P2X7 receptor (P2X7R) activation requires âË?¼100-fold higher\nconcentrations of ATP than other P2X receptor (P2XR) subtypes.\nSuch high levels are found during cellular stress, and\nP2X7Rs consequently contribute to a range of pathophysiological\nconditions.We have used chimeric and mutant P2X7Rs,\ncoupled with molecular modeling, to produce a validated model\nof the binding mode of the subtype-selective antagonist\nA438079 at an intersubunit allosteric site. Within the allosteric\nsite large effects on antagonist action were found for point\nmutants of residues F88A, D92A, F95A, and F103A that were\nconserved or similar between sensitive/insensitive P2XR subtypes,\nsuggesting that these side-chain interactions were not\nsolely responsible for high-affinity antagonist binding. Antagonist\nsensitivity was increased with mutations that remove the\nbulk of side chains around the center of the binding pocket,\nsuggesting that the dimensions of the pocket make a significant\ncontribution to selectivity. Chimeric receptors swapping\nthe left flipper (around the orthosteric site) reduced both ATP\nand antagonist sensitivity. Point mutations within this region\nhighlighted the contribution of a P2X7R-specific aspartic acid\nresidue (D280) that modeling suggests forms a salt bridge with\nthe lower body region of the receptor. The D280A mutant\nremoving this charge increased ATP potency 15-fold providing\na new insight into the low ATP sensitivity of the P2X7R. The\northo- and allosteric binding sites form either side of the\nb-strand Y291-E301 adjacent to the left flipper. This structural\nlinking may explain the contribution of the left flipper to both\nagonist and antagonist action.
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