Skip to main content
SpringerLink
Log in

Direct evidence for functional TRPV1/TRPA1 heteromers

  • Ion channels, receptors and transporters
  • Published:
Pflügers Archiv - European Journal of Physiology Aims and scope Submit manuscript

Abstract

Transient receptor potential cation channel, subfamily V, member 1 (TRPV1) plays a key role in sensing environmental hazards and in enhanced pain sensation following inflammation. A considerable proportion of TRPV1-expressing cells also express transient receptor potential cation channel, subfamily A, member 1 (TRPA1). There is evidence for a TRPV1-TRPA1 interaction that is predominantly calcium-dependent, and it has been suggested that the two proteins might form a heteromeric channel. Here, we constructed subunit concatemers to search for direct evidence for such an interaction. We found that a TRPV1::TRPV1 concatemer and TRPV1 formed channels with similar properties. A TRPV1::TRPA1 concatemer was responsive to TRPV1 agonists capsaicin, acidic pH and ethanol, but not to TRPA1 agonists. Isolated TRPV1 and TRPV1::TRPA1 imaged by atomic force microscopy (AFM) both had molecular volumes consistent with the formation of tetrameric channels. Antibodies decorated epitope tags on TRPV1 with a four-fold symmetry, as expected for a homotetramer. In contrast, pairs of antibodies decorated tags on TRPV1::TRPA1 predominantly at 180°, indicating the formation of a channel consisting of two TRPV1::TRPA1 concatemers arranged face to face. TRPV1::TRPA1 was sensitized by PKC activation and could be inhibited by a TRPV1 antagonist. TRPV1::TRPA1 was activated by heat and displayed a threshold and temperature coefficient similar to TRPV1. However, the channel formed by TRPV1::TRPA1 has only two binding sites for capsaicin and shows less total current and a smaller capsaicin-induced shift in voltage-dependent gating than TRPV1::TRPV1 or TRPV1. We conclude that the presence of TRPA1 exerts a functional inhibition on TRPV1.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9

Similar content being viewed by others

Abbreviations

AFM:

Atomic force microscopy

TRP:

Transient receptor potential cation channel, subfamily and member as noted

References

  1. Caterina MJ, Leffler A, Malmberg AB, Martin WJ, Trafton J, Petersen-Zeitz KR et al (2000) Impaired nociception and pain sensation in mice lacking the capsaicin receptor. Science 288:306–313

    Article  CAS  PubMed  Google Scholar 

  2. Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, Julius D (1997) The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature 389:816–824

    Article  CAS  PubMed  Google Scholar 

  3. Cheng W, Yang F, Liu S, Colton CK, Wang C, Cui Y et al (2012) Heteromeric heat-sensitive transient receptor potential channels exhibit distinct temperature and chemical response. J Biol Chem 287:7279–7288

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  4. Cvetkov TL, Huynh KW, Cohen MR, Moiseenkova-Bell VY (2011) Molecular architecture and subunit organization of TRPA1 ion channel revealed by electron microscopy. J Biol Chem 286:38168–38176

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  5. Dittert I, Vlachová V, Knotková H, Vitásková Z, Vyklicky L, Kress M et al (1998) A technique for fast application of heated solutions of different composition to cultured neurones. J Neurosci Methods 82:195–201

    Article  CAS  PubMed  Google Scholar 

  6. Doerner JF, Gisselmann G, Hatt H, Wetzel CH (2007) Transient receptor potential channel A1 is directly gated by calcium ions. J Biol Chem 282:13180–13189

    Article  CAS  PubMed  Google Scholar 

  7. Grynkiewicz G, Poenie M, Tsien RY (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260:3440–3450

    CAS  PubMed  Google Scholar 

  8. Hellwig N, Albrecht N, Harteneck C, Schultz G, Schaefer M (2005) Homo- and heteromeric assembly of TRPV channel subunits. J Cell Sci 118:917–928

    Article  CAS  PubMed  Google Scholar 

  9. Hoenderop JGJ, Voets T, Hoefs S, Weidema F, Prenen J, Nilius B et al (2003) Homo- and heterotetrameric architecture of the epithelial Ca2+ channels TRPV5 and TRPV6. EMBO J 22:776–785

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  10. Janssens A, Voets T (2011) Ligand stoichiometry of the cold- and menthol-activated channel TRPM8. J Physiol 589:4827–4835

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  11. Kedei N, Szabo T, Lile JD, Treanor JJ, Olah Z, Iadarola MJ et al (2001) Analysis of the native quaternary structure of vanilloid receptor 1. J Biol Chem 276:28613–28619

    Article  CAS  PubMed  Google Scholar 

  12. Kobayashi K, Fukuoka T, Obata K, Yamanaka H, Dai Y, Tokunaga A et al (2005) Distinct expression of TRPM8, TRPA1, and TRPV1 mRNAs in rat primary afferent neurons with adelta/c-fibers and colocalization with trk receptors. J Comp Neurol 493:596–606

    Article  CAS  PubMed  Google Scholar 

  13. Kobori T, Smith GD, Sandford R, Edwardson JM (2009) The transient receptor potential channels TRPP2 and TRPC1 form a heterotetramer with a 2:2 stoichiometry and an alternating subunit arrangement. J Biol Chem 284:35507–35513

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  14. Kwan KY, Allchorne AJ, Vollrath MA, Christensen AP, Zhang D-S, Woolf CJ et al (2006) TRPA1 contributes to cold, mechanical, and chemical nociception but is not essential for hair-cell transduction. Neuron 50:277–289

    Article  CAS  PubMed  Google Scholar 

  15. Liao M, Cao E, Julius D, Cheng Y (2013) Structure of the TRPV1 ion channel determined by electron cryo-microscopy. Nature 504:107–112

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  16. Liu B, Hui K, Qin F (2003) Thermodynamics of heat activation of single capsaicin ion channels VR1. Biophys J 85:2988–3006

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  17. Liu H, Naismith JH (2008) An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol. BMC Biotechnol 8:91

    Article  PubMed Central  PubMed  Google Scholar 

  18. Moiseenkova-Bell VY, Stanciu LA, Serysheva II, Tobe BJ, Wensel TG (2008) Structure of TRPV1 channel revealed by electron cryomicroscopy. Proc Natl Acad Sci U S A 105:7451–7455

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  19. Neaves KJ, Cooper LP, White JH, Carnally SM, Dryden DTF, Edwardson JM et al (2009) Atomic force microscopy of the EcoKI Type I DNA restriction enzyme bound to DNA shows enzyme dimerization and DNA looping. Nucleic Acids Res 37:2053–2063

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  20. Nilius B, Owsianik G (2011) The transient receptor potential family of ion channels. Genome Biol 12:218

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  21. Olah Z, Szabo T, Karai L, Hough C, Fields RD, Caudle RM et al (2001) Ligand-induced dynamic membrane changes and cell deletion conferred by vanilloid receptor 1. J Biol Chem 276:11021–11030

    Article  CAS  PubMed  Google Scholar 

  22. Ruparel NB, Patwardhan AM, Akopian AN, Hargreaves KM (2008) Homologous and heterologous desensitization of capsaicin and mustard oil responses utilize different cellular pathways in nociceptors. Pain 135:271–279

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  23. Schneider SW, Lärmer J, Henderson RM, Oberleithner H (1998) Molecular weights of individual proteins correlate with molecular volumes measured by atomic force microscopy. Pflügers Arch Eur J Physiol 435:362–367

    Article  CAS  Google Scholar 

  24. Scott DW (1979) On optimal and data-based histograms. Biometrika 66:605–610

    Article  Google Scholar 

  25. Sokolov MV, Shamotienko O, Dhochartaigh SN, Sack JT, Dolly JO (2007) Concatemers of brain Kv1 channel alpha subunits that give similar K+ currents yield pharmacologically distinguishable heteromers. Neuropharmacology 53:272–282

    Article  CAS  PubMed  Google Scholar 

  26. Staruschenko A, Jeske NA, Akopian AN (2010) Contribution of TRPV1-TRPA1 interaction to the single channel properties of the TRPA1 channel. J Biol Chem 285:15167–15177

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  27. Stewart AP, Egressy K, Lim A, Edwardson JM (2010) AFM imaging reveals the tetrameric structure of the TRPM8 channel. Biochem Biophys Res Commun 394:383–386

    Article  CAS  PubMed  Google Scholar 

  28. Stewart AP, Smith GD, Sandford RN, Edwardson JM (2010) Atomic force microscopy reveals the alternating subunit arrangement of the TRPP2-TRPV4 heterotetramer. Biophys J 99:790–797

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  29. Storti B, Bizzarri R, Cardarelli F, Beltram F (2012) Intact microtubules preserve transient receptor potential vanilloid 1 (TRPV1) functionality through receptor binding. J Biol Chem 287:7803–7811

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  30. Story GM, Peier AM, Reeve AJ, Eid SR, Mosbacher J, Hricik TR et al (2003) ANKTM1, a TRP-like channel expressed in nociceptive neurons, is activated by cold temperatures. Cell 112:819–829

    Article  CAS  PubMed  Google Scholar 

  31. Tominaga M, Wada M, Masu M (2001) Potentiation of capsaicin receptor activity by metabotropic ATP receptors as a possible mechanism for ATP-evoked pain and hyperalgesia. Proc Natl Acad Sci U S A 98:6951–6956

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  32. Voets T, Droogmans G, Wissenbach U, Janssens A, Flockerzi V, Nilius B (2004) The principle of temperature-dependent gating in cold- and heat-sensitive TRP channels. Nature 430:748–754

    Article  CAS  PubMed  Google Scholar 

  33. Voets T (2012) Quantifying and modeling the temperature-dependent gating of TRP channels. Rev Physiol Biochem Pharmacol 162:91–119

    CAS  PubMed  Google Scholar 

  34. Welch BL (1947) The generalisation of student’s problems when several different population variances are involved. Biometrika 34:28–35

    CAS  PubMed  Google Scholar 

  35. Xiao B, Dubin AE, Bursulaya B, Viswanath V, Jegla TJ, Patapoutian A (2008) Identification of transmembrane domain 5 as a critical molecular determinant of menthol sensitivity in mammalian TRPA1 channels. J Neurosci 28:9640–9651

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  36. Zhang F, Liu S, Yang F, Zheng J, Wang K (2011) Identification of a tetrameric assembly domain in the C terminus of heat-activated TRPV1 channels. J Biol Chem 286:15308–15316

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  37. Zhang X, Li L, McNaughton PA (2008) Proinflammatory mediators modulate the heat-activated ion channel TRPV1 via the scaffolding protein AKAP79/150. Neuron 59:450–461

    Article  PubMed  Google Scholar 

  38. Zurborg S, Yurgionas B, Jira JA, Caspani O, Heppenstall PA (2007) Direct activation of the ion channel TRPA1 by Ca2+. Nat Neurosci 10:277–279

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgement

This work was funded by grants from BBSRC (to PAM), the Newton Trust (to PAM), and Kidney Research UK (to JME). MJMF held a Feodor-Lynen Fellowship. DB holds a David James Bursary from the Department of Pharmacology, University of Cambridge. The authors declare that they have no conflict of interest.

Ethical standards

The authors declare that the experiments comply with the current laws of the countries in which they were performed. The study does not contain experiments using animals or humans or tissues derived from these.

Author contributions

MJMF conceived the study, generated the plasmids, designed, performed and analysed the functional experiments and drafted the manuscript. DB, PJ and TAG performed and analysed the AFM experiments. PAM and PWR helped design experiments and provided critical intellectual support. JME designed atomic force microscopy experiments, interpreted the results, wrote the respective parts of the manuscript and critically revised the manuscript.

Author information

Authors and Affiliations

  1. Institute of Physiology and Pathophysiology, University of Erlangen-Nuremberg, Universitätsstrasse 17, 91052, Erlangen, Germany

    Michael J. M. Fischer & Peter W. Reeh

  2. Department of Pharmacology, University of Cambridge, Cambridge, UK

    Michael J. M. Fischer, Dilshan Balasuriya, Pia Jeggle, Tom A. Goetze, Peter A. McNaughton & J. Michael Edwardson

  3. Department of Biology I—Botany, Biocenter of the LMU Munich, Munich, Germany

    Tom A. Goetze

  4. Wolfson Centre for Age-Related Diseases, King’s College London, London, UK

    Peter A. McNaughton

Authors
  1. Michael J. M. Fischer
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Dilshan Balasuriya
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Pia Jeggle
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Tom A. Goetze
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Peter A. McNaughton
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Peter W. Reeh
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. J. Michael Edwardson
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Michael J. M. Fischer.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Suppl. Fig. 1

Pharmacology of TRPV1::TRPA1. a In contrast to TRPA1 channels, TRPV1::TRPA1 is insensitive to the cysteine-targeting TRPA1 agonists AITC (20 μM) and H2O2 (100 μM). Stimulation with capsaicin 200 nM served as a control. b In contrast to TRPV1 channels, TRPV1::TRPA1 channels had a low sensitivity to TRPV1 agonist ethanol. (JPEG 20 kb)

High resolution image (TIFF 41 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Fischer, M.J.M., Balasuriya, D., Jeggle, P. et al. Direct evidence for functional TRPV1/TRPA1 heteromers. Pflugers Arch - Eur J Physiol 466, 2229–2241 (2014). https://doi.org/10.1007/s00424-014-1497-z

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00424-014-1497-z

Keywords

Search

Navigation