Mapping the Allosteric Action of Antagonists A740003 and A438079 Reveals a Role for the Left Flipper in Ligand Sensitivity at P2X7 Receptors

Rebecca C Allsopp,Anfal Bin Dayel,Richard J Evans,Ralf Schmid,Sudad Dayl

doi:10.1124/mol.117.111021

Abstract

P2X7 receptor (P2X7R) activation requires ∼100-fold higher concentrations of ATP than other P2X receptor (P2XR) subtypes. Such high levels are found during cellular stress, and P2X7Rs consequently contribute to a range of pathophysiological conditions. We have used chimeric and mutant P2X7Rs, coupled with molecular modeling, to produce a validated model of the binding mode of the subtype-selective antagonist A438079 at an intersubunit allosteric site. Within the allosteric site large effects on antagonist action were found for point mutants of residues F88A, D92A, F95A, and F103A that were conserved or similar between sensitive/insensitive P2XR subtypes, suggesting that these side-chain interactions were not solely responsible for high-affinity antagonist binding. Antagonist sensitivity was increased with mutations that remove the bulk of side chains around the center of the binding pocket, suggesting that the dimensions of the pocket make a significant contribution to selectivity. Chimeric receptors swapping the left flipper (around the orthosteric site) reduced both ATP and antagonist sensitivity. Point mutations within this region highlighted the contribution of a P2X7R-specific aspartic acid residue (D280) that modeling suggests forms a salt bridge with the lower body region of the receptor. The D280A mutant removing this charge increased ATP potency 15-fold providing a new insight into the low ATP sensitivity of the P2X7R. The ortho- and allosteric binding sites form either side of the β-strand Y291-E301 adjacent to the left flipper. This structural linking may explain the contribution of the left flipper to both agonist and antagonist action.

Highlights

ATP is released from cells in different ways, including regulated exocytosis from neurons, following platelet activation, and in response to tissue damage/cell death
We propose that P2X7 receptor (P2X7R)-selective antagonism results from a combination of the size of the allosteric pocket determined by residues not in direct interaction with the antagonists and direct interactions of the ligand with the receptor
It is important to stress that the pattern between different inhibitors is not fully consistent at these residues and probably reflects subtle differences in binding modes. We propose that these mutations can serve as a “finger print” for variants of allosteric P2X7R inhibition

Summary

Introduction

ATP is released from cells in different ways, including regulated exocytosis from neurons, following platelet activation, and in response to tissue damage/cell death. It acts as a ligand for P2X-receptor (P2XR) cation channels and a subset of G protein-coupled P2Y receptors (Burnstock, 2012). The contribution of extracellular ATP acting at cell surface P2XRs is well established in physiologic and pathophysiological contexts ranging from taste sensation to blood clotting (Kaczmarek-Hajek et al, 2012). Raised extracellular levels (millimolar ATP), are found as a response to inflammation, cell damage, and necrosis, resulting in stimulation of P2X7Rs on a variety of cell types, including macrophages, neurons, oligodendrocytes, osteoblasts, fibroblasts, and endothelial and epithelial cells (Bartlett et al, 2014). The selective P2X7R antagonist A438079 (3-(5-(2,3-dichlorophenyl)-1H-tetrazol-1-yl)methyl pyridine hydrochloride hydrate) protects against status

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