Article

Tightening of the ATP-binding sites induces the opening of P2X receptor channels.

Faculté de Pharmacie, Laboratoire de Biophysicochimie des Récepteurs Canaux, UMR 7199 CNRS, Conception et Application de Molécules Bioactives, Université de Strasbourg, Illkirch, France.
The EMBO Journal (Impact Factor: 10.75). 03/2012; 31(9):2134-43. DOI: 10.1038/emboj.2012.75
Source: PubMed

ABSTRACT The opening of ligand-gated ion channels in response to agonist binding is a fundamental process in biology. In ATP-gated P2X receptors, little is known about the molecular events that couple ATP binding to channel opening. In this paper, we identify structural changes of the ATP site accompanying the P2X2 receptor activation by engineering extracellular zinc bridges at putative mobile regions as revealed by normal mode analysis. We provide evidence that tightening of the ATP sites shaped like open 'jaws' induces opening of the P2X ion channel. We show that ATP binding favours jaw tightening, whereas binding of a competitive antagonist prevents gating induced by this movement. Our data reveal the inherent dynamic of the binding jaw, and provide new structural insights into the mechanism of P2X receptor activation.

Full-text

Available from: Antoine Taly, Jun 02, 2015
1 Follower
 · 
287 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: P2X receptors are trimeric ATP-activated non-selective cation channels. The ATP binding pocket is positioned between two neighboring subunits. Accompanying ligand binding, subunit-subunit contacts are most likely involved in receptor function and drive a conformational change to open the ion permeation pathway. In this way, we sought to determine the function of side chains of the zebrafish P2X4 receptor ectodomain left-flipper-to-dorsal-fin interface residues in ligand binding. By Combining site-directed mutagenesis and electrophysiology methods, we showed that cysteine substitutions of I212, S215, Y216 and L217 resulted in decreased sensitivity to ATP. In addition, the ATP induced current at L217C was completely inhibited by sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES(-)), indicating a role for this residue in ATP action. Deletion of residues 285-293 from the zebrafish P2X4 receptor abolished channel function. However, insertion of the same sequence frame into a homologous position of the rat P2X6 receptor did not rescue channel function, suggesting that these residues are necessary but not sufficient for achieving the correct ATP-induced conformation.
    Neuroscience Letters 08/2014; 582. DOI:10.1016/j.neulet.2014.08.038 · 2.06 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The P2X receptor is an ATP-gated cation channel expressed on the plasma membrane. The head domain (Gln111-Val167 in the rat P2X4 receptor) regulates ATP-induced cation influx. In this study, we prepared a head domain with three disulfide bonds, such as the intact rat P2X4 receptor contains. NMR analysis showed that the head domain possessed the same fold as in the zebrafish P2X4 receptor previously determined by crystallography. Furthermore, the inhibitory, divalent, metal ion binding sites were determined by NMR techniques. These findings will be useful for the design of specific inhibitors for the P2X receptor family. Copyright © 2015. Published by Elsevier B.V.
    FEBS Letters 02/2015; 589(6). DOI:10.1016/j.febslet.2015.01.034 · 3.34 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Background And PurposeIt is assumed that ATP induces closure of the binding jaw of ligand-gated P2X receptors, which eventually results in the opening of the membrane channel and the flux of cations. Immobilization by cysteine mutagenesis of the binding jaw inhibited ATP-induced current responses, but did not allow discrimination between disturbances of binding, gating, subunit assembly or trafficking to the plasma membrane.Experimental ApproachA molecular model of the pain-relevant human hP2X3 receptor was used to identify amino acid pairs which were located at the lips of the binding jaw and did not participate in agonist binding but strongly approached each other even in the absence of ATP.Key ResultsA series of cysteine double mutant hP2X3 receptors, expressed in HEK293 cells or Xenopus laevis oocytes, exhibited depressed current responses to α,β-methylene ATP (α,β-meATP), due to the formation of spontaneous inter-subunit disulfide bonds. Dithiothreitol (DTT), by reducing these bonds, reversed the blockade of the α,β-meATP transmembrane current. Amino-reactive fluorescence labelling of the His-tagged hP2X3 receptor and its mutants expressed in HEK293 or X. laevis oocytes demonstrated the formation of inter-subunit crosslinks in cysteine double mutants and in addition, confirmed their correct trimeric assembly and cell surface expression.Conclusions And ImplicationsIn conclusion, spontaneous tightening of the binding jaw of the hP2X3 receptor by inter-subunit cross-linking of cysteine residues substituted at positions not directly involved in agonist binding inhibited agonist-evoked currents without interfering with binding, subunit assembly or trafficking.
    British Journal of Pharmacology 07/2014; 171(22). DOI:10.1111/bph.12830 · 4.99 Impact Factor