initial (left), transient (middle) and final (right). Extracellular part of the M2 receptor is up and TM IV and V are in front. Backbone of the receptor is colored by position in red to white to blue gradient. Side chains of D103, E172, E175, N404 and Y430 are displayed. Cyan - carbon, blue – nitrogen, green – carbon of methoctramine, red – oxygen; yellow – hydrogen bonds.