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

Jan Jakubík,Vladimír Doležal,Pavel Zimčík,Esam E El-Fakahany,Alena Randáková

doi:10.1038/srep40381

Abstract

Interaction of orthosteric ligands with extracellular domain was described at several aminergic G protein-coupled receptors, including muscarinic acetylcholine receptors. The orthosteric antagonists quinuclidinyl benzilate (QNB) and N-methylscopolamine (NMS) bind to the binding pocket of the muscarinic acetylcholine receptor formed by transmembrane α-helices. We show that high concentrations of either QNB or NMS slow down dissociation of their radiolabeled species from all five subtypes of muscarinic acetylcholine receptors, suggesting allosteric binding. The affinity of NMS at the allosteric site is in the micromolar range for all receptor subtypes. Using molecular modelling of the M2 receptor we found that E172 and E175 in the second extracellular loop and N419 in the third extracellular loop are involved in allosteric binding of NMS. Mutation of these amino acids to alanine decreased affinity of NMS for the allosteric binding site confirming results of molecular modelling. The allosteric binding site of NMS overlaps with the binding site of some allosteric, ectopic and bitopic ligands. Understanding of interactions of NMS at the allosteric binding site is essential for correct analysis of binding and action of these ligands.

Highlights

Equilibrium dissociation constants (KD) of NMS and quinuclidinyl benzilate (QNB) binding to the orthosteric site are expressed as negative logarithm
N-methylscopolamine ([3H]NMS) and quinuclidinyl benzilate ([3H]QNB) bound to individual subtypes of muscarinic receptors with similar affinity, that was slightly lower at M2 receptors (Table 1)
Similar apparent affinities of NMS for the allosteric site were found by measurement of the effects of various concentrations of NMS on [3H]QNB dissociation (Supplementary data, Figure SI2, Table SI1)

Summary

Introduction

Equilibrium dissociation constants (KD) of NMS and QNB binding to the orthosteric site are expressed as negative logarithm. Dissociation rate constants are expressed in per minute rate and apparent affinity of NMS for the allosteric site (KA) is expressed as negative logarithm. Amino acids at the vestibule (Fig. 1) of the binding pocket between ECL2 and ECL3 with which NMS interacts in an allosteric manner. This finding complicates interpretation of experiments in which radiolabeled antagonists such as NMS and QNB that are presumed to interact exclusively with the receptor orthosteric domain are used as tracers in studying of binding properties of other ligands that are not available in a radioactive form

Methods

Results

Conclusion