Mapping the site of action of the human P2X7 receptor antagonists AZ11645373, brilliant blue G, KN-62, calmidazolium and ZINC58368839 to the inter-subunit allosteric pocket.

The P2X7 receptor (P2X7R) is a trimeric ligand-gated ion channel which is activated by ATP. It is implicated in the cellular response to trauma/disease and considered to have significant therapeutic potential. Using chimeras and point mutants we have mapped the binding site of the P2X7R selective antagonist AZ11645373 to the known allosteric binding pocket at the interface between two subunits, in proximity to, but separated from the ATP binding site. Our structural model of AZ11645373 binding is consistent with effects of mutations on antagonist sensitivity, and the proposed binding mode explains variation in antagonist sensitivity between the human and rat P2X7 receptors. We have also determined the site of action for the P2X7R selective antagonists ZINC58368839, brilliant blue G, KN-62 and calmidazolium. The effect of inter-subunit allosteric pocket "signature mutants" F88A, T90V, D92A, F103A and V312A on antagonist sensitivity suggests that ZINC58368839 comprises a similar binding mode as AZ11645373 and other previously characterised antagonists. For the larger antagonists brilliant blue G, KN-62 and calmidazolium our data imply an overlapping, but distinct binding mode involving the central upper vestibule of the receptor in addition to the inter-subunit allosteric pocket. Our work explains the site of action for a series of P2X7R antagonists, and establishes "signature mutants" for P2X7R binding mode characterization. SIGNIFICANCE STATEMENT: The P2X7 receptor is a trimeric ligand-gated ion channel activated by ATP. The receptor is implicated in the cellular response to trauma/disease, and considered to have significant therapeutic potential. Using chimeras, point mutants and molecular modelling we have mapped the binding site of the P2X7 receptor selective antagonists AZ11645373, ZINC58368839, brilliant blue G, KN-62 and calmidazolium to a known inter-subunit binding pocket distinct from the ATP binding site. Our work explains the site of action for a series of P2X7R antagonists, and establishes signature mutants for P2X7R binding mode characterization.