| Abstract No.: | A-B1054 |
| Country: | Canada |
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| Title: | FUNCTIONAL MODULATION OF P2X PURINERGIC RECEPTORS BY PHOSPHOINOSITIDES |
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| Authors/Affiliations: | 1 Louis-Philippe Bernier*; 1 Gary Mo; 1 Ariel Ase; 1 Stephanie Chevallier; 1 Dominique Blais; 1 Xinkang Tong; 1 Edith Hamel; 2 Eric Boue-Grabot; 1 Philippe Seguela;
1 McGill University, Montreal, QC, Canada; 2 Université Victor Segalen, Bordeaux, France
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| Content: | Objective: P2X receptors are ATP-gated non-selective cation channels involved in diverse physiological mechanisms such as pain signalling, smooth muscle contractions, microglial response and modulation of synaptic transmission. The P2X family consists of seven subunits (P2X1-7), assembling as homo- or hetero-trimers to form functional receptor-channels. Phosphoinositides (PIPn) are anionic lipids known to modulate several ion channels and transporters. Here, we demonstrate that PIPn modulate the function of several P2X receptor subtypes.
Materials and methods: We tested the effects of PIPn using two-electrode voltage clamp recordings on Xenopus oocytes expressing various rP2X subtypes. P2X1-mediated smooth muscle contraction was tested in a rat isolated mesenteric artery preparation. Patch-clamp recordings were done on rat dorsal root ganglion neurons expressing P2X2/3 and P2X3 and on BV-2 microglial cells expressing P2X4. PIPn modulation of P2X4-mediated calcium entry was measured by imaging on BV-2 cells. Direct interactions between PIPn and P2X subunits were tested using a lipid strip binding assay.
Results: P2X1, a subtype expressed in smooth muscle cells, is positively regulated by PI(4,5)P2 (PIP2) in an isolated rat mesenteric artery preparation, as are P2X1 currents in the Xenopus oocyte expression system. The P2X3 subtype, primarily involved in peripheral nociception, is also positively regulated by PIP2 in dorsal root ganglion (DRG) neurons and in Xenopus oocytes. P2X2/3, an heteromeric ATP receptor also expressed in DRG neurons, is regulated by both PIP2 and PI(3,4,5)P3 (PIP3), a product of PI3 kinase. We also investigated the dependence of P2X4 function on PIPn. P2X4 is expressed in spinal microglia where it is involved in neuropathic pain. We found that both PIP2 and PIP3 can modulate P2X4-mediated currents and calcium entry in BV-2 microglial cells. To determine the nature of the interaction between the P2X channels and PIPn, we used a lipid strip binding assay with GST-tagged peptides corresponding to a C-terminal region proximal to the transmembrane domain of each subunit. We found that P2X1, P2X2 and P2X4 can directly bind to PIPn via a semi-conserved 16-amino-acid domain enriched in basic residues.
Conclusion: These data indicate that the P2X receptor-channels can be functionally regulated by metabotropic pathways affecting the levels of intracellular PIPn, through direct channel-lipid interactions.
Acknowledgements: This work was supported by CIHR (PS) and CNRS (EBG). LPB holds a CIHR training grant “Pain M2C” studentship. GM holds a NSERC-IPS (AstraZeneca) studentship.
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