title: Conformational flexibility of the agonist binding jaw of the human P2X3 receptor is a prerequisite for channel opening.
abstract: OBJECTIVE
  It 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.
  
  
  METHODS
  A molecular model of the pain-relevant human (h)P2X3 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.
  
  
  RESULTS
  A 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. Reducing these bonds with dithiothreitol 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 cross links in cysteine double mutants and, in addition, confirmed their correct trimeric assembly and cell surface expression.
  
  
  CONCLUSIONS
  In 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.
authors.affiliation: Rudolf-Boehm-Institute of Pharmacology and Toxicology, University of Leipzig, Leipzig, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Kowalski
authors.forename: R
authors.initials: R
authors.lastname: Hausmann
authors.forename: A
authors.initials: A
authors.lastname: Dopychai
authors.forename: M
authors.initials: M
authors.lastname: Grohmann
authors.forename: H
authors.initials: H
authors.lastname: Franke
authors.forename: K
authors.initials: K
authors.lastname: Nieber
authors.forename: G
authors.initials: G
authors.lastname: Schmalzing
authors.forename: P
authors.initials: P
authors.lastname: Illes
authors.forename: T
authors.initials: T
authors.lastname: Riedel
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating; D008958:Models, Molecular; D009154:Mutation; D009865:Oocytes; D011487:Protein Conformation; D058911:Purinergic P2X Receptor Agonists / Q000494:pharmacology*; D058477:Receptors, Purinergic P2X3 / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D014982:Xenopus laevis
chemical_list: D058911:Purinergic P2X Receptor Agonists; D058477:Receptors, Purinergic P2X3; D000255:Adenosine Triphosphate; C002630:alpha,beta-methyleneadenosine 5'-triphosphate