title: pH-dependent inhibition of native GABA(A)  receptors by HEPES.
abstract: OBJECTIVE
  Artificial buffers such as HEPES are extensively used to control extracellular pH (pH(e) ) to investigate the effect of H(+) ions on GABA(A) receptor function.
  
  
  METHODS
  In neurones cultured from spinal cord dorsal horn (DH), dorsal root ganglia (DRG) and cerebellar granule cells (GC) of neonatal rats, we studied the effect of pH(e) on currents induced by GABA(A) receptor agonists, controlling pH(e) with HCO(3) (-) or different concentrations of HEPES.
  
  
  RESULTS
  Changing HEPES concentration from 1 to 20 mM at constant pH(e) strongly inhibited the currents induced by submaximal GABA applications, but not those induced by glycine or glutamate, on DH, DRG or GC neurones, increasing twofold the EC(50) for GABA in DH neurones and GC. Submaximal GABA(A) receptor-mediated currents were also inhibited by piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), 3-(N-morpholino)propanesulfonic acid, tris(hydroxymethyl)aminomethane or imidazole. PIPES and HEPES, both piperazine derivatives, similarly inhibited GABA(A) receptors, whereas the other buffers had weaker effects and 2-(N-morpholino)ethanesulfonic acid had no effect. HEPES-induced inhibition of submaximal GABA(A) receptor-mediated currents was unaffected by diethylpyrocarbonate, a histidine-modifying reagent. HEPES-induced inhibition of GABA(A) receptors was independent of membrane potential, HCO(3) (-) and intracellular Cl(-) concentration and was not modified by flumazenil, which blocks the benzodiazepine binding site. However, it strongly depended on pH(e) .
  
  
  CONCLUSIONS
  Inhibition of GABA(A) receptors by HEPES depended on pH(e) , leading to an apparent H(+) -induced inhibition of DH GABA(A) receptors, unrelated to the pH sensitivity of these receptors in both low and physiological buffering conditions, suggesting that protonated HEPES caused this inhibition.
authors.affiliation: Nociception et Douleur, INCI, UPR3212 CNRS, Université de Strasbourg, Strasbourg, France. hugels@inci-cnrs.unistra.fr
authors.forename: S
authors.initials: S
authors.lastname: Hugel
authors.forename: N
authors.initials: N
authors.lastname: Kadiri
authors.forename: J L
authors.initials: JL
authors.lastname: Rodeau
authors.forename: S
authors.initials: S
authors.lastname: Gaillard
authors.forename: R
authors.initials: R
authors.lastname: Schlichter
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D016466:CHO Cells; D002531:Cerebellum / Q000166:cytology; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D058787:GABA-A Receptor Antagonists / Q000494:pharmacology*; D005786:Gene Expression Regulation; D006531:HEPES / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015394:Molecular Structure; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D051381:Rats; D011963:Receptors, GABA-A / Q000235:genetics / Q000378:metabolism*; D013116:Spinal Cord / Q000166:cytology; D009435:Synaptic Transmission / Q000187:drug effects
chemical_list: D058787:GABA-A Receptor Antagonists; D011963:Receptors, GABA-A; D006531:HEPES