Department of Molecular and Cell Biology (A.B.D., R.J.E., R.S.) and Leicester Institute of Structural and Chemical Biology (R.S.), University of Leicester, Leicester, United Kingdom.
Department of Molecular and Cell Biology (A.B.D., R.J.E., R.S.) and Leicester Institute of Structural and Chemical Biology (R.S.), University of Leicester, Leicester, United Kingdom
Mol Pharmacol. 2019 Sep;96(3):355-363. doi: 10.1124/mol.119.116715. Epub 2019 Jul 1.
The P2X7 receptor is a trimeric ligand-gated ion channel 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 intersubunit allosteric pocket "signature mutants" F88A, T90V, D92A, F103A, and V312A on antagonist sensitivity suggests that ZINC58368839 comprises a binding mode similar to AZ11645373 and other previously characterized 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 intersubunit allosteric pocket. Our work explains the site of action for a series of P2X7R antagonists and establishes "signature mutants" for P2X7R binding-mode characterization.
P2X7 受体是一种由三磷酸腺苷（ATP）激活的三聚体配体门控离子通道。它与细胞对创伤/疾病的反应有关，被认为具有重要的治疗潜力。我们使用嵌合体和点突变体将 P2X7R 选择性拮抗剂 AZ11645373 的结合位点定位到两个亚基之间的已知变构结合口袋，该结合口袋靠近但与 ATP 结合位点分离。我们的 AZ11645373 结合结构模型与突变对拮抗剂敏感性的影响一致，并且所提出的结合模式解释了人源和大鼠 P2X7 受体之间拮抗剂敏感性的差异。我们还确定了 P2X7R 选择性拮抗剂 ZINC58368839、亮蓝 G、KN-62 和卡马西平的作用部位。亚基间变构口袋“特征突变体”F88A、T90V、D92A、F103A 和 V312A 对拮抗剂敏感性的影响表明，ZINC58368839 的结合模式类似于 AZ11645373 和其他先前表征的拮抗剂。对于较大的拮抗剂亮蓝 G、KN-62 和卡马西平，我们的数据表明，除了亚基间变构口袋外，它们还涉及受体的中央上前庭，存在重叠但不同的结合模式。我们的工作解释了一系列 P2X7R 拮抗剂的作用部位，并为 P2X7R 结合模式表征建立了“特征突变体”。
