Molecular Mechanisms of Methoctramine Binding and Selectivity at Muscarinic Acetylcholine Receptors

Abstract

Methoctramine (N,-methyl]hexyl]-1,8-octane] diamine) is an M 2 -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 M 2 and M 3 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 M 2 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 NMSoccupied M 2 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 M 3 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 M 3 receptor.

Binding of N-methylscopolamine to the extracellular domain of muscarinic acetylcholine receptors

“…The extracellular domain of muscarinic receptors has been shown to be essential in binding of many allosteric modulators7891011121314, ectopic ligands15 and bitopic ligands161718. For example, glutamates E172 and E175 with which NMS interacts are part of the EDGE motif that has been shown to be essential for binding of the prototypic allosteric modulator gallamine7.…”

“…Therefore, attention of researchers has turned to ligands that interact with the extracellular domain of the muscarinic receptor that differs among receptor subtypes. The extracellular domain of muscarinic receptors has been shown to bind a wide variety of selective ligands including allosteric modulators7891011121314, ectopic ligands15 and bitopic ligands161718.…”

Distinctive binding properties of the negative allosteric modulator, [ 3 H]SB269,652, at recombinant dopamine D 3 receptors

“…binds to one protomer in a dimer, it does not alter the binding of molecules of SB269,652 to the other protomer. A similar mechanism of binding and allostery has been described for the antagonist methoctramine at muscarinic M 2 receptors (Jakubík et al, 2014). refer to specific and unspecific binding in the absence and presence of 2 mM dopamine set at 100%…”

Current Advances in Allosteric Modulation of Muscarinic Receptors

“…Detailed analysis of methoctramine binding has revealed two modes of interaction with the receptor. Transient binding to the common allosteric site center 1 (namely E175 in the o2 loop) is followed by stable binding that occurs concurrently to both the allosteric and orthosteric sites [61]. Methoctramine is thus a bitopic dualsteric antagonist.…”