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

Mo Gary, Bernier Louis-Philippe, Zhao Qi, Chabot-Doré Anne-Julie, Ase, Logothetis Diomedes, Cao Chang-Qing, Séguéla, Séguéla Philippe

Description

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 (PIP 2 ) and phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ) 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 μM induced a dramatic decrease in the amplitude of ATP- or α,β-meATP-evoked P2X3 currents, while incubation with 100 nM wortmannin (selective PI3K inhibition) produced no significant effect. Intracellular application of PIP 2 was able to fully reverse the inhibition of P2X3 currents induced by wortmannin. In Xenopus oocytes and in HEK293 cells expressing recombinant P2X3, 35 μM 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 μM and 100 nM. The decrease of P2X2/3 current amplitude induced by wortmannin could be partially reversed by application of PIP 2 or PIP 3 , 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 PIP 2 , PIP 3 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.

Informations

• Publié par: biomed
• Publié le: 01 janvier 2009
• Nombre de lectures: 5
• Langue: English

Extrait

Pga e 1fo1  ( 3 apegum nr bet nor foaticnoitrup esops)

Published: 11 August 2009 Received: 21 May 2009 009 Molecular Pain 2009, 5 :47 doi:10.1186/1744-8069-5-47  Accepted: 11 August 2 This article is available from: http ://www.molecularpain.com/content/5/1/47 © 2009 Mo et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons. org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the orig inal work is properly cited.

Abstract Background: P2X3 and P2X2/3 purinergic receptor-c hannels, expressed in primary sensory neurons that mediate nociception, have been im plicated in neuropathic and inflammatory pain responses. The phospholipids phosphati dylinositol 4,5-bisphosphate (PIP 2 ) and phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ) are involved in functional modulati on of several types of ion channels. We report here evidence that th ese phospholipids are able to modu late 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 neuron s, incubation with the PI3K /PI4K inhibitor wortmannin at 35 μ M induced a dramatic decrease in the amplitude of ATP- or α , β -meATP-evoked P2X3 currents, while incubation with 100 nM wortmann in (selective PI3K inhibition) produced no significant effect. Int cation o P was able to fully revers e the inhibition of P2X3 racellular appli f PI 2 currents induced by wortmannin. In Xenopus oocytes and in HEK293 cells expressing recombinant P2X3, 35 μ M wortmannin incubation induced a signific ant decrease in the rate of receptor recovery. Native and recombinant P2X2/3 re ceptor-mediated current s were inhibited by incubation with wortmannin both at 35 μ M and 100 nM. The decrease of P2X2/3 current amplitude induced by wortmannin could be partia lly reversed by application of PIP 2 or PIP 3 , indicating a sensitivity to both phosphoin ositides in DRG neurons and Xenopus oocytes. Using a lipid binding assay, we demonstrate that the C-terminus of the P2X2 subunit binds directly to PIP 2 , PIP 3 and other phosphoinositides. In cont rast, no direct binding was dete cted between the C-terminus of P2X3 subunit and phosphoinositides. Conclusion: Our findings indicate a functional regula tion of homomeric P2X3 and heteromeric P2X2/3 ATP receptors by phosphoinositides in th e plasma membrane of DRG nociceptors, based on subtype-specific mechanisms of direct and indirect lipid sensing.

Address: 1 Montreal Neurological Institute, Department of Neurol ogy & Neurosurgery, McGill University, Montreal, Canada, 2 Department of Structural and Chemical Biology, Mount Sinai School of Medicine, New York, USA and 3 Department of Bioscience, AstraZeneca R&D Montreal, Montreal, Canada Email: Gary Mo - [e-mail]; Louis-Philippe Bernier - [e-mail]; Qi Zhao - [e-mail]; Anne-Julie Chabot-Doré - a [e-mail]; Ariel R Ase - [e-mail]; Diomedes Logothetis - [e-mail]; Ch ang-Qing Cao - [e-mail]; Philippe Séguéla* - phili [e-mail] * Corresponding author †Equal contributors

Molecular Pain

Bio Med Central

Research Open Access Subtype-specific regulation of P2X3 and P2X2/3 receptors by phosphoinositides in peripheral nociceptors Gary Mo †1,3 , Louis-Philippe Bernier †1 , Qi Zhao 2 , Anne-Julie Chabot-Doré 1 , Ariel R Ase 1 , Diomedes Logothetis 2 , Chang-Qing Cao 3 and Philippe Séguéla* 1