title: Regulation of human recombinant P2X3 receptors by ecto-protein kinase C.
abstract: 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 alpha,beta-methylene ATP (alpha,beta-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 alpha,beta-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 alpha,beta-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. alpha,beta-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.
authors.affiliation: Rudolf-Boehm-Institute of Pharmacology and Toxicology, University of Leipzig, D-04107 Leipzig, Germany.
authors.forename: Kerstin
authors.initials: K
authors.lastname: Wirkner
authors.forename: Doychin
authors.initials: D
authors.lastname: Stanchev
authors.forename: Laszlo
authors.initials: L
authors.lastname: Köles
authors.forename: Markus
authors.initials: M
authors.lastname: Klebingat
authors.forename: Hassan
authors.initials: H
authors.lastname: Dihazi
authors.forename: Gesine
authors.initials: G
authors.lastname: Flehmig
authors.forename: Catherine
authors.initials: C
authors.lastname: Vial
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Evans
authors.forename: Susanna
authors.initials: S
authors.lastname: Fürst
authors.forename: Peter P
authors.initials: PP
authors.lastname: Mager
authors.forename: Klaus
authors.initials: K
authors.lastname: Eschrich
authors.forename: Peter
authors.initials: P
authors.lastname: Illes
mesh_terms: D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D006801:Humans; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism / Q000502:physiology*; D058910:Purinergic P2 Receptor Agonists; D051381:Rats; D017208:Rats, Wistar; D018048:Receptors, Purinergic P2 / Q000235:genetics / Q000378:metabolism*; D058477:Receptors, Purinergic P2X3; D011994:Recombinant Proteins / Q000819:agonists / Q000378:metabolism; D014544:Uridine Triphosphate / Q000494:pharmacology
chemical_list: C546407:P2RX3 protein, human; C546410:P2rx3 protein, rat; D047428:Protein Kinase Inhibitors; D058910:Purinergic P2 Receptor Agonists; D018048:Receptors, Purinergic P2; D058477:Receptors, Purinergic P2X3; D011994:Recombinant Proteins; D011494:Protein Kinases; C034454:ectoprotein kinase; D011493:Protein Kinase C; D014544:Uridine Triphosphate