Decrease of current responses at human recombinant P2X3 receptors after substitution by Asp of Ser/Thr residues in protein kinase C phosphorylation sites of their ecto-domains

Doychin Stanchev, Gesine Flehmig, Zoltan Gerevich, Wolfgang Nörenberg, Hassan Dihazi, Susanna Fürst, Klaus Eschrich, Peter Illes, Kerstin Wirkner

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

The whole-cell patch-clamp technique was used to record current responses to nucleotides in HEK 293 cells transiently transfected with the human (h) P2X3 receptor. When GDP-β-S was included into the pipette solution, UTP at concentrations which did not alter the holding current, facilitated the α,β-methylene ATP (α,β-meATP)-induced current. The substitution of Ser/Thr residues situated within protein kinase C (PKC) consensus phosphorylation sites of the P2X3 receptor ecto-domain by the neutral amino acid Ala either abolished (T134A, S178A) or did not alter (T196A, S269A) the UTP-induced potentiation of the α,β-meATP current. The substitution of the same Ser/Thr residues in all four PKC sites by the negatively charged Asp prevented the potentiation by UTP. The Asp mutations abolished the first, fast offset time-constant, but did not alter, or in the case of S269D even increased, the second, slow offset time-constant; at the same time such mutations invariably increased the onset time-constant and massively depressed the peak current amplitude. None of the Ala mutations (with the exception of S269A) influenced the time-course of desensitisation or the peak current amplitude. It is concluded that constitutive activation of PKC sites at the ecto-domain of the hP2X3 receptor both abolishes the UTP-induced potentiation of the α,β-meATP current and accelerates its rate of desensitisation.

Original languageEnglish
Pages (from-to)78-83
Number of pages6
JournalNeuroscience Letters
Volume393
Issue number1
DOIs
Publication statusPublished - Jan 23 2006

Keywords

  • Ecto-protein kinase C
  • Nucleotides
  • P2X receptor-phosphorylation
  • Site-directed mutagenesis

ASJC Scopus subject areas

  • Neuroscience(all)

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