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The Journal of Neuroscience, August 24, 2005, 25(34):7734-7742; doi:10.1523/JNEUROSCI.2028-05.2005

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Cellular/Molecular
Regulation of Human Recombinant P2X₃ Receptors by Ecto-Protein Kinase C

Kerstin Wirkner,¹ Doychin Stanchev,¹ Laszlo Köles,^1,3 Markus Klebingat,¹ Hassan Dihazi,² Gesine Flehmig,² Catherine Vial,⁵ Richard J. Evans,⁵ Susanna Fürst,^3,4 Peter P. Mager,¹ Klaus Eschrich,² and Peter Illes¹

¹Rudolf-Boehm-Institute of Pharmacology and Toxicology, University of Leipzig, D-04107 Leipzig, Germany, ²Department of Biochemistry, University of Leipzig, D-04103 Leipzig, Germany, ³Department of Pharmacology and Pharmacotherapy and ⁴Group of Neuropsychopharmacology, Semmelweis University, H-1445 Budapest, Hungary, and ⁵Department of Cell Physiology and Pharmacology, University of Leicester, Leicester LE1 9HN, United Kingdom

The whole-cell patch-clamp technique was used to record current responses to nucleotides and nucleosides in human embryonic kidney HEK293 cells transfected with the human purinergic P2X₃ receptor. When guanosine 5'-O-(3-thiodiphosphate) was included into the pipette solution, UTP at concentrations that did not alter the holding current facilitated the ,-methylene ATP (,-meATP)-induced current. ATP and GTP, but not UDP or uridine, had an effect similar to that of UTP. Compounds known to activate protein kinase C (PKC) acted like the nucleoside triphosphates investigated, whereas various PKC inhibitors invariably reduced the effects of both PKC activators and UTP. The substitution by Ala of Ser/Thr residues situated within PKC consensus sites of the P2X₃ receptor ectodomain either abolished (PKC2 and PKC3; T134A, S178A) or did not alter (PKC4 and PKC6; T196A, S269A) the UTP-induced potentiation of the ,-meATP current. Both the blockade of ecto-protein kinase C activity and the substitution of Thr-134 or Ser-178 by Ala depressed the maximum of the concentration-response curve for ,-meATP without altering the EC₅₀ values. Molecular simulation of the P2X₃ receptor structure indicated no overlap between assumed nucleotide binding domains and the relevant phosphorylation sites PKC2 and PKC3. ,-meATP-induced currents through native homomeric P2X₃ receptors of rat dorsal root ganglia were also facilitated by UTP. In conclusion, it is suggested that low concentrations of endogenous nucleotides in the extracellular space may prime the sensitivity of P2X₃ receptors toward the effect of subsequently applied (released) higher agonistic concentrations. The priming effect of nucleotides might be attributable to a phosphorylation of PKC sites at the ectodomain of P2X₃ receptors.

Key words: nucleotides; ecto-protein kinase C; P2X₃ receptor phosphorylation; site-directed mutagenesis; mutant; analgesia

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Received Feb 9, 2005; revised July 4, 2005; accepted July 5, 2005.

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