Advertisement

Heterologous Expression and Patch-Clamp Recording of P2X Receptors in HEK293 Cells

  • Lin-Hua JiangEmail author
  • Sébastien Roger
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2041)

Abstract

P2X receptors (P2XRs) are ligand-gated ion channels gated by extracellular adenosine 5′-triphosphate (ATP) and play a critical role in mediating ATP-induced purinergic signaling in physiological and pathological processes. Heterologous expression of P2XR in human embryonic kidney 293 (HEK293) cells and measurement of P2XR-mediated currents using patch-clamp recording technique have been widely used to study the biophysical and pharmacological properties of these receptors. Combination of electrophysiology with site-directed mutagenesis and structural information has shed light on the molecular basis for receptor activation and mechanisms of actions by receptor antagonists and modulators. It is anticipated that such methodologies will continue helping us to provide more mechanistic understanding of P2XRs and to test novel receptor antagonists and allosteric modulators for therapeutical purposes. In this chapter, we describe protocols of transiently or stably expressing the P2XR in HEK293 cells and measuring P2XR-mediated currents by using whole-cell recording.

Key words

P2X receptors HEK293 cells Heterologous expression Patch-clamp recording 

Notes

Acknowledgments

The work from Jiang’s laboratory was supported by Biotechnology and Biological Sciences Research Council and Wellcome Trust.

References

  1. 1.
    North RA (2002) Molecular physiology of P2X receptors. Physiol Rev 82:1013–1067CrossRefGoogle Scholar
  2. 2.
    Khakh BS, North RA (2012) Neuromodulation by extracellular ATP and P2X receptors in the CNS. Neuron 76:51–69CrossRefGoogle Scholar
  3. 3.
    Di Virgilio F, Sarti AC, Grassi F (2018) Modulation of innate and adaptive immunity by P2X ion channels. Curr Opin Immunol 52:51–59CrossRefGoogle Scholar
  4. 4.
    Oliveira Á, Illes P, Ulrich H (2016) Purinergic receptors in embryonic and adult neurogenesis. Neuropharmacology 104:272–281CrossRefGoogle Scholar
  5. 5.
    Jiang LH, Hao Y, Mousawi F, Peng H, Yang X (2017) Expression of P2 purinergic receptors in mesenchymal stem cells and their roles in extracellular nucleotide regulation of cell functions. J Cell Physiol 232:287–297CrossRefGoogle Scholar
  6. 6.
    Bernier LP, Ase AR, Séguéla P (2018) P2X receptor channels in chronic pain pathways. Br J Pharmacol 175:2219–2230CrossRefGoogle Scholar
  7. 7.
    Tewari M, Seth P (2015) Emerging role of P2X7 receptors in CNS health and disease. Ageing Res Rev 24:328–342CrossRefGoogle Scholar
  8. 8.
    Wei L, Syed Mortadza SA, Yan J, Zhang L, Wang L, Yin Y, Li C, Chalon S, Emond P, Belzung C, Li D, Lu C, Roger S, Jiang LH (2018) ATP-activated P2X7 receptor in the pathophysiology of mood disorders and as an emerging target for the development of novel antidepressant therapeutics. Neurosci Biobehav Rev 87:192–205CrossRefGoogle Scholar
  9. 9.
    Burnstock G (2016) P2X ion channel receptors and inflammation. Purinergic Signal 12:59–67CrossRefGoogle Scholar
  10. 10.
    Tozzi M, Novak I (2017) Purinergic receptors in adipose tissue as potential targets in metabolic disorders. Front Pharmacol 8:878CrossRefGoogle Scholar
  11. 11.
    Roger S, Jelassi B, Couillin I, Pelegrin P, Besson P, Jiang LH (2015) Understanding the roles of the P2X7 receptor in solid tumour progression and therapeutic perspectives. Biochim Biophys Acta 1848:2584–2602CrossRefGoogle Scholar
  12. 12.
    Di Virgilio F, Sarti AC, Falzoni S, De Marchi E, Adinolfi E (2018) Extracellular ATP and P2 purinergic signalling in the tumour microenvironment. Nat Rev Cancer 18:601–618CrossRefGoogle Scholar
  13. 13.
    Hamill O, Marty A, Neher E, Sakmann B, Sigworth F (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch 391:85–100CrossRefGoogle Scholar
  14. 14.
    Rassendren F, Buell G, Newbolt A, North RA, Surprenant A (1997) Identification of amino acid residues contributing to the pore of a P2X receptor. EMBO J 16:3446–3454CrossRefGoogle Scholar
  15. 15.
    Marquez-Klaka B, Rettinger J, Bhargava Y, Eisele T, Nicke A (2007) Identification of an intersubunit cross-link between substituted cysteine residues located in the putative ATP binding site of the P2X1 receptor. J Neurosci 27:1456–1466CrossRefGoogle Scholar
  16. 16.
    Stelmashenko O, Lalo U, Yang Y, Bragg L, North RA, Compan V (2012) Activation of trimeric P2X2 receptors by fewer than three ATP molecules. Mol Pharmacol 82:760–766CrossRefGoogle Scholar
  17. 17.
    Stelmashenko O, Compan V, Browne LE, North RA (2014) Ectodomain movements of an ATP-gated ion channel (P2X2 receptor) probed by disulfide locking. J Biol Chem 289:9909–9917CrossRefGoogle Scholar
  18. 18.
    Browne LE, Nunes JP, Sim JA, Chudasama V, Bragg L, Caddick S, North RA (2014) Optical control of trimeric P2X receptors and acid-sensing ion channels. Proc Natl Acad Sci U S A 111:521–526CrossRefGoogle Scholar
  19. 19.
    Zhao WS, Wang J, Ma XJ, Yang Y, Liu Y, Huang LD, Fan YZ, Cheng XY, Chen HZ, Wang R, Yu Y (2014) Relative motions between left flipper and dorsal fin domains favour P2X4 receptor activation. Nat Commun 5:4189CrossRefGoogle Scholar
  20. 20.
    Wang J, Sun LF, Cui WW, Zhao WS, Ma XF, Li B, Liu Y, Yang Y, Hu YM, Huang LD, Cheng XY, Li L, Lu XY, Tian Y, Yu Y (2017) Intersubunit physical couplings fostered by the left flipper domain facilitate channel opening of P2X4 receptors. J Biol Chem 292:7619–7635CrossRefGoogle Scholar
  21. 21.
    Caseley EA, Muench SP, Jiang LH (2017) Conformational changes during human P2X7 receptor activation examined by structural modelling and cysteine-based cross-linking studies. Purinergic Signal 13:135–141CrossRefGoogle Scholar
  22. 22.
    Jiang R, Taly A, Lemoine D, Martz A, Cunrath O, Grutter T (2012) Tightening of the ATP-binding sites induces the opening of P2X receptor channels. EMBO J 31:2134–2143CrossRefGoogle Scholar
  23. 23.
    Sim JA, Broomhead HE, North RA (2008) Ectodomain lysines and suramin block of P2X1 receptors. J Biol Chem 283:29841–29846CrossRefGoogle Scholar
  24. 24.
    Karasawa A, Kawate T (2016) Structural basis for subtype-specific inhibition of the P2X7 receptor. Elife 5:e22153CrossRefGoogle Scholar
  25. 25.
    Kasuya G, Yamaura T, Ma XB, Nakamura R, Takemoto M, Nagumo H, Tanaka E, Dohmae N, Nakane T, Yu Y, Ishitani R, Matsuzaki O, Hattori M, Nureki O (2017) Structural insights into the competitive inhibition of the ATP-gated P2X receptor channel. Nat Commun 8:876CrossRefGoogle Scholar
  26. 26.
    Wang J, Wang Y, Cui WW, Huang Y, Yang Y, Liu Y, Zhao WS, Cheng XY, Sun WS, Cao P, Zhu MX, Wang R, Hattori M, Yu Y (2018) Druggable negative allosteric site of P2X3 receptors. Proc Natl Acad Sci U S A 115:4939–4944CrossRefGoogle Scholar
  27. 27.
    Browne LE, Jiang LH, North RA (2010) New structure enlivens interest in P2X receptors. Trends Pharmacol Sci 31:229–237CrossRefGoogle Scholar
  28. 28.
    Coddou C, Yan Z, Obsil T, Huidobro-Toro JP, Stojilkovic SS (2011) Activation and regulation of purinergic P2X receptor channels. Pharmacol Rev 63:641–683CrossRefGoogle Scholar
  29. 29.
    Pasqualetto G, Brancale A, Young MT (2018) The molecular determinants of small-molecule ligand binding at P2X receptors. Front Pharmacol 9:58CrossRefGoogle Scholar
  30. 30.
    Schmid R, Evans RJ (2018) ATP-gated P2X receptor channels: molecular insights into functional roles. Annu Rev Physiol.  https://doi.org/10.1146/annurev-physiol-020518-114259
  31. 31.
    Roger S, Mei ZZ, Baldwin JM, Dong L, Bradley H, Baldwin SA, Surprenant A, Jiang LH (2010) Single nucleotide polymorphisms that were identified in affective mood disorders affect ATP-activated P2X7 receptor functions. J Psychiatr Res 44:347–355CrossRefGoogle Scholar
  32. 32.
    Aprile-Garcia F, Metzger MW, Paez-Pereda M, Stadler H, Acuña M, Liberman AC, Senin SA, Gerez J, Hoijman E, Refojo D, Mitkovski M, Panhuysen M, Stühmer W, Holsboer F, Deussing JM, Arzt E (2016) Co-expression of wild-type P2X7R with Gln460Arg variant alters receptor function. PLoS One 11:e0151862CrossRefGoogle Scholar
  33. 33.
    Metzger MW, Walser SM, Dedic N, Aprile-Garcia F, Jakubcakova V, Adamczyk M, Webb KJ, Uhr M, Refojo D, Schmidt MV, Friess E, Steiger A, Kimura M, Chen A, Holsboer F, Arzt E, Wurst W, Deussing JM (2017) Heterozygosity for the mood disorder-associated variant Gln460Arg alters P2X7 receptor function and sleep quality. J Neurosci 37:11688–11700CrossRefGoogle Scholar
  34. 34.
    Bradley HJ, Browne LE, Yang W, Jiang LH (2011) Pharmacological properties of the rhesus macaque monkey P2X7 receptor. Br J Pharmacol 164:743–754CrossRefGoogle Scholar
  35. 35.
    Caseley EA, Muench SP, Fishwick CW, Jiang LH (2016) Structure-based identification and characterisation of structurally novel human P2X7 receptor antagonists. Biochem Pharmacol 116:130–139CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.School of Biomedical Sciences, Faculty of Biological SciencesUniversity of LeedsLeedsUK
  2. 2.EA4245, Transplantation, Immunology, Inflammation, Faculty of MedicineUniversity of ToursToursFrance

Personalised recommendations