Identification of Key Residues Coordinating Functional Inhibition of P2X 7 Receptors by Zinc and Copper □ S Xing Liu, Annmarie Surprenant, Hong-Ju Mao, Sebastien Roger, Rong Xia, Helen Bradley, and Lin-Hua Jiang Institute of Membrane and Systems Biology, Faculty of Biological Sciences, University of Leeds, Leeds, United Kingdom (X.L., H.-J.M., R.X., H.B., L.-H.J.); and Department of Biomedical Science, University of Sheffield, United Kingdom (A.S., S.R.) Received July 4, 2007; accepted October 23, 2007 ABSTRACT P2X 7 receptors are distinct from other ATP-gated P2X recep- tors in that they are potently inhibited by submicromolar con- centrations of zinc and copper. The molecular basis for the strong functional inhibition by zinc and copper at this purinergic ionotropic receptor is controversial. We hypothesized that it involves a direct interaction of zinc and copper with residues in the ectodomain of the P2X 7 receptor. Fourteen potential metal interacting residues are conserved in the ectodomain of all mammalian P2X 7 receptors, none of which is homologous to previously identified sites in other P2X receptors shown to be important for functional potentiation by zinc. We introduced alanine substitutions into each of these residues, expressed wild-type and mutated receptors in human embryonic kidney 293 cells, and recorded resulting ATP and BzATP-evoked membrane currents. Agonist concentration-response curves were similar for all 12 functional mutant receptors. Alanine substitution at His 62 or Asp 197 strongly attenuated both zinc and copper inhibition, and the double mutant [H62A/D197A] mutant receptor was virtually insensitive to inhibition by zinc or copper. Thus, we conclude that zinc and copper inhibition is due to a direct interaction of these divalent cations with ectodo- main residues of the P2X 7 receptor, primarily involving com- bined interaction with His 62 and Asp 197 residues. The P2X 7 receptor (previously termed P2Z) is the last member (Surprenant et al., 1996) of the ATP-gated P2X receptor family and is found primarily on hematopoietic lin- eage cells, such as lymphocytes, monocytes, and macro- phages, as well as brain microglial cells and astrocytes (North, 2002; Duan and Neary, 2006). Studies using different approaches, including gene knockout, have underscored P2X 7 receptors as a crucial component in a diversity of bio- logical processes, such as inflammation, elimination of intra- cellular pathogens, release of pro-inflammatory cytokines by macrophages, and release of neurotransmitters by astro- cytes, and chronic inflammatory and neuropathic pain (e.g., Lammas et al., 1997; Solle et al., 2001; Chessell et al., 2005; Duan and Neary, 2006; Ferrari et al., 2006). P2X 7 receptors show several unique functional features that differ strikingly from those of other members of the P2X receptor family (North, 2002). P2X 7 receptors display re- markable functional plasticity; brief activation opens small cation permeable channels, whereas prolonged stimulation induces formation of “large molecule” permissive pores, rapid membrane and mitochondrial morphological changes, cy- toskeletal rearrangement, and eventual cell death (Sur- prenant et al., 1996; North, 2002; Morelli et al., 2003). The most upstream signaling event consequent to the P2X 7 re- ceptor activation, the ionic current, also shows properties uniquely different from those of other P2X receptors. Cur- rents are activated by submillimolar concentrations of ATP, which are 10 to 100 times higher than those required for activation of other P2X receptors. Furthermore, P2X 7 recep- tors are potently inhibited by submicromolar concentrations of zinc and copper, whereas other P2X receptors are strongly potentiated or unaffected (Wildman et al., 1998, 1999a,b; Xiong et al., 1999; Acun ˜ a-Castillo et al., 2000; Clyne et al., 2002; Lorca et al., 2005). P2X 7 receptors are also substan- tially inhibited by magnesium and calcium, albeit with much less potency than zinc and copper. Earlier studies examining the effects of calcium and magnesium have led to the view that ATP 4- is the effective agonist form of ATP and these divalent cations inhibit the P2X 7 receptors by reducing the effective agonist concentrations (Cockcroft and Gomperts, This work was supported by the Biotechnology and Biological Sciences Research Council (BBSRC) (L.-H.J., A.S.), the Royal Society (H.-J. M., L.-H.J.), a UK Overseas Research Scholarship (R.X.), and a BBSRC doctoral training grant (H.B.). Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.107.039651. □ S The online version of this article (available at http://molpharm. aspetjournals.org) contains supplemental material. ABBREVIATIONS: BzATP, 2',3'-O-(4-benzoylbenzoyl) adenosine 5'-triphosphate; HEK, human embryonic kidney. 0026-895X/08/7301-252–259$20.00 MOLECULAR PHARMACOLOGY Vol. 73, No. 1 Copyright © 2008 The American Society for Pharmacology and Experimental Therapeutics 39651/3291971 Mol Pharmacol 73:252–259, 2008 Printed in U.S.A. 252