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Rab5 regulates internalisation of P2X4 receptors and potentiation by ivermectin

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Abstract

The P2X4 receptor is an ATP-gated ion channel expressed in neurons, endothelia and immune cells. Plasma membrane expression of P2X4 is regulated by dynamin-dependent endocytosis, and this study identifies a Rab5-dependent pathway of receptor internalisation. Expression of Rab5 constructs altered the distribution of P2X4 in HEK-293 cells, and both constitutive internalisation and agonist-induced desensitisation of P2X4 were increased by co-expression of wild-type Rab5 or constitutively active Rab5 (Q79L). Expression of inactive dynamin K44A and Rab5 S34N constructs abolished agonist-induced desensitisation, suggesting internalisation as the underlying mechanism. Blocking P2X4 internalisation in this way also abolished potentiation of ATP-induced currents by the allosteric modulator ivermectin. This suggests that the dynamin-Rab5 internalisation pathway is essential for the ivermectin potentiation effect. In agreement with this hypothesis, the co-expression of wild-type dynamin, wild-type Rab5 or active Rab5 (Q79L) could increase the potentiation of the ATP-induced P2X4 response by ivermectin. These findings highlight Rab5 GTPase as a key regulator of P2X4 receptor cell surface expression and internalisation.

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References

  1. North RA (2002) Molecular physiology of P2X receptors. Physiol Rev 82(4):1013–1067

    PubMed  CAS  Google Scholar 

  2. Surprenant A, North RA (2009) Signaling at purinergic P2X receptors. Annu Rev Physiol 71:333–359

    Article  PubMed  CAS  Google Scholar 

  3. Yamamoto K, Sokabe T, Matsumoto T, Yoshimura K, Shibata M, Ohura N, Fukuda T, Sato T, Sekine K, Kato S, Isshiki M, Fujita T, Kobayashi M, Kawamura K, Masuda H, Kamiya A, Ando J (2006) Impaired flow-dependent control of vascular tone and remodeling in P2X4-deficient mice. Nat Med 12(1):133–137

    Article  PubMed  CAS  Google Scholar 

  4. Sim JA (2006) Altered hippocampal synaptic potentiation in P2X4 knock-out mice. J Neurosci 26(35):9006–9009

    Article  PubMed  CAS  Google Scholar 

  5. Ulmann L, Hatcher J, Hughes J, Chaumont S, Green P, Conquet F, Buell G, Reeve A, Chessell I, Rassendren F (2008) Up-regulation of P2X4 receptors in spinal microglia after peripheral nerve injury mediates BDNF release and neuropathic pain. J Neurosci 28(44):11263–11268

    Article  PubMed  CAS  Google Scholar 

  6. Khakh BS, Proctor WR, Dunwiddie TV, Labarca C, Lester HA (1999) Allosteric control of gating and kinetics at P2X(4) receptor channels. J Neurosci 19(17):7289–7299

    PubMed  CAS  Google Scholar 

  7. Dawson GR, Wafford KA, Smith AV, Marshall GR, Bayley PJ, Schaeffer JM, Meinke PT, McKernan RM (2000) Anticonvulsant and adverse effects of avermectin analogs in mice are mediated through the gamma-aminobutyric acid(A) receptor. J Pharmacol Exp Ther 295(3):1051–1060

    PubMed  CAS  Google Scholar 

  8. Shan Q (2001) Ivermectin, an unconventional agonist of the glycine receptor chloride channel. J Biol Chem 276(16):12556–12564

    Article  PubMed  CAS  Google Scholar 

  9. Toulmé E, Soto F, Garret M, Boué-Grabot E (2006) Functional properties of internalization-deficient P2X4 receptors reveal a novel mechanism of ligand-gated channel facilitation by ivermectin. Mol Pharmacol 69(2):576–587

    Article  PubMed  Google Scholar 

  10. Silberberg S, Li M, Swartz K (2007) Ivermectin interaction with transmembrane helices reveals widespread rearrangements during opening of P2X receptor channels. Neuron 54(2):263–274

    Article  PubMed  CAS  Google Scholar 

  11. Royle SJ, Qureshi OS, Bobanović LK, Evans PR, Owen DJ, Murrell-Lagnado RD (2005) Non-canonical YXXGPhi endocytic motifs: recognition by AP2 and preferential utilization in P2X4 receptors. J Cell Sci 118:3073–3080

    Article  PubMed  CAS  Google Scholar 

  12. Royle SJ, Murrell-Lagnado RD (2002) Constitutive cycling: a general mechanism to regulate cell surface proteins. Bioessays 25(1):39–46

    Article  CAS  Google Scholar 

  13. Stokes L, Surprenant A (2009) Dynamic regulation of the P2X 4receptor in alveolar macrophages by phagocytosis and classical activation. Eur J Immunol 39(4):986–995

    Article  PubMed  CAS  Google Scholar 

  14. Qureshi OS, Paramasivam A, Yu JCH, Murrell-Lagnado RD (2007) Regulation of P2X4 receptors by lysosomal targeting, glycan protection and exocytosis. J Cell Sci 120(21):3838–3849

    Article  PubMed  CAS  Google Scholar 

  15. Schafer DA (2004) Regulating actin dynamics at membranes: a focus on dynamin. Traffic 5(7):463–469

    Article  PubMed  CAS  Google Scholar 

  16. Zerial M, McBride H (2001) Rab proteins as membrane organizers. Nat Rev Mol Cell Biol 2(2):107–117

    Article  PubMed  CAS  Google Scholar 

  17. Bucci C, Parton RG, Mather IH, Stunnenberg H, Simons K, Hoflack B, Zerial M (1992) The small GTPase rab5 functions as a regulatory factor in the early endocytic pathway. Cell 70(5):715–728

    Article  PubMed  CAS  Google Scholar 

  18. Lakadamyali M, Rust MJ, Zhuang X (2006) Ligands for clathrin-mediated endocytosis are differentially sorted into distinct populations of early endosomes. Cell 124(5):997–1009

    Article  PubMed  CAS  Google Scholar 

  19. Dinneen J (2004) Expression of dominant negative rab5 in HeLa cells regulates endocytic trafficking distal from the plasma membrane. Exp Cell Res 294(2):509–522

    Article  PubMed  CAS  Google Scholar 

  20. Hunker CM, Kruk I, Hall J, Giambini H, Veisaga ML, Barbieri MA (2006) Role of Rab5 in insulin receptor-mediated endocytosis and signaling. Arch Biochem Biophys 449(1–2):130–142

    Article  PubMed  CAS  Google Scholar 

  21. Zadeh AD, Xu H, Loewen ME, Noble GP, Steele DF, Fedida D (2008) Internalized Kv1.5 traffics via Rab-dependent pathways. J Physiol 586:4793–4813

    Article  PubMed  CAS  Google Scholar 

  22. Brown TC, Tran IC, Backos DS, Esteban JA (2005) NMDA receptor-dependent activation of the small GTPase Rab5 drives the removal of synaptic AMPA receptors during hippocampal LTD. Neuron 45(1):81–94

    Article  PubMed  CAS  Google Scholar 

  23. Bobanovic LK, Royle SJ, Murrell-Lagnado RD (2002) P2X receptor trafficking in neurons is subunit specific. J Neurosci 22(12):4814–4824

    PubMed  CAS  Google Scholar 

  24. Fountain SJ, North RA (2006) A C-terminal lysine that controls human P2X4 receptor desensitization. J Biol Chem 281(22):15044–15049

    Article  PubMed  CAS  Google Scholar 

  25. Gold ES, Underhill DM, Morrissette NS, Guo J, McNiven MA, Aderem A (1999) Dynamin 2 is required for phagocytosis in macrophages. J Exp Med 190(12):1849–1856

    Article  PubMed  CAS  Google Scholar 

  26. Kitano M, Nakaya M, Nakamura T, Nagata S, Matsuda M (2008) Imaging of Rab5 activity identifies essential regulators for phagosome maturation. Nature 453(7192):241–245

    Article  PubMed  CAS  Google Scholar 

  27. Herskovits JS, Burgess CC, Obar RA, Vallee RB (1993) Effects of mutant rat dynamin on endocytosis. J Cell Biol 122(3):565–578

    Article  PubMed  CAS  Google Scholar 

  28. Bowler JW, Bailey RJ, North RA, Surprenant A (2003) P2X4, P2Y1 and P2Y2 receptors on rat alveolar macrophages. Br J Pharmacol 140(3):567–575

    Article  PubMed  CAS  Google Scholar 

  29. Pataki G, Czopf L, Jilling T, Marczin N, Catravas J, Matalon S (1995) Regulation of fluid-phase endocytosis in alveolar macrophages. Am J Physiology 269(4 Pt 1):L520–L526

    CAS  Google Scholar 

  30. Ulmann L, Hirbec H, Rassendren F (2010) P2X4 receptors mediate PGE2 release by tissue-resident macrophages and initiate inflammatory pain. EMBO J 29(14):2290–2300

    Article  PubMed  CAS  Google Scholar 

  31. Soto F, Garcia-Guzman M, Gomez-Hernandez JM, Hollmann M, Karschin C, Stühmer W (1996) P2X4: an ATP-activated ionotropic receptor cloned from rat brain. Proc Natl Acad Sci USA 93(8):3684–3688

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

The author gratefully acknowledges the support of Professor Annmarie Surprenant during this project and would like to thank Elizabeth Martin and Weihong Ma for their help with cell culture and transfections at the University of Sheffield, Jeremy Sanderson (University of Sheffield) for helping with confocal microscopy and Professor Elizabeth Smythe (University of Sheffield) for the Rab5 constructs.

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  1. Department of Biomedical Science, University of Sheffield, Sheffield, UK

    Leanne Stokes

  2. Sydney Medical School Nepean, University of Sydney, Level 5 South Block, Nepean Hospital, Penrith, NSW, 2750, Australia

    Leanne Stokes

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Correspondence to Leanne Stokes.

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Stokes, L. Rab5 regulates internalisation of P2X4 receptors and potentiation by ivermectin. Purinergic Signalling 9, 113–121 (2013). https://doi.org/10.1007/s11302-012-9336-1

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  • DOI: https://doi.org/10.1007/s11302-012-9336-1

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