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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
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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) P2X(3) receptor. When GDP-beta-S was included into the pipette solution, UTP at concentrations which did not alter the holding current, facilitated the alpha,beta-methylene ATP (alpha,beta-meATP)-induced current. The substitution of Ser/Thr residues situated within protein kinase C (PKC) consensus phosphorylation sites of the P2X(3) 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 alpha,beta-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 hP2X(3) receptor both abolishes the UTP-induced potentiation of the alpha,beta-meATP current and accelerates its rate of desensitisation.

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Adenosine TriphosphateAspartic AcidCell LineDose-Response Relationship, DrugElectric StimulationHumansMembrane PotentialsMutagenesisPatch-Clamp TechniquesPhosphorylationProtein Kinase CPurinergic P2 Receptor AgonistsPurinergic P2 Receptor AntagonistsReceptors, Purinergic P2Receptors, Purinergic P2X2Recombinant ProteinsSerineThreonineTransfectionUridine TriphosphateComparative StudyJournal ArticleResearch Support, Non-U.S. Gov't

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Doychin Stanchev, Gesine Flehmig, Zoltan Gerevich, Wolfgang Nörenberg, Hassan Dihazi, Susanna Fürst, Klaus Eschrich, Peter Illes, Kerstin Wirkner. 2005-10-13. 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.. https://doi.org/10.1016/j.neulet.2005.09.045

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