P2X Electrophysiology and Surface Trafficking in Xenopus Oocytes

  • Eléonore Bertin
  • Audrey Martínez
  • Eric Boué-GrabotEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2041)


Xenopus oocytes serve as a standard heterologous expression system for the study of various ligand-gated ion channels including ATP P2X receptors. Here we describe the whole-cell two-electrode voltage clamp and biotinylation/Western blotting techniques to investigate the functional properties and surface trafficking from P2X-expressing oocytes.

Key words

P2X ATP-gated channels Double-electrode patch clamp Electrophysiology Receptor function Surface trafficking Xenopus Oocytes 


  1. 1.
    Gurdon JB, Woodland HR, Lingrel JB (1974) The translation of mammalian globin mRNA injected into fertilized eggs of Xenopus laevis. I. Message stability in development. Dev Biol 39:125–133PubMedCrossRefGoogle Scholar
  2. 2.
    Gundersen CB, Miledi R, Parker I (1983) Serotonin receptors induced by exogenous messenger RNA in Xenopus oocytes. Proc R Soc Lond B Biol Sci 219:103–109PubMedCrossRefGoogle Scholar
  3. 3.
    Sakmann B, Methfessel C, Mishina M, Takahashi T, Takai T, Kurasaki M, Fukuda K, Numa S (1985) Role of acetylcholine receptor subunits in gating of the channel. Nature 318:538–543PubMedCrossRefGoogle Scholar
  4. 4.
    Burnstock G (2012) Discovery of purinergic signalling, the initial resistance and current explosion of interest. Br J Pharmacol 167:238–255PubMedPubMedCentralCrossRefGoogle Scholar
  5. 5.
    Roberts JA, Vial C, Digby HR, Agboh KC, Wen H, Atterbury-Thomas A, Evans RJ (2006) Molecular properties of P2X receptors. Pflugers Arch 452:486–500PubMedCrossRefGoogle Scholar
  6. 6.
    Sigel E (1990) Use of Xenopus oocytes for the functional expression of plasma membrane proteins. J Membr Biol 117:201–221PubMedCrossRefGoogle Scholar
  7. 7.
    Valera S, Hussy N, Evans RJ, Adami N, North RA, Surprenant A, Buell G (1994) A new class of ligand-gated ion channel defined by P2x receptor for extracellular ATP. Nature 371:516–519PubMedCrossRefGoogle Scholar
  8. 8.
    Le KT, Boue-Grabot E, Archambault V, Seguela P (1999) Functional and biochemical evidence for heteromeric ATP-gated channels composed of P2X1 and P2X5 subunits. J Biol Chem 274:15415–15419PubMedCrossRefGoogle Scholar
  9. 9.
    Brown SG, Townsend-Nicholson A, Jacobson KA, Burnstock G, King BF (2002) Heteromultimeric P2X(1/2) receptors show a novel sensitivity to extracellular pH. J Pharmacol Exp Ther 300:673–680PubMedPubMedCentralCrossRefGoogle Scholar
  10. 10.
    Liu M, King BF, Dunn PM, Rong W, Townsend-Nicholson A, Burnstock G (2001) Coexpression of P2X(3) and P2X(2) receptor subunits in varying amounts generates heterogeneous populations of P2X receptors that evoke a spectrum of agonist responses comparable to that seen in sensory neurons. J Pharmacol Exp Ther 296:1043–1050PubMedGoogle Scholar
  11. 11.
    Xiong K, Li C, Weight FF (2000) Inhibition by ethanol of rat P2X(4) receptors expressed in Xenopus oocytes. Br J Pharmacol 130:1394–1398PubMedPubMedCentralCrossRefGoogle Scholar
  12. 12.
    Boue-Grabot E, Akimenko MA, Seguela P (2000) Unique functional properties of a sensory neuronal P2X ATP-gated channel from zebrafish. J Neurochem 75:1600–1607PubMedCrossRefGoogle Scholar
  13. 13.
    Bo X, Schoepfer R, Burnstock G (2000) Molecular cloning and characterization of a novel ATP P2X receptor subtype from embryonic chick skeletal muscle. J Biol Chem 275:14401–14407PubMedCrossRefGoogle Scholar
  14. 14.
    Wang CZ, Namba N, Gonoi T, Inagaki N, Seino S (1996) Cloning and pharmacological characterization of a fourth P2X receptor subtype widely expressed in brain and peripheral tissues including various endocrine tissues. Biochem Biophys Res Commun 220:196–202PubMedCrossRefGoogle Scholar
  15. 15.
    Seguela P, Haghighi A, Soghomonian JJ, Cooper E (1996) A novel neuronal P2x ATP receptor ion channel with widespread distribution in the brain. J Neurosci 16:448–455PubMedCrossRefGoogle Scholar
  16. 16.
    Evans RJ, Lewis C, Buell G, Valera S, North RA, Surprenant A (1995) Pharmacological characterization of heterologously expressed ATP-gated cation channels (P2x purinoceptors). Mol Pharmacol 48:178–183PubMedGoogle Scholar
  17. 17.
    Lynch KJ, Touma E, Niforatos W, Kage KL, Burgard EC, van Biesen T, Kowaluk EA, Jarvis MF (1999) Molecular and functional characterization of human P2X(2) receptors. Mol Pharmacol 56:1171–1181PubMedCrossRefGoogle Scholar
  18. 18.
    Le KT, Paquet M, Nouel D, Babinski K, Seguela P (1997) Primary structure and expression of a naturally truncated human P2X ATP receptor subunit from brain and immune system. FEBS Lett 418:195–199PubMedCrossRefGoogle Scholar
  19. 19.
    Schneider M, Prudic K, Pippel A, Klapperstück M, Braam U, Müller CE, Schmalzing G, Markwardt F (2017) Interaction of purinergic P2X4 and P2X7 receptor subunits. Front Pharmacol 8:860PubMedPubMedCentralCrossRefGoogle Scholar
  20. 20.
    Wen H, Evans RJ (2009) Regions of the amino terminus of the P2X receptor required for modification by phorbol ester and mGluR1alpha receptors. J Neurochem 108:331–340PubMedPubMedCentralCrossRefGoogle Scholar
  21. 21.
    Codocedo JF, Rodriguez FE, Huidobro-Toro JP (2009) Neurosteroids differentially modulate P2X ATP-gated channels through non-genomic interactions. J Neurochem 110:734–744PubMedCrossRefGoogle Scholar
  22. 22.
    Low SE, Kuwada JY, Hume RI (2008) Amino acid variations resulting in functional and nonfunctional zebrafish P2X(1) and P2X (5.1) receptors. Purinergic Signal 4:383–392PubMedPubMedCentralCrossRefGoogle Scholar
  23. 23.
    Locovei S, Scemes E, Qiu F, Spray DC, Dahl G (2007) Pannexin1 is part of the pore forming unit of the P2X(7) receptor death complex. FEBS Lett 581:483–488PubMedPubMedCentralCrossRefGoogle Scholar
  24. 24.
    Roberts JA, Evans RJ (2005) Mutagenesis studies of conserved proline residues of human P2X receptors for ATP indicate that proline 272 contributes to channel function. J Neurochem 92:1256–1264PubMedCrossRefGoogle Scholar
  25. 25.
    Davies DL, Kochegarov AA, Kuo ST, Kulkarni AA, Woodward JJ, King BF, Alkana RL (2005) Ethanol differentially affects ATP-gated P2X(3) and P2X(4) receptor subtypes expressed in Xenopus oocytes. Neuropharmacology 49:243–253PubMedCrossRefGoogle Scholar
  26. 26.
    Kanjhan R, Raybould NP, Jagger DJ, Greenwood D, Housley GD (2003) Allosteric modulation of native cochlear P2X receptors: insights from comparison with recombinant P2X2 receptors. Audiol Neurootol 8:115–128PubMedCrossRefGoogle Scholar
  27. 27.
    Paukert M, Hidayat S, Grunder S (2002) The P2X(7) receptor from Xenopus laevis: formation of a large pore in Xenopus oocytes. FEBS Lett 513:253–258PubMedCrossRefGoogle Scholar
  28. 28.
    Nakazawa K, Ojima H, Ohno Y (2002) A highly conserved tryptophane residue indispensable for cloned rat neuronal P2X receptor activation. Neurosci Lett 324:141–144PubMedCrossRefGoogle Scholar
  29. 29.
    Ennion SJ, Evans RJ (2002) Conserved cysteine residues in the extracellular loop of the human P2X(1) receptor form disulfide bonds and are involved in receptor trafficking to the cell surface. Mol Pharmacol 61:303–311PubMedCrossRefGoogle Scholar
  30. 30.
    Ennion SJ, Evans RJ (2002) P2X(1) receptor subunit contribution to gating revealed by a dominant negative PKC mutant. Biochem Biophys Res Commun 291:611–616PubMedCrossRefGoogle Scholar
  31. 31.
    Eickhorst AN, Berson A, Cockayne D, Lester HA, Khakh BS (2002) Control of P2X(2) channel permeability by the cytosolic domain. J Gen Physiol 120:119–131PubMedPubMedCentralCrossRefGoogle Scholar
  32. 32.
    Clyne JD, LaPointe LD, Hume RI (2002) The role of histidine residues in modulation of the rat P2X(2) purinoceptor by zinc and pH. J Physiol 539:347–359PubMedPubMedCentralCrossRefGoogle Scholar
  33. 33.
    Ennion SJ, Ritson J, Evans RJ (2001) Conserved negatively charged residues are not required for ATP action at P2X(1) receptors. Biochem Biophys Res Commun 289:700–704PubMedCrossRefGoogle Scholar
  34. 34.
    Ennion S, Hagan S, Evans RJ (2000) The role of positively charged amino acids in ATP recognition by human P2X(1) receptors. J Biol Chem 275:29361–29367PubMedCrossRefGoogle Scholar
  35. 35.
    Dutton JL, Poronnik P, Li GH, Holding CA, Worthington RA, Vandenberg RJ, Cook DI, Barden JA, Bennett MR (2000) P2X(1) receptor membrane redistribution and down-regulation visualized by using receptor-coupled green fluorescent protein chimeras. Neuropharmacology 39:2054–2066PubMedCrossRefGoogle Scholar
  36. 36.
    Boue-Grabot E, Archambault V, Seguela P (2000) A protein kinase C site highly conserved in P2X subunits controls the desensitization kinetics of P2X(2) ATP-gated channels. J Biol Chem 275:10190–10195PubMedCrossRefGoogle Scholar
  37. 37.
    Newbolt A, Stoop R, Virginio C, Surprenant A, North RA, Buell G, Rassendren F (1998) Membrane topology of an ATP-gated ion channel (P2X receptor). J Biol Chem 273:15177–15182PubMedCrossRefGoogle Scholar
  38. 38.
    Werner P, Seward EP, Buell GN, North RA (1996) Domains of P2X receptors involved in desensitization. Proc Natl Acad Sci U S A 93:15485–15490PubMedPubMedCentralCrossRefGoogle Scholar
  39. 39.
    Becker D, Woltersdorf R, Boldt W, Schmitz S, Braam U, Schmalzing G, Markwardt F (2008) The P2X7 carboxyl tail is a regulatory module of P2X7 receptor channel activity. J Biol Chem 283:25725–25734PubMedCrossRefGoogle Scholar
  40. 40.
    Emerit MB, Baranowski C, Diaz J, Martinez A, Areias J, Alterio J, Masson J, Boue-Grabot E, Darmon M (2016) A new mechanism of receptor targeting by interaction between two classes of ligand-gated ion channels. J Neurosci 36:1456–1470PubMedPubMedCentralCrossRefGoogle Scholar
  41. 41.
    Jo YH, Donier E, Martinez A, Garret M, Toulme E, Boue-Grabot E (2011) Cross-talk between P2X4 and gamma-aminobutyric acid, type A receptors determines synaptic efficacy at a central synapse. J Biol Chem 286:19993–20004PubMedPubMedCentralCrossRefGoogle Scholar
  42. 42.
    Toulme E, Blais D, Leger C, Landry M, Garret M, Seguela P, Boue-Grabot E (2007) An intracellular motif of P2X(3) receptors is required for functional cross-talk with GABA(A) receptors in nociceptive DRG neurons. J Neurochem 102:1357–1368PubMedCrossRefGoogle Scholar
  43. 43.
    Khakh BS, Fisher JA, Nashmi R, Bowser DN, Lester HA (2005) An angstrom scale interaction between plasma membrane ATP-gated P2X2 and alpha4beta2 nicotinic channels measured with fluorescence resonance energy transfer and total internal reflection fluorescence microscopy. J Neurosci 25:6911–6920PubMedPubMedCentralCrossRefGoogle Scholar
  44. 44.
    Boue-Grabot E, Toulme E, Emerit MB, Garret M (2004) Subunit-specific coupling between gamma-aminobutyric acid type A and P2X2 receptor channels. J Biol Chem 279:52517–52525PubMedCrossRefGoogle Scholar
  45. 45.
    Khakh BS, Zhou X, Sydes J, Galligan JJ, Lester HA (2000) State-dependent cross-inhibition between transmitter-gated cation channels. Nature 406:405–410PubMedCrossRefGoogle Scholar
  46. 46.
    Boue-Grabot E, Barajas-Lopez C, Chakfe Y, Blais D, Belanger D, Emerit MB, Seguela P (2003) Intracellular cross talk and physical interaction between two classes of neurotransmitter-gated channels. J Neurosci 23:1246–1253PubMedPubMedCentralCrossRefGoogle Scholar
  47. 47.
    Boue-Grabot E, Emerit MB, Toulme E, Seguela P, Garret M (2004) Cross-talk and co-trafficking between rho1/GABA receptors and ATP-gated channels. J Biol Chem 279:6967–6975PubMedCrossRefGoogle Scholar
  48. 48.
    Pougnet JT, Compans B, Martinez A, Choquet D, Hosy E, Boue-Grabot E (2016) P2X-mediated AMPA receptor internalization and synaptic depression is controlled by two CaMKII phosphorylation sites on GluA1 in hippocampal neurons. Sci Rep 6:31836PubMedPubMedCentralCrossRefGoogle Scholar
  49. 49.
    Pougnet JT, Toulme E, Martinez A, Choquet D, Hosy E, Boue-Grabot E (2014) ATP P2X receptors downregulate AMPA receptor trafficking and postsynaptic efficacy in hippocampal neurons. Neuron 83:417–430PubMedCrossRefGoogle Scholar
  50. 50.
    Boue-Grabot E, Pankratov Y (2017) Modulation of central synapses by astrocyte-released ATP and postsynaptic P2X receptors. Neural Plast 2017:9454275PubMedPubMedCentralCrossRefGoogle Scholar
  51. 51.
    Marsal J, Tigyi G, Miledi R (1995) Incorporation of acetylcholine receptors and Cl− channels in Xenopus oocytes injected with Torpedo electroplaque membranes. Proc Natl Acad Sci U S A 92:5224–5228PubMedPubMedCentralCrossRefGoogle Scholar
  52. 52.
    Miledi R, Eusebi F, Martinez-Torres A, Palma E, Trettel F (2002) Expression of functional neurotransmitter receptors in Xenopus oocytes after injection of human brain membranes. Proc Natl Acad Sci U S A 99:13238–13242PubMedPubMedCentralCrossRefGoogle Scholar
  53. 53.
    Bernareggi A, Reyes-Ruiz JM, Lorenzon P, Ruzzier F, Miledi R (2011) Microtransplantation of acetylcholine receptors from normal or denervated rat skeletal muscles to frog oocytes. J Physiol 589:1133–1142PubMedPubMedCentralCrossRefGoogle Scholar
  54. 54.
    Belujon P, Baufreton J, Grandoso L, Boue-Grabot E, Batten TF, Ugedo L, Garret M, Taupignon AI (2009) Inhibitory transmission in locus coeruleus neurons expressing GABAA receptor epsilon subunit has a number of unique properties. J Neurophysiol 102:2312–2325PubMedCrossRefGoogle Scholar
  55. 55.
    North RA, Jarvis MF (2013) P2X receptors as drug targets. Mol Pharmacol 83:759–769PubMedPubMedCentralCrossRefGoogle Scholar
  56. 56.
    Dumont JN (1972) Oogenesis in Xenopus laevis (Daudin). I. Stages of oocyte development in laboratory maintained animals. J Morphol 136:153–179PubMedCrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Eléonore Bertin
    • 1
  • Audrey Martínez
    • 1
  • Eric Boué-Grabot
    • 1
    Email author
  1. 1.Institut des Maladies Neurodégénératives, CNRS UMR 5293Université de BordeauxBordeauxFrance

Personalised recommendations