TY - JOUR T1 - Mapping the allosteric action of antagonists A740003 and A438079 reveals a role of the left flipper in ligand sensitivity at P2X7 receptors JF - Molecular Pharmacology JO - Mol Pharmacol DO - 10.1124/mol.117.111021 SP - mol.117.111021 AU - Rebecca C Allsopp AU - Sudad Dayl AU - Anfal Bin Dayel AU - Ralf Schmid AU - Richard J. Evans Y1 - 2018/01/01 UR - http://molpharm.aspetjournals.org/content/early/2018/03/13/mol.117.111021.abstract N2 - P2X7 receptor (P2X7Rs) activation requires ~ 100 fold higher concentrations of ATP than other 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 modelling, to produce a validated model of the binding mode of the subtype selective antagonist A438079 at an inter-subunit 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 centre 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 modelling 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. ER -