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Structural Insights into the Function of P2X4: An ATP-Gated Cation Channel of Neuroendocrine Cells

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Abstract

The P2X4 receptor (P2X4R) is a member of a family of ATP-gated cation channels that are composed of three subunits. Each subunit has two transmembrane (TM) domains linked by a large extracellular loop and intracellularly located N- and C-termini. The receptors are expressed in excitable and non-excitable cells and have been implicated in the modulation of membrane excitability, calcium signaling, neurotransmitter and hormone release, and pain physiology. P2X4Rs activate rapidly and desensitize within the seconds of agonist application, both with the rates dependent on ATP concentrations, and deactivate rapidly and independently of ATP concentration. Disruption of conserved cysteine ectodomain residues affects ATP binding and gating. Several ectodomain residues of P2X4R were identified as critical for ATP binding, including K67, K313, and R295. Ectodomain residues also account for the allosteric regulation of P2X4R; H140 is responsible for copper binding and H286 regulates receptor functions with protons. Ivermectin sensitized receptors, amplified the current amplitude, and slowed receptor deactivation by binding in the TM region. Scanning mutagenesis of TMs revealed the helical topology of both domains, and suggested that receptor function is critically dependent on the conserved Y42 residue. In this brief article, we summarize this study and re-interpret it using a model based on crystallization of the zebrafish P2X4.1 receptor.

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References

  • Acuna-Castillo C, Morales B, Huidobro-Toro JP (2000) Zinc and copper modulate differentially the P2X4 receptor. J Neurochem 74:1529–1537

    Article  CAS  PubMed  Google Scholar 

  • Alqallaf SM, Evans BA, Kidd EJ (2009) Atypical P2X receptor pharmacology in two human osteoblast-like cell lines. Br J Pharmacol 156:1124–1135

    Article  CAS  PubMed  Google Scholar 

  • Bo X, Zhang Y, Nassar M, Burnstock G, Schoepfer R (1995) A P2X purinoceptor cDNA conferring a novel pharmacological profile. FEBS Lett 375:129–133

    Article  CAS  PubMed  Google Scholar 

  • Bobanovic LK, Royle SJ, Murrell-Lagnado RD (2002) P2X receptor trafficking in neurons is subunit specific. J Neurosci 22:4814–4824

    CAS  PubMed  Google Scholar 

  • Boumechache M, Masin M, Edwardson JM, Gorecki DC, Murrell-Lagnado R (2009) Analysis of assembly and trafficking of native P2X4 and P2X7 receptor complexes in rodent immune cells. J Biol Chem 284:13446–13454

    Article  CAS  PubMed  Google Scholar 

  • Bowler JW, Bailey RJ, North RA, Surprenant A (2003) P2X4, P2Y1 and P2Y2 receptors on rat alveolar macrophages. Br J Pharmacol 140:567–575

    Article  CAS  PubMed  Google Scholar 

  • Browne LE, Jiang LH, North RA (2010) New structure enlivens interest in P2X receptors. Trends Pharmacol Sci 31:229–237

    Article  CAS  PubMed  Google Scholar 

  • Buell G, Lewis C, Collo G, North RA, Surprenant A (1996) An antagonist-insensitive P2X receptor expressed in epithelia and brain. EMBO J 15:55–62

    CAS  PubMed  Google Scholar 

  • Cao L, Broomhead HE, Young MT, North RA (2009) Polar residues in the second transmembrane domain of the rat P2X2 receptor that affect spontaneous gating, unitary conductance, and rectification. J Neurosci 29:14257–14264

    Article  CAS  PubMed  Google Scholar 

  • Clarke CE, Benham CD, Bridges A, George AR, Meadows HJ (2000) Mutation of histidine 286 of the human P2X4 purinoceptor removes extracellular pH sensitivity. J Physiol 523 Pt 3:697–703

    Google Scholar 

  • Clyne JD, Wang LF, Hume RI (2002) Mutational analysis of the conserved cysteines of the rat P2X2 purinoceptor. J Neurosci 22:3873–3880

    CAS  PubMed  Google Scholar 

  • Coddou C, Morales B, Gonzalez J, Grauso M, Gordillo F, Bull P, Rassendren F, Huidobro-Toro JP (2003) Histidine 140 plays a key role in the inhibitory modulation of the P2X4 nucleotide receptor by copper but not zinc. J Biol Chem 278:36777–36785

    Article  CAS  PubMed  Google Scholar 

  • Coddou C, Lorca RA, Acuna-Castillo C, Grauso M, Rassendren F, Huidobro-Toro JP (2005) Heavy metals modulate the activity of the purinergic P2X4 receptor. Toxicol Appl Pharmacol 202:121–131

    Article  CAS  PubMed  Google Scholar 

  • Coddou C, Acuna-Castillo C, Bull P, Huidobro-Toro JP (2007) Dissecting the facilitator and inhibitor allosteric metal sites of the P2X4 receptor channel: critical roles of CYS132 for zinc potentiation and ASP138 for copper inhibition. J Biol Chem 282:36879–36886

    Article  CAS  PubMed  Google Scholar 

  • Egan TM, Khakh BS (2004) Contribution of calcium ions to P2X channel responses. J Neurosci 24:3413–3420

    Article  CAS  PubMed  Google Scholar 

  • Egan TM, Haines WR, Voigt MM (1998) A domain contributing to the ion channel of ATP-gated P2X2 receptors identified by the substituted cysteine accessibility method. J Neurosci 18:2350–2359

    CAS  PubMed  Google Scholar 

  • Ennion SJ, Evans RJ (2002) Conserved cysteine residues in the extracellular loop of the human P2X(1) receptor form disulfide bonds and are involved in receptor trafficking to the cell surface. Mol Pharmacol 61:303–311

    Article  CAS  PubMed  Google Scholar 

  • Ennion S, Hagan S, Evans RJ (2000) The role of positively charged amino acids in ATP recognition by human P2X1 receptors. J Biol Chem 275:35656

    Article  CAS  PubMed  Google Scholar 

  • Evans RJ (2009) Orthosteric and allosteric binding sites of P2X receptors. Eur Biophys J 38:319–327

    Article  CAS  PubMed  Google Scholar 

  • Fountain SJ, North RA (2006) A C-terminal lysine that controls human P2X4 receptor desensitization. J Biol Chem 281:15044–15049

    Article  CAS  PubMed  Google Scholar 

  • Guex N, Peitsch MC (1997) SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 18:2714–2723

    Article  CAS  PubMed  Google Scholar 

  • Haines WR, Migita K, Cox JA, Egan TM, Voigt MM (2001a) The first transmembrane domain of the P2X receptor subunit participates in the agonist-induced gating of the channel. J Biol Chem 276:32793–32798

    Article  CAS  PubMed  Google Scholar 

  • Haines WR, Voigt MM, Migita K, Torres GE, Egan TM (2001b) On the contribution of the first transmembrane domain to whole-cell current through an ATP-gated ionotropic P2X receptor. J Neurosci 21:5885–5892

    CAS  PubMed  Google Scholar 

  • He ML, Koshimizu TA, Tomic M, Stojilkovic SS (2002) Purinergic P2X(2) receptor desensitization depends on coupling between ectodomain and C-terminal domain. Mol Pharmacol 62:1187–1197

    Article  CAS  PubMed  Google Scholar 

  • He ML, Zemkova H, Stojilkovic SS (2003a) Dependence of purinergic P2X receptor activity on ectodomain structure. J Biol Chem 278:10182–10188

    Article  CAS  PubMed  Google Scholar 

  • He ML, Zemkova H, Koshimizu TA, Tomic M, Stojilkovic SS (2003b) Intracellular calcium measurements as a method in studies on activity of purinergic P2X receptor channels. Am J Physiol Cell Physiol 285:C467–C479

    CAS  PubMed  Google Scholar 

  • Jarvis MF, Khakh BS (2009) ATP-gated P2X cation-channels. Neuropharmacology 56:208–215

    Article  CAS  PubMed  Google Scholar 

  • Jelinkova I, Yan Z, Liang Z, Moonat S, Teisinger J, Stojilkovic SS, Zemkova H (2006) Identification of P2X4 receptor-specific residues contributing to the ivermectin effects on channel deactivation. Biochem Biophys Res Commun 349:619–625

    Article  CAS  PubMed  Google Scholar 

  • Jelinkova I, Vavra V, Jindrichova M, Obsil T, Zemkova HW, Zemkova H, Stojilkovic SS (2008) Identification of P2X(4) receptor transmembrane residues contributing to channel gating and interaction with ivermectin. Pflugers Arch 456:939–950

    Article  CAS  PubMed  Google Scholar 

  • Jiang LH, Rassendren F, Surprenant A, North RA (2000) Identification of amino acid residues contributing to the ATP-binding site of a purinergic P2X receptor. J Biol Chem 275:34190–34196

    Article  CAS  PubMed  Google Scholar 

  • Jiang LH, Rassendren F, Spelta V, Surprenant A, North RA (2001) Amino acid residues involved in gating identified in the first membrane-spanning domain of the rat P2X(2) receptor. J Biol Chem 276:14902–14908

    Article  CAS  PubMed  Google Scholar 

  • Jindrichova M, Vavra V, Obsil T, Stojilkovic SS, Zemkova H (2009) Functional relevance of aromatic residues in the first transmembrane domain of P2X receptors. J Neurochem 109:923–934

    Article  CAS  PubMed  Google Scholar 

  • Kawate T, Michel JC, Birdsong WT, Gouaux E (2009) Crystal structure of the ATP-gated P2X(4) ion channel in the closed state. Nature 460:592–598

    Article  CAS  PubMed  Google Scholar 

  • Khakh BS, Egan TM (2005) Contribution of transmembrane regions to ATP-gated P2X2 channel permeability dynamics. J Biol Chem 280:6118–6129

    Article  CAS  PubMed  Google Scholar 

  • Khakh BS, North RA (2006) P2X receptors as cell-surface ATP sensors in health and disease. Nature 442:527–532

    Article  CAS  PubMed  Google Scholar 

  • Khakh BS, Bao XR, Labarca C, Lester HA (1999a) Neuronal P2X transmitter-gated cation channels change their ion selectivity in seconds. Nat Neurosci 2:322–330

    Article  CAS  PubMed  Google Scholar 

  • Khakh BS, Proctor WR, Dunwiddie TV, Labarca C, Lester HA (1999b) Allosteric control of gating and kinetics at P2X(4) receptor channels. J Neurosci 19:7289–7299

    CAS  PubMed  Google Scholar 

  • Kracun S, Chaptal V, Abramson J, Khakh BS (2010) Gated access to the pore of a P2X receptor: structural implications for closed-open transitions. J Biol Chem 285:10110–10121

    Article  CAS  PubMed  Google Scholar 

  • Le KT, Babinski K, Seguela P (1998) Central P2X4 and P2X6 channel subunits coassemble into a novel heteromeric ATP receptor. J Neurosci 18:7152–7159

    CAS  PubMed  Google Scholar 

  • Li Z, Migita K, Samways DS, Voigt MM, Egan TM (2004) Gain and loss of channel function by alanine substitutions in the transmembrane segments of the rat ATP-gated P2X2 receptor. J Neurosci 24:7378–7386

    Article  CAS  PubMed  Google Scholar 

  • Li M, Chang TH, Silberberg SD, Swartz KJ (2008) Gating the pore of P2X receptor channels. Nat Neurosci 11:883–887

    Article  CAS  PubMed  Google Scholar 

  • Lorca RA, Coddou C, Gazitua MC, Bull P, Arredondo C, Huidobro-Toro JP (2005) Extracellular histidine residues identify common structural determinants in the copper/zinc P2X2 receptor modulation. J Neurochem 95:499–512

    Article  CAS  PubMed  Google Scholar 

  • Marquez-Klaka B, Rettinger J, Bhargava Y, Eisele T, Nicke A (2007) Identification of an intersubunit cross-link between substituted cysteine residues located in the putative ATP binding site of the P2X1 receptor. J Neurosci 27:1456–1466

    Article  CAS  PubMed  Google Scholar 

  • Migita K, Haines WR, Voigt MM, Egan TM (2001) Polar residues of the second transmembrane domain influence cation permeability of the ATP-gated P2X(2) receptor. J Biol Chem 276:30934–30941

    Article  CAS  PubMed  Google Scholar 

  • Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK, Goodsell DS, Olson AJ (2009) AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility. J Comput Chem 30:2785–2791

    Article  CAS  PubMed  Google Scholar 

  • Nicke A (2008) Homotrimeric complexes are the dominant assembly state of native P2X7 subunits. Biochem Biophys Res Commun 377:803–808

    Article  CAS  PubMed  Google Scholar 

  • Nicke A, Kerschensteiner D, Soto F (2005) Biochemical and functional evidence for heteromeric assembly of P2X1 and P2X4 subunits. J Neurochem 92:925–933

    Article  CAS  PubMed  Google Scholar 

  • North RA (2002) Molecular physiology of P2X receptors. Physiol Rev 82:1013–1067

    CAS  PubMed  Google Scholar 

  • Ormond SJ, Barrera NP, Qureshi OS, Henderson RM, Edwardson JM, Murrell-Lagnado RD (2006) An uncharged region within the N terminus of the P2X6 receptor inhibits its assembly and exit from the endoplasmic reticulum. Mol Pharmacol 69:1692–1700

    Article  CAS  PubMed  Google Scholar 

  • Popova M, Asatryan L, Ostrovskaya O, Wyatt LR, Li K, Alkana RL, Davies DL (2010) A point mutation in the ectodomain-transmembrane 2 interface eliminates the inhibitory effects of ethanol in P2X4 receptors. J Neurochem 112:307–317

    Article  CAS  PubMed  Google Scholar 

  • Qureshi OS, Paramasivam A, Yu JC, Murrell-Lagnado RD (2007) Regulation of P2X4 receptors by lysosomal targeting, glycan protection and exocytosis. J Cell Sci 120:3838–3849

    Article  CAS  PubMed  Google Scholar 

  • Rassendren F, Buell G, Newbolt A, North RA, Surprenant A (1997) Identification of amino acid residues contributing to the pore of a P2X receptor. EMBO J 16:3446–3454

    Article  CAS  PubMed  Google Scholar 

  • Rokic M, Tvrdonova V, Vavra V, Jindrichova M, Obsil T, Stojilkovic SS, Zemkova H (2010) Roles of conserved ectodomain cysteines of the rat P2X4 purinoreceptor in agonist binding and channel gating. Physiol Res

  • Royle SJ, Bobanovic LK, Murrell-Lagnado RD (2002) Identification of a non-canonical tyrosine-based endocytic motif in an ionotropic receptor. J Biol Chem 277:35378–35385

    Article  CAS  PubMed  Google Scholar 

  • Rubio ME, Soto F (2001) Distinct Localization of P2X receptors at excitatory postsynaptic specializations. J Neurosci 21:641–653

    CAS  PubMed  Google Scholar 

  • Sali A, Blundell TL (1993) Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol 234:779–815

    Article  CAS  PubMed  Google Scholar 

  • Samways DS, Egan TM (2007) Acidic amino acids impart enhanced Ca2+ permeability and flux in two members of the ATP-gated P2X receptor family. J Gen Physiol 129:245–256

    Article  CAS  PubMed  Google Scholar 

  • Samways DS, Migita K, Li Z, Egan TM (2008) On the role of the first transmembrane domain in cation permeability and flux of the ATP-gated P2X2 receptor. J Biol Chem 283:5110–5117

    Article  CAS  PubMed  Google Scholar 

  • Silberberg SD, Chang TH, Swartz KJ (2005) Secondary structure and gating rearrangements of transmembrane segments in rat P2X4 receptor channels. J Gen Physiol 125:347–359

    Article  CAS  PubMed  Google Scholar 

  • Silberberg SD, Li M, Swartz KJ (2007) Ivermectin Interaction with transmembrane helices reveals widespread rearrangements during opening of P2X receptor channels. Neuron 54:263–274

    Article  CAS  PubMed  Google Scholar 

  • Sim JA, Chaumont S, Jo J, Ulmann L, Young MT, Cho K, Buell G, North RA, Rassendren F (2006) Altered hippocampal synaptic potentiation in P2X4 knock-out mice. J Neurosci 26:9006–9009

    Article  CAS  PubMed  Google Scholar 

  • Soto F, Garcia-Guzman M, Gomez-Hernandez JM, Hollmann M, Karschin C, Stuhmer W (1996) P2X4: an ATP-activated ionotropic receptor cloned from rat brain. Proc Natl Acad Sci USA 93:3684–3688

    Article  CAS  PubMed  Google Scholar 

  • Stokes L, Surprenant A (2009) Dynamic regulation of the P2X4 receptor in alveolar macrophages by phagocytosis and classical activation. Eur J Immunol 39:986–995

    Article  CAS  PubMed  Google Scholar 

  • Stoop R, Surprenant A, North RA (1997) Different sensitivities to pH of ATP-induced currents at four cloned P2X receptors. J Neurophysiol 78:1837–1840

    CAS  PubMed  Google Scholar 

  • Sun JH, Cai GJ, Xiang ZH (2007) Expression of P2X purinoceptors in PC12 phaeochromocytoma cells. Clin Exp Pharmacol Physiol 34:1282–1286

    Article  CAS  PubMed  Google Scholar 

  • Surprenant A, North RA (2009) Signaling at purinergic P2X receptors. Annu Rev Physiol 71:333–359

    Article  CAS  PubMed  Google Scholar 

  • Toulme E, Garcia A, Samways D, Egan TM, Carson MJ, Khakh BS (2010) P2X4 receptors in activated C8–B4 cells of cerebellar microglial origin. J Gen Physiol 135:333–353

    Article  CAS  PubMed  Google Scholar 

  • Tsuda M, Shigemoto-Mogami Y, Koizumi S, Mizokoshi A, Kohsaka S, Salter MW, Inoue K (2003) P2X4 receptors induced in spinal microglia gate tactile allodynia after nerve injury. Nature 424:778–783

    Article  CAS  PubMed  Google Scholar 

  • Ulmann L, Hatcher JP, Hughes JP, Chaumont S, Green PJ, Conquet F, Buell GN, Reeve AJ, Chessell IP, Rassendren F (2008) Up-regulation of P2X4 receptors in spinal microglia after peripheral nerve injury mediates BDNF release and neuropathic pain. J Neurosci 28:11263–11268

    Article  CAS  PubMed  Google Scholar 

  • Virginio C, Church D, North RA, Surprenant A (1997) Effects of divalent cations, protons and calmidazolium at the rat P2X7 receptor. Neuropharmacology 36:1285–1294

    Article  CAS  PubMed  Google Scholar 

  • Wareham K, Vial C, Wykes RC, Bradding P, Seward EP (2009) Functional evidence for the expression of P2X1, P2X4 and P2X7 receptors in human lung mast cells. Br J Pharmacol 157:1215–1224

    Article  CAS  PubMed  Google Scholar 

  • Wilkinson WJ, Jiang LH, Surprenant A, North RA (2006) Role of ectodomain lysines in the subunits of the heteromeric P2X2/3 receptor. Mol Pharmacol 70:1159–1163

    Article  CAS  PubMed  Google Scholar 

  • Xiong K, Li C, Weight FF (2000) Inhibition by ethanol of rat P2X(4) receptors expressed in Xenopus oocytes. Br J Pharmacol 130:1394–1398

    Article  CAS  PubMed  Google Scholar 

  • Yamamoto K, Sokabe T, Matsumoto T, Yoshimura K, Shibata M, Ohura N, Fukuda T, Sato T, Sekine K, Kato S, Isshiki M, Fujita T, Kobayashi M, Kawamura K, Masuda H, Kamiya A, Ando J (2006) Impaired flow-dependent control of vascular tone and remodeling in P2X4-deficient mice. Nat Med 12:133–137

    Article  CAS  PubMed  Google Scholar 

  • Yan Z, Liang Z, Tomic M, Obsil T, Stojilkovic SS (2005) Molecular determinants of the agonist binding domain of a P2X receptor channel. Mol Pharmacol 67:1078–1088

    Article  CAS  PubMed  Google Scholar 

  • Yan Z, Liang Z, Obsil T, Stojilkovic SS (2006) Participation of the Lys313-Ile333 sequence of the purinergic P2X4 receptor in agonist binding and transduction of signals to the channel gate. J Biol Chem 281:32649–32659

    Article  CAS  PubMed  Google Scholar 

  • Zemkova H, Kucka M, Li S, Gonzalez-Iglesias AE, Tomic M, Stojilkovic SS (2010) Characterization of purinergic P2X4 receptor channels expressed in anterior pituitary cells. Am J Physiol Endocrinol Metab 298:E644–E651

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This study was supported by the Intramural Research Program of the NICHD, the NIH and the Internal Grant Agency of the Academy of Sciences (Grants No. IAA500110910 and IAA501110801).

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Authors and Affiliations

  1. Section on Cellular Signaling, Program in Developmental Neuroscience, NICHD, National Institutes of Health, Bldg. 49, Room 6A-36, 49 Convent Drive, Bethesda, MD, 20892-4510, USA

    Stanko S. Stojilkovic & Zonghe Yan

  2. Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University, Prague, Czech Republic

    Tomas Obsil

  3. Department of Cellular and Molecular Neuroendocrinology, Institute of Physiology of the Academy of Sciences of the Czech Republic, Prague 4, Czech Republic

    Tomas Obsil & Hana Zemkova

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  1. Stanko S. Stojilkovic
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  2. Zonghe Yan
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  3. Tomas Obsil
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  4. Hana Zemkova
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Correspondence to Stanko S. Stojilkovic.

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A commentary to this article can be found at doi:10.1007/s10571-010-9611-z.

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Stojilkovic, S.S., Yan, Z., Obsil, T. et al. Structural Insights into the Function of P2X4: An ATP-Gated Cation Channel of Neuroendocrine Cells. Cell Mol Neurobiol 30, 1251–1258 (2010). https://doi.org/10.1007/s10571-010-9568-y

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