Mo G et al. (2009), Subtype-specific regulation of P2X3 and P2X2/3 ...

XB-ART-40150

Mol Pain 2009 Aug 11;5:47. doi: 10.1186/1744-8069-5-47.

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Subtype-specific regulation of P2X3 and P2X2/3 receptors by phosphoinositides in peripheral nociceptors.

Mo G , Bernier LP , Zhao Q , Chabot-Doré AJ , Ase AR , Logothetis D , Cao CQ , Séguéla P .

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P2X3 and P2X2/3 purinergic receptor-channels, expressed in primary sensory neurons that mediate nociception, have been implicated in neuropathic and inflammatory pain responses. The phospholipids phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3) are involved in functional modulation of several types of ion channels. We report here evidence that these phospholipids are able to modulate the function of homomeric P2X3 and heteromeric P2X2/3 purinoceptors expressed in dorsal root ganglion (DRG) nociceptors and in heterologous expression systems. In dissociated rat DRG neurons, incubation with the PI3K/PI4K inhibitor wortmannin at 35 microM induced a dramatic decrease in the amplitude of ATP- or alpha,beta-meATP-evoked P2X3 currents, while incubation with 100 nM wortmannin (selective PI3K inhibition) produced no significant effect. Intracellular application of PIP2 was able to fully reverse the inhibition of P2X3 currents induced by wortmannin. In Xenopus oocytes and in HEK293 cells expressing recombinant P2X3, 35 microM wortmannin incubation induced a significant decrease in the rate of receptor recovery. Native and recombinant P2X2/3 receptor-mediated currents were inhibited by incubation with wortmannin both at 35 microM and 100 nM. The decrease of P2X2/3 current amplitude induced by wortmannin could be partially reversed by application of PIP2 or PIP3, indicating a sensitivity to both phosphoinositides in DRG neurons and Xenopus oocytes. Using a lipid binding assay, we demonstrate that the C-terminus of the P2X2 subunit binds directly to PIP2, PIP3 and other phosphoinositides. In contrast, no direct binding was detected between the C-terminus of P2X3 subunit and phosphoinositides. Our findings indicate a functional regulation of homomeric P2X3 and heteromeric P2X2/3 ATP receptors by phosphoinositides in the plasma membrane of DRG nociceptors, based on subtype-specific mechanisms of direct and indirect lipid sensing.

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Species referenced: Xenopus
Genes referenced: drg1 p2rx2 pik3ca pik3cg

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	Figure 1. Sensitivity of native P2X3 receptor activity to PIP2 depletion in DRG neurons. A) Typical traces of P2X3 response to 10 μM α,β-meATP in DRG nociceptors (left), to 10 μM α,β-meATP after 2 h incubation with wortmannin (middle), to 10 μM α,β-meATP after 2 h incubation with wortmannin and intracellular application of 200 μM PIP2 (right). B) Quantitative results. P2X3 responses to 10 μM α,β-meATP under control conditions, after 2 h incubation with 100 nM wortmannin, 35 μM wortmannin, or 35 μM wortmannin with 200 μM PIP2 in the pipette solution (N = 6–13). C) Quantitative results. P2X3 responses to 10 μM ATP under control conditions, after 2 h incubation with 100 nM wortmannin, 35 μM wortmannin, or 35 μM wortmannin with 200 μM PIP2 with in the pipette solution (N = 7–18). (***, P < 0.001).
	Figure 2. Sensitivity of native P2X2/3 receptor activity to PIP3 depletion in DRG neurons. A) Sample traces demonstrating the effects of 35 μM (middle) and 100 nM wortmannin incubation (2 h) (right) on P2X2/3 response to 10 μM α,β-meATP in DRG (control on left). B) Pooled data of P2X2/3 responses to 10 μM α,β-meATP under control condition, after 2 h incubation with 35 μM or 100 nM wortmannin (N = 5–6). (**, P < 0.01)
	Figure 3. Sensitivity of P2X2/3 currents to changes in PIP2 and PIP3 levels in Xenopus oocytes. A) Sample traces showing the effect of 35 μM wortmannin incubation and the injection of PBS, PIP2 or PIP3 as compared to an initial current recorded prior to incubation. B) Pooled data of P2X2/3 responses to 2 h incubation in control solution (Barth's solution with DMSO), in 35 μM wortmannin, 100 nM wortmannin, or 35 μM LY294002 (N = 10–33). C) Pooled data of P2X2/3 current inhibition by 2 h incubation with 35 μM wortmannin in oocytes injected with vehicle, PIP2, and PIP3 (N = 5–17). (, P < 0.01; *, P < 0.001)
	Figure 4. PIP2-activated P2X2 and P2X3 currents in excised inside-out macropatches from Xenopus oocytes. The pipette solution contained ATP at 100 μM for P2X2 and at 30 μM for P2X3. The currents were evoked by voltage ramps from -100 mV to +100 mV. The traces shown were constructed by connecting the current points at -80 mV. (**, P < 0.01)
	Figure 5. The C-terminus of P2X2, but not of P2X3, directly binds phosphoinositides. A) Alignment of the proximal C-termini of rat P2X2 and P2X3 subunits showing intracellular basic residues candidates for lipid binding. The dashed lines delineate the sequences of the peptides used in the lipid binding assay. B) Positions of the different lipids spotted on the membrane. C) Binding pattern of GST protein alone, GST-P2X2 (F355-T372) and GST-P2X3 (F346-T364) shows that the sequence from P2X2, but not from P2X3, directly binds several anionic phospholipids including PIP2 and PIP3.
	Figure 6. Sensitivity of P2X3 current to PIP2 depletion in HEK293 cells is reversed by R356Q mutation. A) Pooled data of wild-type and mutant P2X3 responses to 10 μM ATP expressed in HEK293 cells (N = 5–11). B) Representative surface biotinylation data of wild-type and mutant P2X3 receptor expression. C) Sample traces showing 35 μM wortmannin-induced rundown of wild-type P2X3 currents in HEK293 cells. D) Quantitative results. Rundown of wild-type and R356Q mutant P2X3 current in response to 35 μM wortmannin incubation (N = 5–6). (**, P < 0.01).

References [+] :

Bernier, Direct modulation of P2X1 receptor-channels by the lipid phosphatidylinositol 4,5-bisphosphate. 2008, Pubmed, Xenbase