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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 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)
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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)

Mentions: Although P2X3 is the predominant P2X receptor subunit expressed in DRG nociceptors, about one over ten neurons responded with a rapidly-activating and slowly-desensitizing response (Fig. 2A), attributed to the activation of heteromeric P2X2/3 receptor channels [30]. Contrary to homomeric P2X3 channels expressed in DRG neurons, α,β-meATP-evoked responses of native heteromeric P2X2/3 channels were sensitive to both high as well as low wortmannin concentration. As shown in Fig. 2A and 2B, incubation with 35 μM and 100 nM wortmannin decreased P2X2/3 current responses by 53% and 59% of control level respectively (control: 8.8 ± 1.1 pF/pA; 35 μM wortmannin: 4.2 ± 0.6 pF/pA; 100 nM wortmannin: 3.6 ± 0.6 pF/pA, N = 5–6).


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

License
Show All Figures
getmorefigures.php?uid=PMC2734547&req=5

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)
Mentions: Although P2X3 is the predominant P2X receptor subunit expressed in DRG nociceptors, about one over ten neurons responded with a rapidly-activating and slowly-desensitizing response (Fig. 2A), attributed to the activation of heteromeric P2X2/3 receptor channels [30]. Contrary to homomeric P2X3 channels expressed in DRG neurons, α,β-meATP-evoked responses of native heteromeric P2X2/3 channels were sensitive to both high as well as low wortmannin concentration. As shown in Fig. 2A and 2B, incubation with 35 μM and 100 nM wortmannin decreased P2X2/3 current responses by 53% and 59% of control level respectively (control: 8.8 ± 1.1 pF/pA; 35 μM wortmannin: 4.2 ± 0.6 pF/pA; 100 nM wortmannin: 3.6 ± 0.6 pF/pA, N = 5–6).

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