title: Identification of the amino acid residues in the extracellular domain of rat P2X(7) receptor involved in functional inhibition by acidic pH.
abstract: OBJECTIVE
  P2X(7), receptors are potently inhibited by extracellular acidification. The underlying molecular basis remains unknown. This study aimed to examine the role of extracellular histidine, lysine, aspartic acid and glutamic acid residues in the functional inhibition of rat P2X(7) receptors by acidic pH.
  
  
  METHODS
  We introduced point mutations into rat P2X(7) receptor by site-directed mutagenesis, expressed wild type (WT) and mutant receptors in human embryonic kidney (HEK293) cells and, using patch clamp recording, characterized the effects of acidic pH on BzATP [2'-3'O-(4-benzoylbenzoyl) adenosine 5'-triphosphate]-evoked ionic currents.
  
  
  RESULTS
  Reducing extracellular pH, that is, increasing extracellular proton concentrations, inhibited BzATP-evoked currents in cells expressing WT P2X(7) receptors, with IC(50) value (half-maximal antagonist or inhibitor concentration) for protons of 0.2 mumol.L(-1). The major effect of acidification was suppression of the maximal current response without altering the agonist sensitivity. five residues in the receptor extracellular domain (His(85), Lys(110), Lys(137), Asp(197) and His(219)) were mutated to alanine and current inhibition by protons assessed. Compared with WT, the H85A, H219A, K137A mutants were two- to threefold more sensitive, whereas the K110A and D197A mutants were 2.5- and 9-fold less sensitive. Double-alanine substitution of Lys(110) and Asp(197) resulted in 23-fold decreased sensitivity to inhibition by protons. Furthermore, charge neutralization (K110M, K110F, D197N and D197F), but not charge conserving mutation (K110R and D197E), attenuated the inhibition of currents by protons.
  
  
  CONCLUSIONS
  Functional inhibition of rat P2X(7) receptors by acidic pH was variably affected by the extra-cellular His(85), Lys(110), Lys(137), Asp(197) and His(219) residues, with the Asp(197) residue being most critical for this inhibition.
authors.affiliation: Institute of Membrane and Systems Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK.
authors.forename: X
authors.initials: X
authors.lastname: Liu
authors.forename: W
authors.initials: W
authors.lastname: Ma
authors.forename: A
authors.initials: A
authors.lastname: Surprenant
authors.forename: L-H
authors.initials: LH
authors.lastname: Jiang
mesh_terms: D000596:Amino Acids / Q000235:genetics*; D000818:Animals; D002460:Cell Line; D005110:Extracellular Space / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D051379:Mice; D016297:Mutagenesis, Site-Directed; D018408:Patch-Clamp Techniques; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D058919:Purinergic P2 Receptor Antagonists; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000235:genetics / Q000502:physiology; D058486:Receptors, Purinergic P2X7
chemical_list: D000596:Amino Acids; C546468:P2RX7 protein, human; C546469:P2rx7 protein, mouse; C546470:P2rx7 protein, rat; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7