Regulation of Human Recombinant P2X₃Receptors 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 P2X 3 receptor. When guanosine 5Ј-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 P2X 3 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 EC 50 values. Molecular simulation of the P2X 3 receptor structure indicated no overlap between assumed nucleotide binding domains and the relevant phosphorylation sites PKC2 and PKC3. ␣,␤-meATP-induced currents through native homomeric P2X 3 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 P2X 3 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 P2X 3 receptors.

Activation and Regulation of Purinergic P2X Receptor Channels

“…These observations do not exclude the potential phosphorylation of other residues by PKC. In hP2X3R, the substitution of Ser/Thr residues situated within the PKC consensus phosphorylation sites with Ala either abolished (T134A, S178A) or did not alter (T196A, S269A) the UTP-induced potentiation of current (Wirkner et al, 2005;Stanchev et al, 2006).…”

Neutralization of Nerve Growth Factor Induces Plasticity of ATP-Sensitive P2X3Receptors of Nociceptive Trigeminal Ganglion Neurons

“…The P2X 3 receptor possesses a large number of intracellular threonine residues that can be the target of different kinases, including ectokinases (Pawlowska et al, 1993;Wirkner et al, 2005), allowing for a complex modulation of receptor expression and function by NGF. Our present data demonstrating block of NGF effects on P2X 3 receptors by the PKC inhibitor chelerythrine further points to a role for this kinase.…”

The Leishmania-Macrophage Interactions: Role of E-NTPDases and Purinergic Signaling

“…However, it is important to note that the variability of the population and expression of receptors in each cell type is crucial to determine the physiological response for each stimulus [82,83,[86][87][88][89][90][91][92][93][94] .…”