Kasuya G , Fujiwara Y , Tsukamoto H , Morinaga S , Ryu S , Touhara K , Ishitani R , Furutani Y , Hattori M , Nureki O .

	Figure 1. Current responses of zebrafish P2X4 evoked by nucleoside triphosphates.(A–D) Representative currents of zebrafish P2X4 WT, evoked first by 100 μM ATP and then by (A) 1 mM ATP, (B) 1 mM CTP, (C) 1 mM GTP and (D) 1 mM UTP. Each nucleotide was applied for 10 sec. The holding potential was −70 mV.
	Figure 2. Overall comparisons of the CTP-bound and ATP-bound ΔP2X4-C structures.(A,B) The CTP-bound ΔP2X4-C structure viewed parallel to the membrane (A) and from the extracellular side (B). (C) The omit Fo − Fc map contoured at 4σ, showing the electron density of CTP. CTP is depicted by a stick model. (D,E) The ATP-bound ΔP2X4-C structure viewed parallel to the membrane (D) and from the extracellular side (E). (F) The omit Fo − Fc map contoured at 4σ, showing the electron density of ATP. ATP is depicted by a stick model.
	Figure 3. Close-up views of the CTP and ATP binding sites.(A–D) Close-up views of the CTP binding site in the CTP-bound ΔP2X4-C structure (A,B), and the ATP binding site in the ATP-bound ΔP2X4-C structure (C,D). Side chains of amino acid residues and nucleoside triphosphates are depicted by stick models. The molecule is colored according to the previously proposed dolphin-like model. Each dotted black line and number indicates a hydrogen bond and its length (<3.3 Å) respectively. (E) Sequence alignment around agonist binding site of P2X receptors. Amino acid sequences were aligned using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) and are shown using ESPript3 (http://espript.ibcp.fr/ESPript/ESPript/). For the sequence alignment, zebrafish P2X4 (zfP2X4, GI: 12656589) and the following P2X receptors were used: human (hP2X1, 4505545; hP2X2, 25092719; hP2X3, 28416925; hP2X4, 116242696; hP2X5, 209572778; hP2X6, 6469324; and hP2X7, 29294631), rat (rP2X1, 1352689; rP2X2, 18093098; rP2X3, 1030065; rP2X4, 1161345; rP2X5, 1279659; rP2X6, 1279661; and rP2X7, 1322005), Gulf Coast tick (amP2X, GI: 346469461), and blood fluke (smP2X, 51988420).
	Figure 4. Current responses of rWT, rT186S, rH140A and rH140R evoked by ATP and CTP.(A,B) Representative currents of rWT evoked by ATP (1–300 μM) (A) and CTP (10–10,000 μM) (B). (C,D) Representative currents of the rT186S mutant evoked by ATP (1–300 μM) (C) and CTP (10–10,000 μM) (D). (E,F) Representative currents of the rH140A mutant evoked by ATP (1–300 μM) (E) and CTP (10–10,000 μM) (F). (G,H) Representative currents of the rH140R mutant evoked by ATP (1–300 μM) (G) and CTP (10–10,000 μM) (H). (I–K) Concentration-response curves evoked by ATP and CTP in the rWT and the rT186S mutant (I), in the rWT and the rH140A mutant (J) and in the rWT and the rH140R mutant (K). Symbols are defined in the figure, and bars depict means ± SEM (n = 7–9).
	Figure 5. ATR-FTIR spectroscopic analysis of zfP2X4 for the ATP and CTP bindings.(A) The reduction of the ligand binding induced difference spectra in the ΔP2X4-C zfWT, zfT189S and zfT189V mutants around the P-O stretching region (1250–800 cm−1) after washing treatment for 15–30 min. (B) The residual nucleotides after the washing treatment estimated from the ATR-FTIR analysis in (A).
	Figure 6. Model for the nucleotide base specificity of P2X receptors.(A–D) Schematic representations of the interactions between zfP2X4 and ATP (A), CTP (B), GTP (C) or UTP (D). Residues involved in nucleotide base recognition and each nucleoside triphosphate are depicted by stick models. Black dashed lines and numbers indicate hydrogen bond interactions, and red crosses indicate non-complementary hydrogen bond partners, despite reasonable hydrogen bonding distances.

Adams, PHENIX: a comprehensive Python-based system for macromolecular structure solution. 2010, Pubmed

Adams, PHENIX: a comprehensive Python-based system for macromolecular structure solution. 2010, Pubmed
Brake, New structural motif for ligand-gated ion channels defined by an ionotropic ATP receptor. 1994, Pubmed , Xenbase
Browne, P2X receptor intermediate activation states have altered nucleotide selectivity. 2013, Pubmed
Burnstock, Purinergic nerves. 1972, Pubmed
Burnstock, Is there a basis for distinguishing two types of P2-purinoceptor? 1985, Pubmed
Burnstock, Pathophysiology and therapeutic potential of purinergic signaling. 2006, Pubmed
Burnstock, Purinergic signaling and blood vessels in health and disease. 2014, Pubmed
Chen, A P2X purinoceptor expressed by a subset of sensory neurons. 1995, Pubmed , Xenbase
Emsley, Features and development of Coot. 2010, Pubmed
Fujiwara, Voltage- and [ATP]-dependent gating of the P2X(2) ATP receptor channel. 2009, Pubmed , Xenbase
Furutani, ATR-FTIR Spectroscopy Revealing the Different Vibrational Modes of the Selectivity Filter Interacting with K(+) and Na(+) in the Open and Collapsed Conformations of the KcsA Potassium Channel. 2012, Pubmed
Furutani, Specific interactions between alkali metal cations and the KcsA channel studied using ATR-FTIR spectroscopy. 2015, Pubmed
Furutani, Sodium or lithium ion-binding-induced structural changes in the K-ring of V-ATPase from Enterococcus hirae revealed by ATR-FTIR spectroscopy. 2011, Pubmed
Garcia-Guzman, Characterization of recombinant human P2X4 receptor reveals pharmacological differences to the rat homologue. 1997, Pubmed , Xenbase
Garcia-Guzman, Molecular characterization and pharmacological properties of the human P2X3 purinoceptor. 1997, Pubmed , Xenbase
Gever, Pharmacology of P2X channels. 2006, Pubmed
Haines, Properties of the novel ATP-gated ionotropic receptor composed of the P2X(1) and P2X(5) isoforms. 1999, Pubmed
Hattori, Molecular mechanism of ATP binding and ion channel activation in P2X receptors. 2012, Pubmed
Karasawa, Structural basis for subtype-specific inhibition of the P2X7 receptor. 2016, Pubmed
Kasuya, Structural Insights into Divalent Cation Modulations of ATP-Gated P2X Receptor Channels. 2016, Pubmed , Xenbase
Kawate, Crystal structure of the ATP-gated P2X(4) ion channel in the closed state. 2009, Pubmed
King, Effects of extracellular pH on agonism and antagonism at a recombinant P2X2 receptor. 1997, Pubmed , Xenbase
Lewis, Lack of run-down of smooth muscle P2X receptor currents recorded with the amphotericin permeabilized patch technique, physiological and pharmacological characterization of the properties of mesenteric artery P2X receptor ion channels. 2000, Pubmed
Liman, Subunit stoichiometry of a mammalian K+ channel determined by construction of multimeric cDNAs. 1992, Pubmed , Xenbase
Mansoor, X-ray structures define human P2X(3) receptor gating cycle and antagonist action. 2016, Pubmed
McCoy, Phaser crystallographic software. 2007, Pubmed
North, P2X receptors as drug targets. 2013, Pubmed
North, Molecular physiology of P2X receptors. 2002, Pubmed
Ohta, P2X2 receptors are essential for [Ca2+]i increases in response to ATP in cultured rat myenteric neurons. 2005, Pubmed
Roberts, ATP binding at human P2X1 receptors. Contribution of aromatic and basic amino acids revealed using mutagenesis and partial agonists. 2004, Pubmed , Xenbase
Soto, P2X4: an ATP-activated ionotropic receptor cloned from rat brain. 1996, Pubmed , Xenbase
Surprenant, Signaling at purinergic P2X receptors. 2009, Pubmed
Tvrdonova, Identification of functionally important residues of the rat P2X4 receptor by alanine scanning mutagenesis of the dorsal fin and left flipper domains. 2014, Pubmed
Valera, A new class of ligand-gated ion channel defined by P2x receptor for extracellular ATP. 1994, Pubmed , Xenbase
Williams, Purinergic and pyrimidinergic receptors as potential drug targets. 2000, Pubmed
Xia, The specific vibrational modes of GTP in solution and bound to Ras: a detailed theoretical analysis by QM/MM simulations. 2011, Pubmed
Xiong, Role of extracellular histidines in antagonist sensitivity of the rat P2X4 receptor. 2004, Pubmed , Xenbase
Zhang, Involvement of ectodomain Leu 214 in ATP binding and channel desensitization of the P2X4 receptor. 2014, Pubmed
Zhao, Relative motions between left flipper and dorsal fin domains favour P2X4 receptor activation. 2014, Pubmed