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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 - Mol Pain (2009)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Montreal Neurological Institute, Department of Neurology & Neurosurgery, McGill University, Montreal, Canada. gary.mo@mail.mcgill.ca

ABSTRACT

Background: 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.

Results: 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.

Conclusion: 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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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).
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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).

Mentions: To further investigate the modulation of P2X3 receptor channels by phosphoinositides observed in DRG, mutation studies were conducted to identify residues on the P2X3 subunit that are critical for the interaction with PIP2. Interestingly, we observed that the initial current amplitude of recombinant P2X3 receptors expressed in HEK293 cells and Xenopus oocytes was not sensitive to PIP2 and PIP3 depletion by incubation with 35 μM wortmannin (Table 1). We previously reported that basic residues play a key role in modulation of several P2X receptor subtypes by phospholipids [20,25,26] so five point mutations (K348Q, K354Q, R356Q, K357Q, and R367Q) were generated to target candidate lysine and arginine residues involved in direct or indirect modulation of P2X3 by phosphoinositides. Of the five mutant P2X3 channel subunits produced, only the K348Q, R356Q and R367Q mutants responded to 10 μM α,β-meATP. K348Q and R367Q were indistinguishable from wild-type P2X3 channels while the mutants K354Q and K357Q were silent (Fig. 6A). The R356Q mutant displayed a 48% (64.7 ± 14.3 pA/pF) reduction in activity compared to wild-type P2X3 channel responses (125.2 ± 11.6 pA/pF). To check if the decreased responses of the R356Q mutant were actually due to a decreased receptor function rather than a decreased receptor trafficking, a surface biotinylation study was performed with all the mutants and the wild-type P2X3 constructs. Surface protein expression for HEK293 cells transfected with wild-type P2X3 was not significantly different from the one in HEK293 cells transfected with any of the mutants (Fig. 6B).


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 - Mol Pain (2009)

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).
© Copyright Policy - open-access
Related In: Results  -  Collection

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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).
Mentions: To further investigate the modulation of P2X3 receptor channels by phosphoinositides observed in DRG, mutation studies were conducted to identify residues on the P2X3 subunit that are critical for the interaction with PIP2. Interestingly, we observed that the initial current amplitude of recombinant P2X3 receptors expressed in HEK293 cells and Xenopus oocytes was not sensitive to PIP2 and PIP3 depletion by incubation with 35 μM wortmannin (Table 1). We previously reported that basic residues play a key role in modulation of several P2X receptor subtypes by phospholipids [20,25,26] so five point mutations (K348Q, K354Q, R356Q, K357Q, and R367Q) were generated to target candidate lysine and arginine residues involved in direct or indirect modulation of P2X3 by phosphoinositides. Of the five mutant P2X3 channel subunits produced, only the K348Q, R356Q and R367Q mutants responded to 10 μM α,β-meATP. K348Q and R367Q were indistinguishable from wild-type P2X3 channels while the mutants K354Q and K357Q were silent (Fig. 6A). The R356Q mutant displayed a 48% (64.7 ± 14.3 pA/pF) reduction in activity compared to wild-type P2X3 channel responses (125.2 ± 11.6 pA/pF). To check if the decreased responses of the R356Q mutant were actually due to a decreased receptor function rather than a decreased receptor trafficking, a surface biotinylation study was performed with all the mutants and the wild-type P2X3 constructs. Surface protein expression for HEK293 cells transfected with wild-type P2X3 was not significantly different from the one in HEK293 cells transfected with any of the mutants (Fig. 6B).

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Montreal Neurological Institute, Department of Neurology & Neurosurgery, McGill University, Montreal, Canada. gary.mo@mail.mcgill.ca

ABSTRACT

Background: 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.

Results: 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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus