Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Tracking transmitter-gated P2X cation channel activation in vitro and in vivo

Nature Methods volume 5, pages 87–93 (2008)Cite this article

Abstract

We present a noninvasive approach to track activation of ATP-gated P2X receptors and potentially other transmitter-gated cation channels that show calcium fluxes. We genetically engineered rat P2X receptors to carry calcium sensors near the channel pore and tested this as a reporter for P2X2 receptor opening. The method has several advantages over previous attempts to image P2X channel activation by fluorescence resonance energy transfer (FRET): notably, it reports channel opening rather than a conformation change in the receptor protein. Our FRET-based imaging approach can be used as a general method to track, in real time, the location, regional expression variation, mobility and activation of transmitter-gated P2X channels in living neurons in vitro and in vivo. This approach should help to determine when, where and how different receptors are activated during physiological processes.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: P2X2-cam receptors are functional and show ATP-evoked FRET changes.
Figure 2: Concentration-dependent ATP-evoked FRET changes for P2X2-cam channels expressed in HEK cells.
Figure 3: P2X2-cam is targeted to the plasma membrane of hippocampal neurons and can move to recover from photobleaching.
Figure 4: ATP-evoked FRET changes for P2X2-cam channels expressed in hippocampal neurons.
Figure 5: Activation of P2X2-cam receptors by endogenous ATP in hippocampal neurons.
Figure 6: In vivo FRET experiments for P2X2-cam channels.

Similar content being viewed by others

References

  1. Hille, B. Ion Channels of Excitable Membranes 3rd edn (Sinauer Associates, Sunderland, MA, 2001).

  2. Burnstock, G. Purinergic nerves. Pharmacol. Rev. 24, 509–581 (1972).

    CAS  PubMed  Google Scholar 

  3. Burnstock, G. Physiology and pathophysiology of purinergic neurotransmission. Physiol. Rev. 87, 659–797 (2007).

    Article  CAS  PubMed  Google Scholar 

  4. Khakh, B.S. & North, R.A. P2X receptors as cell surface ATP sensors in health and disease. Nature 442, 527–532 (2006).

    Article  CAS  PubMed  Google Scholar 

  5. North, R.A. Molecular physiology of P2X receptors. Physiol. Rev. 82, 1013–1067 (2002).

    Article  CAS  PubMed  Google Scholar 

  6. Chiu, C.-S., Kartalov, E., Unger, M., Quake, S. & Lester, H.A. Single-molecule measurements calibrate green fluorescent protein surface densities on transparent beads for use with 'knock-in' animals and other expression systems. J. Neurosci. Methods 105, 55–63 (2001).

    Article  CAS  PubMed  Google Scholar 

  7. Sugiyama, Y., Kawabata, I., Sobue, K. & Okabe, S. Determination of absolute protein numbers in single synapses by a GFP-based calibration technique. Nat. Methods 2, 677–684 (2005).

    Article  CAS  PubMed  Google Scholar 

  8. Fisher, J.A., Girdler, G. & Khakh, B.S. Time resolved measurement of state specific P2X ion channel cytosolic gating motions. J. Neurosci. 24, 10475–10487 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Egan, T.M. & Khakh, B.S. Contribution of calcium ions to P2X channel responses. J. Neurosci. 24, 3413–3420 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Guerrero, G. et al. Heterogeneity in synaptic transmission along a Drosophila larval motor axon. Nat. Neurosci. 8, 1188–1196 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Lee, M.Y. et al. Local subplasma membrane Ca2+ signals detected by a tethered Ca2+ sensor. Proc. Natl. Acad. Sci. USA 103, 13232–13237 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Tour, O. et al. Calcium Green FlAsH as a genetically targeted small-molecule calcium indicator. Nat. Chem. Biol. 3, 423–431 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Miyawaki, A. Innovations in the imaging of brain functions using fluorescent proteins. Neuron 48, 189–199 (2005).

    Article  CAS  PubMed  Google Scholar 

  14. Palmer, A.E. & Tsien, R.Y. Measuring calcium signaling using genetically targetable fluorescent indicators. Nat. Protoc. 1, 1057–1065 (2006).

    Article  CAS  PubMed  Google Scholar 

  15. Demuro, A. & Parker, I. 'Optical patch-clamping': single-channel recording by imaging Ca2+ flux through individual muscle acetylcholine receptor channels. J. Gen. Physiol. 126, 179–192 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Miyawaki, A., Griesbeck, O., Heim, R. & Tsien, R.Y. Dynamic and quantitative Ca2+ measurements using improved cameleons. Proc. Natl. Acad. Sci. USA 96, 2135–2140 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Khakh, B.S. et al. Activation-dependent changes in receptor distribution and dendritic morphology in hippocampal neurons expressing P2X2–green fluorescent protein receptors. Proc. Natl. Acad. Sci. USA 98, 5288–5293 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Egan, T.M., Samways, D.S. & Li, Z. Biophysics of P2X receptors. Pflugers Arch. 452, 501–512 (2006).

    Article  CAS  PubMed  Google Scholar 

  19. Kanjhan, R. et al. Distribution of the P2X2 receptor subunit of the ATP-gated ion channels in the rat central nervous system. J. Comp. Neurol. 407, 11–32 (1999).

    Article  CAS  PubMed  Google Scholar 

  20. Rubio, M.E. & Soto, F. Distinct localisation of P2X receptors at excitatory postsynaptic specializations. J. Neurosci. 21, 641–653 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Lippincott-Schwartz, J., Snapp, E. & Kenworthy, A. Studying protein dynamics in living cells. Nat. Rev. Mol. Cell Biol. 2, 444–456 (2001).

    Article  CAS  PubMed  Google Scholar 

  22. Wieraszko, A., Goldsmith, G. & Seyfried, T.N. Stimulation-dependent release of adenosine triphosphate from hippocampal slices. Brain Res. 485, 244–250 (1989).

    Article  CAS  PubMed  Google Scholar 

  23. Pankratov, Y., Castro, E., Miras-Portugal, M.T. & Krishtal, O. A purinergic component of the excitatory postsynaptic current mediated by P2X receptors in the CA1 neurons of the rat hippocampus. Eur. J. Neurosci. 10, 3898–3902 (1998).

    Article  CAS  PubMed  Google Scholar 

  24. Pankratov, Y.V., Lalo, U.V. & Krishtal, O.A. Role for P2X receptors in long-term potentiation. J. Neurosci. 22, 8363–8369 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Harata, N.C., Aravanis, A.M. & Tsien, R.W. Kiss-and-run and full-collapse fusion as modes of exo-endocytosis in neurosecretion. J. Neurochem. 97, 1546–1570 (2006).

    Article  CAS  PubMed  Google Scholar 

  26. Norton, W.H., Rohr, K.B. & Burnstock, G. Embryonic expression of a P2X(3) receptor encoding gene in zebrafish. Mech. Dev. 99, 149–152 (2000).

    Article  CAS  PubMed  Google Scholar 

  27. Egan, T.M., Cox, J.A. & Voigt, M.M. Molecular cloning and functional characterization of the zebrafish ATP-gated ionotropic receptor P2X3 subunit. FEBS Lett. 475, 287–290 (2000).

    Article  CAS  PubMed  Google Scholar 

  28. Boué-Grabot, E., Akimenko, M.A. & Séguéla, P. Unique functional properties of a sensory neuronal P2X ATP-gated channel from zebrafish. J. Neurochem. 75, 1600–1607 (2000).

    Article  PubMed  Google Scholar 

  29. Diaz-Hernandez, M. et al. Cloning and characterization of two novel zebrafish P2X receptor subunits. Biochem. Biophys. Res. Commun. 295, 849–853 (2002).

    Article  CAS  PubMed  Google Scholar 

  30. Kucenas, S., Li, Z., Cox, J.A., Egan, T.M. & Voigt, M.M. Molecular characterization of the zebrafish P2X receptor subunit gene family. Neuroscience 121, 935–945 (2003).

    Article  CAS  PubMed  Google Scholar 

  31. Kucenas, S., Soto, F., Cox, J.A. & Voigt, M.M. Selective labeling of central and peripheral sensory neurons in the developing zebrafish using P2X3 receptor subunit transgenes. Neuroscience 138, 641–652 (2006).

    Article  CAS  PubMed  Google Scholar 

  32. Appelbaum, L., Skariah, G., Mourrain, P. & Mignot, E. Comparative expression of P2X receptors and ecto-nucleoside triphosphate diphosphohydrolase 3 in hypocretin and sensory neurons in zebrafish. Brain Res. 1174, 66–75 (2007).

    Article  CAS  PubMed  Google Scholar 

  33. Sagasti, A., Guido, M.R., Raible, D.W. & Schier, A.F. Repulsive interactions shape the morphologies and functional arrangement of zebrafish peripheral sensory arbors. Curr. Biol. 15, 804–814 (2005).

    Article  CAS  PubMed  Google Scholar 

  34. Khakh, B.S. et al. International Union of Pharmacology. XXIV. Current status of the nomenclature and properties of P2X receptors and their subunits. Pharmacol. Rev. 53, 107–118 (2001).

    CAS  PubMed  Google Scholar 

  35. Greenwood, D. et al. P2X receptor signaling inhibits BDNF-mediated spiral ganglion neuron development in the neonatal rat cochlea. Development 134, 1407–1417 (2007).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank A.F. Schier (Harvard University) for cloning of the Fugu P2X3 enhancer; R.Y. Tsien (University of California San Diego) for providing YC3.1 cDNA; H. Singh and E. Toulme for comments and discussions; J. Fisher for an early version of Supplementary Figure 3; and F. Schweizer and G. David for advice on working with hippocampal neurons and reagents. E.R. was supported by a Molecular Cellular Integrative Physiology Predoctoral Fellowship, a Buschwald Fellowship and a Neural Repair Training Grant Predoctoral Fellowship. E.S. was supported by the Uehara Memorial Foundation (Japan). Zebrafish work was supported by a Burroughs-Wellcome Career Award in the Biomedical Sciences to A.S. The laboratory (B.S.K.) was supported by the National Institutes of Health, the Human Frontiers Science Program, the Whitehall Foundation and a Stein/Oppenheimer Endowment Award.

Author information

Author notes

  1. Esther Richler, Severine Chaumont and Eiji Shigetomi: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Physiology, University of California Los Angeles, 10833 LeConte Avenue, Los Angeles, 90095, California, USA

    Esther Richler, Severine Chaumont, Eiji Shigetomi & Baljit S Khakh

  2. Department of Neurobiology, David Geffen School of Medicine, University of California Los Angeles, 10833 LeConte Avenue, Los Angeles, 90095, California, USA

    Baljit S Khakh

  3. Department of Molecular, Cellular and Developmental Biology, University of California Los Angeles, 10833 LeConte Avenue, Los Angeles, 90095, California, USA

    Alvaro Sagasti

Authors
  1. Esther Richler
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Severine Chaumont
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Eiji Shigetomi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Alvaro Sagasti
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Baljit S Khakh
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

E.R., S.C., E.S. and B.S.K. designed and did the experiments, contributed data, helped to make the figures, and wrote the paper. A.S. contributed laboratory space, made the vectors for the zebrafish work, and helped to make the figures and to write the paper.

Corresponding author

Correspondence to Baljit S Khakh.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8, Supplementary Notes 1–2, Supplementary Methods (PDF 2961 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Richler, E., Chaumont, S., Shigetomi, E. et al. Tracking transmitter-gated P2X cation channel activation in vitro and in vivo. Nat Methods 5, 87–93 (2008). https://doi.org/10.1038/nmeth1144

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth1144

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing