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:

A pentameric TRPV3 channel with a dilated pore

Abstract

Transient receptor potential (TRP) channels are a large, eukaryotic ion channel superfamily that control diverse physiological functions, and therefore are attractive drug targets1,2,3,4,5. More than 210 structures from more than 20 different TRP channels have been determined, and all are tetramers4. Despite this wealth of structures, many aspects concerning TRPV channels remain poorly understood, including the pore-dilation phenomenon, whereby prolonged activation leads to increased conductance, permeability to large ions and loss of rectification6,7. Here, we used high-speed atomic force microscopy (HS-AFM) to analyse membrane-embedded TRPV3 at the single-molecule level and discovered a pentameric state. HS-AFM dynamic imaging revealed transience and reversibility of the pentamer in dynamic equilibrium with the canonical tetramer through membrane diffusive protomer exchange. The pentamer population increased upon diphenylboronic anhydride (DPBA) addition, an agonist that has been shown to induce TRPV3 pore dilation. On the basis of these findings, we designed a protein production and data analysis pipeline that resulted in a cryogenic-electron microscopy structure of the TRPV3 pentamer, showing an enlarged pore compared to the tetramer. The slow kinetics to enter and exit the pentameric state, the increased pentamer formation upon DPBA addition and the enlarged pore indicate that the pentamer represents the structural correlate of pore dilation. We thus show membrane diffusive protomer exchange as an additional mechanism for structural changes and conformational variability. Overall, we provide structural evidence for a non-canonical pentameric TRP-channel assembly, laying the foundation for new directions in TRP channel research.

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

Access options

Buy this article

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

Fig. 1: Membrane reconstitution of TRPV3.
Fig. 2: TRPV3 pentamers coexist with canonical TRPV3 tetramers in membranes.
Fig. 3: TRPV3 tetramer–pentamer and pentamer–tetramer transitions, and observations of complete reversibility between the tetramer and pentamer states.
Fig. 4: DPBA leads to an increase of TRPV3 pentamers.
Fig. 5: Cryo-EM structures of the TRPV3 channel tetramer and pentamer.

Similar content being viewed by others

Data availability

All data and materials to draw the conclusions in this paper are presented in the main text, figures and the extended data figures and supplementary videos. The cryo-EM maps of the TRPV3 tetramer and pentamer have been deposited in the Electron Microscopy Data Bank with accession codes EMD-40181 and EMD-40183, respectively, and their structural models have been deposited in the PDB with accession codes 8GKA and 8GKG, respectively (Extended Data Table 1). Further data can be received from the corresponding author upon reasonable request.

References

  1. Peng, G., Shi, X. & Kadowaki, T. Evolution of TRP channels inferred by their classification in diverse animal species. Mol. Phylogenet. Evol. 84, 145–157 (2015).

    Article  CAS  PubMed  Google Scholar 

  2. Himmel, N. J. & Cox, D. N. Transient receptor potential channels: current perspectives on evolution. Proc. R. Soc. B. Biol. Sci. 287, 20201309 (2020).

    Article  Google Scholar 

  3. Khalil, M. et al. Functional role of transient receptor potential channels in immune cells and epithelia. Front. Immunol. 9, 174 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  4. Huffer, K. E., Aleksandrova, A. A., Jara-Oseguera, A., Forrest, L. R. & Swartz, K. J. Global alignment and assessment of trp channel transmembrane domain structures to explore functional mechanisms. eLife 9, e58660 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Moran, M. M. TRP channels as potential drug targets. Annu. Rev. Pharmacol. Toxicol. 58, 309–330 (2018).

    Article  CAS  PubMed  Google Scholar 

  6. Ferreira, L. G. B. & Faria, R. X. TRPing on the pore phenomenon: what do we know about transient receptor potential ion channel-related pore dilation up to now? J. Bioenerg. Biomembr. 48, 1–12 (2016).

    Article  CAS  PubMed  Google Scholar 

  7. Zheng, J. & Ma, L. Structure and function of the ThermoTRP channel pore. Curr. Top. Membr. 74, 233–257 (2014).

    Article  CAS  PubMed  Google Scholar 

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

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  9. Bai, X. C., Fernandez, I. S., McMullan, G. & Scheres, S. H. W. Ribosome structures to near-atomic resolution from thirty thousand cryo-EM particles. eLife 2013, 2–13 (2013).

    Google Scholar 

  10. Caterina, M. J. et al. The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature 389, 816–824 (1997).

    Article  ADS  CAS  PubMed  Google Scholar 

  11. Kashio, M. & Tominaga, M. TRP channels in thermosensation. Curr. Opin. Neurobiol. 75, 102591 (2022).

    Article  CAS  PubMed  Google Scholar 

  12. van Goor, M. K. C., Hoenderop, J. G. J. & van der Wijst, J. TRP channels in calcium homeostasis: from hormonal control to structure-function relationship of TRPV5 and TRPV6. Biochim. Biophys. Acta Mol. Cell Res. 1864, 883–893 (2017).

    Article  PubMed  Google Scholar 

  13. Pumroy, R. A. et al. Molecular mechanism of TRPV2 channel modulation by cannabidiol. eLife 8, e48792 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Xu, H., Delling, M., Jun, J. C. & Clapham, D. E. Oregano, thyme and clove-derived flavors and skin sensitizers activate specific TRP channels. Nat. Neurosci. 9, 628–635 (2006).

    Article  CAS  PubMed  Google Scholar 

  15. Deng, Z. et al. Cryo-EM and X-ray structures of TRPV4 reveal insight into ion permeation and gating mechanisms. Nat. Struct. Mol. Biol. 25, 252–260 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Deng, Z. et al. Gating of human TRPV3 in a lipid bilayer. Nat. Struct. Mol. Biol. 27, 635–644 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Nadezhdin, K. D. et al. Structural mechanism of heat-induced opening of a temperature-sensitive TRP channel. Nat. Struct. Mol. Biol. 28, 564–572 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Zubcevic, L. et al. Conformational ensemble of the human TRPV3 ion channel. Nat. Commun. 9, 4773 (2018).

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  19. Nilius, B., Bíró, T. & Owsianik, G. TRPV3: time to decipher a poorly understood family member! J. Physiol. 592, 295–304 (2014).

    Article  CAS  PubMed  Google Scholar 

  20. Bautista, D. & Julius, D. Fire in the hole: pore dilation of the capsaicin receptor TRPV1. Nat. Neurosci. 11, 528–529 (2008).

    Article  CAS  PubMed  Google Scholar 

  21. Chung, M. K., Güler, A. D. & Caterina, M. J. TRPV1 shows dynamic ionic selectivity during agonist stimulation. Nat. Neurosci. 11, 555–564 (2008).

    Article  CAS  PubMed  Google Scholar 

  22. Zhang, K., Julius, D. & Cheng, Y. Structural snapshots of TRPV1 reveal mechanism of polymodal functionality. Cell 184, 5138–5150.e12 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Canul-Sánchez, J. A. et al. Different agonists induce distinct single-channel conductance states in TRPV1 channels. J. Gen. Physiol. 150, 1735–1746 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  24. Nieto-Posadas, A. et al. Lysophosphatidic acid directly activates TRPV1 through a C-terminal binding site. Nat. Chem. Biol. 8, 78–85 (2012).

    Article  CAS  Google Scholar 

  25. Chung, M. K., Güler, A. D. & Caterina, M. J. Biphasic currents evoked by chemical or thermal activation of the heat-gated ion channel, TRPV3. J. Biol. Chem. 280, 15928–15941 (2005).

    Article  CAS  PubMed  Google Scholar 

  26. Chen, J. et al. Pore dilation occurs in TRPA1 but not in TRPM8 channels. Mol. Pain 5, 2–7 (2009).

    Article  Google Scholar 

  27. Banke, T. G., Chaplan, S. R. & Wickenden, A. D. Dynamic changes in the TRPA1 selectivity filter lead to progressive but reversible pore dilation. Am. J. Physiol. Cell Physiol. 298, 1457–1468 (2010).

    Article  Google Scholar 

  28. Zubcevic, L., Le, S., Yang, H. & Lee, S. Y. Conformational plasticity in the selectivity filter of the TRPV2 ion channel. Nat. Struct. Mol. Biol. 25, 405–415 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Uchihashi, T. & Scheuring, S. Applications of high-speed atomic force microscopy to real-time visualization of dynamic biomolecular processes. Biochim. Biophys. Acta Gen. Subj. 1862, 229–240 (2018).

    Article  CAS  PubMed  Google Scholar 

  30. Heath, G. R. & Scheuring, S. Advances in high-speed atomic force microscopy (HS-AFM) reveal dynamics of transmembrane channels and transporters. Curr. Opin. Struct. Biol. 57, 93–102 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Misetic, V., Reiners, O., Krauss, U. & Jaeger, K.-E. NanoDSF thermal unfolding analysis of proteins without tryptophan residues (Application Note NT‐PR‐007). NanoTemperTech https://resources.nanotempertech.com/application-notes/application-note-nt-pr-007-unfolding-without-tryptophan (2016).

  32. Real-Hohn, A., Groznica, M., Löffler, N., Blaas, D. & Kowalski, H. nanoDSF: in vitro label-free method to monitor picornavirus uncoating and test compounds affecting particle stability. Front. Microbiol. 11, 1442 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  33. Grubisha, O. et al. Pharmacological profiling of the TRPV3 channel in recombinant and native assays. Br. J. Pharmacol. 171, 2631–2644 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Yu, F. H., Yarov-Yarovoy, V., Gutman, G. A. & Catterall, W. A. Overview of molecular relationships in the voltage-gated ion channel superfamily. Pharmacol. Rev. 57, 387–395 (2005).

    Article  CAS  PubMed  Google Scholar 

  35. Nadezhdin, K. D. et al. Extracellular cap domain is an essential component of the TRPV1 gating mechanism. Nat. Commun. 12, 4–11 (2021).

    Article  Google Scholar 

  36. Singh, A. K., Saotome, K. & Sobolevsky, A. I. Swapping of transmembrane domains in the epithelial calcium channel TRPV6. Sci. Rep. 7, 10669 (2017).

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  37. Yelshanskaya, M. V. & Sobolevsky, A. I. Ligand-binding sites in vanilloid-subtype TRP channels. Front. Pharmacol. 13, 900623 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Singh, A. K., McGoldrick, L. L. & Sobolevsky, A. I. Structure and gating mechanism of the transient receptor potential channel TRPV3. Nat. Struct. Mol. Biol. 25, 805–813 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Zubcevic, L., Borschel, W. F., Hsu, A. L., Borgnia, M. J. & Lee, S. Y. Regulatory switch at the cytoplasmic interface controls trpv channel gating. eLife 8, e47746 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Ni, C. et al. A novel mutation in TRPV3 gene causes atypical familial Olmsted syndrome. Sci. Rep. 6, 21815 (2016).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  41. Duchatelet, S. et al. A new TRPV3 missense mutation in a patient with Olmsted syndrome and erythromelalgia. JAMA Dermatol. 150, 303–306 (2014).

    Article  PubMed  Google Scholar 

  42. Jiang, Y. et al. Membrane-mediated protein interactions drive membrane protein organization. Nat. Commun. 13, 7373 (2022).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  43. Hazan, A., Kumar, R., Matzner, H. & Priel, A. The pain receptor TRPV1 displays agonist-dependent activation stoichiometry. Sci. Rep. 5, 12278 (2015).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  44. Sente, A. et al. Differential assembly diversifies GABAA receptor structures and signalling. Nature 604, 190–194 (2022).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  45. Noviello, C. M., Kreye, J., Teng, J., Prüss, H. & Hibbs, R. E. Structural mechanisms of GABAA receptor autoimmune encephalitis. Cell 185, 2469–2477 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Cheng, W., Yang, F., Takanishi, C. L. & Zheng, J. Thermosensitive TRPV channel subunits coassemble into heteromeric channels with intermediate conductance and gating properties. J. Gen. Physiol. 129, 191–207 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Bleakman, D., Broroson, J. R. & Miller, R. J. The effects of capsaicin on voltage-gated calcium currents and calcium signals in cultured dorsal root ganglion cells. Br. J. Pharmacol. 101, 423–431 (1990).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Evans, A. R., Nicol, G. D. & Vasko, M. R. Differential regulation of evoked peptide release by voltage-sensitive calcium channels in rat sensory neurons. Brain Res. 712, 265–273 (1996).

    Article  CAS  PubMed  Google Scholar 

  49. Jancso, G. Pathobiological reactions of C‐fibre primary sensory neurones to peripheral nerve injury. Exp. Physiol. 77, 405–431 (1992).

    Article  CAS  PubMed  Google Scholar 

  50. Goehring, A. et al. Screening and large-scale expression of membrane proteins in mammalian cells for structural studies. Nat. Protoc. 9, 2574–2585 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Sanganna Gari, R. R. et al. Correlation of membrane protein conformational and functional dynamics. Nat. Commun. 12, 4363 (2021).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  52. Heath, G. R. et al. Localization atomic force microscopy. Nature 594, 385–390 (2021).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  53. Matin, T. R., Heath, G. R., Scheuring, S. & Boudker, O. Millisecond dynamics of an unlabeled amino acid transporter. Nat. Commun. 11, 5016 (2020).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  54. Rangl, M., Schmandt, N., Perozo, E. & Scheuring, S. Real time dynamics of gating-related conformational changesin CorA. eLife 8, e47322 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Lin, Y. C. et al. Force-induced conformational changes in PIEZO1. Nature 573, 230–234 (2019).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  56. Ruan, Y. et al. Structural titration of receptor ion channel GLIC gating by HS-AFM. Proc. Natl Acad. Sci. USA 115, 10333–10338 (2018).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  57. Ruan, Y. et al. Direct visualization of glutamate transporter elevator mechanism by high-speed AFM. Proc. Natl Acad. Sci. USA 114, 1584–1588 (2017).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  58. Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Suloway, C. et al. Automated molecular microscopy: the new Leginon system. J. Struct. Biol. 151, 41–60 (2005).

    Article  CAS  PubMed  Google Scholar 

  60. Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. CryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).

    Article  CAS  PubMed  Google Scholar 

  61. Bepler, T. et al. Positive-unlabeled convolutional neural networks for particle picking in cryo-electron micrographs. Nat. Methods 16, 1153–1160 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Chen, S. et al. High-resolution noise substitution to measure overfitting and validate resolution in 3D structure determination by single particle electron cryomicroscopy. Ultramicroscopy 135, 24–35 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Oh, S. et al. Differential ion dehydration energetics explains selectivity in the non-canonical lysosomal K+ channel TMEM175. eLife 11, e75122 (2022).

  64. Kimanius, D., Dong, L., Sharov, G., Nakane, T. & Scheres, S. H. W. New tools for automated cryo-EM single-particle analysis in RELION-4.0. Biochem. J 478, 4169–4185 (2021).

    Article  CAS  PubMed  Google Scholar 

  65. Terwilliger, T. C., Sobolev, O. V., Afonine, P. V., Adams, P. D. & Read, R. J. Density modification of cryo-EM maps. Acta Crystallogr. Sect. D Struct. Biol. 76, 912–925 (2020).

    Article  CAS  Google Scholar 

  66. Pettersen, E. F. et al. UCSF Chimera–a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  PubMed  Google Scholar 

  67. Zwart, P. H. et al. Automated structure solution with the PHENIX suite. Methods Mol. Biol. 426, 419–435 (2008).

    Article  CAS  PubMed  Google Scholar 

  68. Emsley, P. & Cowtan, K. Coot: Model-building tools for molecular graphics. Acta Crystallogr. Sect. D: Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  Google Scholar 

  69. Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. Sect. D: Biol. Crystallogr. 66, 12–21 (2010).

    Article  CAS  Google Scholar 

  70. Afonine, P. V. et al. Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr. Sect. D Struct. Biol. 74, 531–544 (2018).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank A. Accardi and J. Dittman for important discussions. Negative-stain EM data were collected in the Electron Microscopy & Histology services of the Weill Cornell Medicine Microscopy & Image Analysis Core using a transmission electron microscope purchased with funds from an National Institutes of Health (NIH) Shared Instrumentation grant (no. S10RR027699) for Shared Resources. Cryo-EM data were collected at the Simons Electron Microscopy Center at the New York Structural Biology Center, with support from the Simons Foundation (grant no. SF349247). Work in the Scheuring laboratory is partly supported by grants from the NIH, National Center for Complementary and Integrative Health (NCCIH), grant no. DP1AT010874 (to S.S.) and National Institute of Neurological Disorders and Stroke (NINDS), grant no. R01NS110790 (to S.S.). Work in the Yuan laboratory is partly supported by grant no. NIH NINDS R01NS099341 (to P.Y.). Work in the Nimigean laboratory is partly supported by grant no. NIH NIMGS R01 GM088352 (to C.M.N.) and grant no. NIH NIMGS F32 GM145091 (to E.D.K.). S.L. is an awardee of the Weizmann Institute of Science Women’s Postdoctoral Career Development Award.

Author information

Authors and Affiliations

Authors

Contributions

S.L. and S.S. designed the study. J.Z. and P.Y. expressed and purified protein from P. pastoris cells. S.L., J.M.B. and E.D.K. expressed and purified protein from HEK GnTI cells. S.L. and J.M.B. reconstituted protein. S.L. performed negative-stain EM imaging. S.L., E.D.K. and C.M.N. performed and analysed electrophysiology measurements. S.L. and J.M.B. performed HS-AFM imaging. S.L. and Y.J. performed HS-AFM data analysis. S.L. and J.M.B. performed nanoDSF experiments and analysis. S.L. and J.M.B. performed cryo-EM sample preparation and data collection. S.L., E.D.K. and N.P. analysed single-particle cryo-EM data. S.L. and S.S. performed channel structure analysis. Y.J. performed and analysed oligomer simulation. S.L. and S.S. wrote the paper. All authors edited the manuscript. S.S. supervised the project.

Corresponding author

Correspondence to Simon Scheuring.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature thanks Ute Hellmich, Thomas Voets and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 TRP-channel structures.

Representative structures (out of >210 structures) of the 23 TRP-channels solved so far. All structures are tetramers, with the four subunits coloured in wheat, green, purple and yellow. Each structure is depicted in surface representation, shown from the intracellular (top) and side (bottom) views. The question mark in the crTRP1 panel signifies that the subfamily to which crTRP1 belongs is yet unknown.

Extended Data Fig. 2 Single-channel recordings of TRPV3.

(a) and (b) Representative single-channel recordings of TRPV3 in the absence (a) and presence (b) of 100 μM DPBA, at −50 and 50 mV. (c) Since-channel current-voltage (IV) curves of TRPV3 obtained from −100 to 100 mV, in the absence and presence of 100 μM DPBA. (d) Single-channel open probabilities determined from recordings obtained at −50 mV, in the absence and presence of 100 μM DPBA. Open probability values (0.27 ± 0.01 and 0.78 ± 0.05 respectively) were derived as the mean values +/− s.e.m. from n ≥ 3 independent experiments (circles). Statistical significance was assessed with the one-tailed Welch’s T-test, yielding a significant (p-value = 0.0007) increase in open channel probability following DPBA addition. All recordings were performed on TRPV3 channels from one purification following the same protein expression and purification protocol as for the cryo-EM analysis, and reconstituted following the same protocol as for the HS-AFM analysis though at higher lipid-to-protein ratio (LPR between 5 and 20 for electrophysiology recordings vs. LPR between 0.5 and 2.5 for HS-AFM experiments). *** p-value < 0.005.

Extended Data Fig. 3 The TRPV3 tetramer and pentamer are reversible.

(a) to (f) Tetramer to pentamer transitions. (g) to (k) Pentamer to tetramer transitions. (l) Tetramer-pentamer-tetramer transition. (m) Pentamer-tetramer-pentamer-tetramer-pentamer-tetramer transition. White arrowheads indicate the occasionally observed monomers ‘attacking’ and inserting into tetramers to yield pentamers, and the observed monomers dissociating from pentamers to yield tetramers.

Extended Data Fig. 4 TRPV3 tetramers can breakup into fragments and reform.

(a) TRPV3 tetramers and pentamers coexist alongside TRPV3 ≤ 3 protomer fragments. Grey arrowheads indicate monomer (1), dimer (2), and trimer (3) fragments. (b) and (c) TRPV3 tetramer breakups into trimer and dimer. (d) TRPV3 fragments form a stable tetramer, which then breaks apart.

Extended Data Fig. 5 Workflow for cryo-EM reconstruction of the TRPV3 tetramer and pentamer.

Flowchart for the cryo-EM data processing, particle picking, classification, and reconstruction, enabling map reconstruction of the tetramer at 2.55Å resolution and for the pentamer at 4.38Å resolution. Unless otherwise stated, all processing steps were conducted in cryoSPARC version 3.3.2. Dashed lines indicate the inputs used for the iterative cycles of heterogenous refinement.

Extended Data Fig. 6 Cryo-EM density maps of the TRPV3 tetramer and pentamer.

(a) and (b) Cryo-EM reconstructed maps of the TRPV3 tetramer (a) and pentamer (b), colored according to local resolution using a rainbow colour scale. (c) and (d) Representative cryo-EM densities of the tetramer (contour level at 5.5 RMSD) (c) and pentamer (contour level at 4.16 RMSD) (d), at 2.55Å and 4.38Å resolution, respectively (the TMDs in the pentamer map are of ~5.0-5.5 Å resolution, see local resolution color scheme in (b)).

Extended Data Fig. 7 Structural comparison of the TRPV3 tetramer and pentamer cryo-EM structures.

(a) and (b) TRPV3 tetramer (a) and pentamer (b) structures, colored according to domains: ARD in purple, VSLD in yellow, PD in pink, SF in green, TRP helix in wheat, coupling domain in light blue. (c) to (e) Superposition of a pentamer subunit (purple) onto the tetramer subunit (green), aligned with respect to the PD, indicating a hinge-motion in the pentamer monomer by 18º, as manifested by rotation of the ARD (c), VSLD, and TRP helix (d). This hinge-motion enables preservation of the inter-subunit interactions of S5 with S1 and S4, and the SF-SF and S6-S6 interactions (e). Neighboring subunits in gray. Open book graphics of the tetramer (f, green) and pentamer (g, purple) inter-subunit contact areas. Contact areas are colored in pink (in the tetramer) and yellow (in the pentamer).

Extended Data Fig. 8 Model of the reversible transition between the tetrameric and pentameric TRPV3 states.

TRPV3 tetramers may dissociate. Dissociation is favored by activation, due to destabilization of the interprotomer interaction, notably the VSLD-PD domain-swap interface, by helix-intercalating molecules (e.g., capsaicin or LPA in TRPV1, DPBA in TRPV3, temperature). Monomers may ‘attack’ and insert into tetramers to yield pentamers with an estimated ~2.4-fold enlarged pore diameter at the SF. Pentamers, lifetime of ~3 min, are less stable than tetramers due to more fragile VSLD-PD interfaces, and shed subunits to regain the tetrameric state (figure created with BioRender.com).

Extended Data Fig. 9 Simulation of oligomeric state transitions.

(a) Visualization of simulated traces. Each column represents an independent space that can be either empty (0) or occupied by a molecule with a specific oligomeric state (1, 2, 3, 4 or 5). Total space: 5250. Total time step: 2000. Initial setup (time step = 1): 5000 out of 5250 spaces had a value of 4 (tetramers) and 250 out of the 5250 spaces had a value of 0 (empty). (b) and (c) Close-up views of the simulated traces in (a) as indicated by the dashed boxes. 1: Empty → monomer → dimer → trimer → tetramer → pentamer transition. 2: Transitions between tetramer and pentamer states (with short pentamer dwell-times). 3: A long pentamer state event. 4: pentamer → tetramer → trimer → dimer → monomer → empty transition. (d) Time-evolution of oligomer counts. Top panel: Tetramers. Middle panel: Pentamers and lower oligomers (trimer, dimer, and monomer) aggregated. Bottom panel: Trimers, dimers and monomers. (e) Oligomer state dwell-times. Left to right: Lower oligomers (n = 34984), tetramer (n = 278141), and pentamer (n = 331579).

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics

Supplementary information

Supplementary Information

This file contains Supplementary Figs. 1–6, Tables 1–3 and Code.

Reporting Summary

Peer Review File

Supplementary Video 1

Overview HS-AFM video of TRPV3 reconstitution. Frame rate, 1 s per frame. Pixel sampling, 0.80 nm per pixel.

Supplementary Video 2

Overview HS-AFM video of TRPV3 reconstitution. Frame rate, 2 s per frame. Pixel sampling, 0.80 nm per pixel.

Supplementary Video 3

Overview HS-AFM video of TRPV3 reconstitution. Frame rate, 1 s per frame. Pixel sampling, 0.27 nm per pixel.

Supplementary Video 4

Overview HS-AFM video of TRPV3 reconstitution. Frame rate, 1 s per frame. Pixel sampling, 0.40 nm per pixel.

Supplementary Video 5

Overview HS-AFM video of TRPV3 reconstitution revealing several channels with pentameric oligomeric states. Frame rate, 1.5 s per frame. Pixel sampling, 0.33 nm per pixel.

Supplementary Video 6

Overview HS-AFM video of TRPV3 reconstitution revealing several channels with pentameric oligomeric states. Frame rate, 1 s per frame. Pixel sampling, 0.50 nm per pixel.

Supplementary Video 7

Overview HS-AFM video of TRPV3 reconstitution revealing several channels with pentameric oligomeric states. Frame rate, 1 s per frame. Pixel sampling, 0.40 nm per pixel.

Supplementary Video 8

Overview HS-AFM video of TRPV3 reconstitution revealing several channels with pentameric oligomeric states. Frame rate, 0.5 s per frame. Pixel sampling, 0.48 nm per pixel.

Supplementary Video 9

Overview HS-AFM video of TRPV3 reconstitution revealing several channels with pentameric oligomeric states. Frame rate, 2 s per frame. Pixel sampling, 0.40 nm per pixel.

Supplementary Video 10

High-resolution HS-AFM videos of tetrameric TRPV3 channels. Frame rate, 1 s per frame. Pixel sampling, 0.20 nm per pixel.

Supplementary Video 11

High-resolution HS-AFM videos of a tetrameric and pentameric TRPV3 channel. Frame rate, 0.3 s per frame. Pixel sampling, 0.12 nm per pixel.

Supplementary Video 12

High-resolution HS-AFM videos of pentameric TRPV3 channels. Frame rate, 1.5 s per frame. Pixel sampling, 0.25 nm per pixel.

Supplementary Video 13

High-resolution HS-AFM videos of a tetrameric and pentameric TRPV3 channel. Frame rate, 1 s per frame. Pixel sampling, 0.35 nm per pixel.

Supplementary Video 14

High-resolution HS-AFM videos of a tetrameric and pentameric TRPV3 channel. Frame rate, 1 s per frame. Pixel sampling, 0.20 nm per pixel.

Supplementary Video 15

HS-AFM video of TRPV3 reconstitution revealing a tetramer–pentamer transition. Left panel shows an overview of TRPV3 reconstitution. Top right panel shows single-molecule tetramer–pentamer transition. Bottom right panel shows the average (t,t + 2) of single-molecule tetramer–pentamer transition. Frame rate, 2 s per frame. Pixel sampling, 0.28 nm per pixel.

Supplementary Video 16

HS-AFM video of TRPV3 reconstitution revealing a tetramer–pentamer transition. Left panel shows an overview of TRPV3 reconstitution. Top right panel shows single-molecule tetramer–pentamer transition. Bottom right panel shows the average (t,t + 2) of single-molecule tetramer–pentamer transition. Frame rate, 2 s per frame. Pixel sampling, 0.67 nm per pixel.

Supplementary Video 17

HS-AFM video of TRPV3 reconstitution revealing a tetramer–pentamer transition. Left panel shows an overview of TRPV3 reconstitution. Top right panel shows single-molecule tetramer–pentamer transition. Bottom right panel shows the average (t,t + 2) of single-molecule tetramer–pentamer transition. Frame rate, 1 s per frame. Pixel sampling, 0.32 nm per pixel.

Supplementary Video 18

HS-AFM video of TRPV3 reconstitution revealing two pentamer–tetramer transitions. Left panel shows an overview of TRPV3 reconstitution. Top right panel shows single-molecule pentamer–tetramer transitions. Bottom right panel shows the verage (t,t + 2) of single-molecule pentamer–tetramer transitions. Frame rate, 1 s per frame. Pixel sampling, 0.25 nm per pixel.

Supplementary Video 19

HS-AFM video of TRPV3 reconstitution revealing a pentamer–tetramer transitions. Left panel shows an overview of TRPV3 reconstitution. Top right panel shows the single-molecule pentamer–tetramer transition. Bottom right panel shows the average (t,t + 2) of single-molecule pentamer–tetramer transition. Frame rate, 1 s per frame. Pixel sampling, 0.35 nm per pixel.

Supplementary Video 20

HS-AFM video of TRPV3 reconstitution revealing a pentamer–tetramer transitions. Left panel shows an overview of TRPV3 reconstitution. Top right panel shows the single-molecule pentamer–tetramer transition. Bottom right panel shows the average (t,t + 2) of single-molecule pentamer–tetramer transition. Frame rate, 1 s per frame. Pixel sampling, 0.32 nm per pixel.

Supplementary Video 21

HS-AFM video of TRPV3 reconstitution revealing a complete tetramer–pentamer–tetramer transitions. Left panel shows an overview of TRPV3 reconstitution. Top right panel shows the single-molecule complete tetramer–pentamer–tetramer transition. Bottom right panel shows the average (t,t + 2) of single-molecule complete tetramer–pentamer–tetramer transition. Frame rate, 1.5 s per frame. Pixel sampling, 0.33 nm per pixel.

Supplementary Video 22

Overview HS-AFM video of TRPV3 reconstitution in the presence of 320 μM DPBA, revealing several channels with pentameric oligomeric states. Frame rate, 1 s per frame. Pixel sampling, 0.40 nm per pixel.

Supplementary Video 23

Overview HS-AFM video of TRPV3 reconstitution in the presence of 320 μM DPBA, revealing several channels with pentameric oligomeric states. Frame rate, 2 s per frame. Pixel sampling, 0.67 nm per pixel.

Supplementary Video 24

Overview HS-AFM video of TRPV3 reconstitution in the presence of 320 μM DPBA, revealing several channels with pentameric oligomeric states. Frame rate, 1 s per frame. Pixel sampling, 0.33 nm per pixel.

Supplementary Video 25

Overview HS-AFM video of TRPV3 reconstitution in the presence of 320 μM DPBA, revealing several channels with pentameric oligomeric states. Frame rate, 1 s per frame. Pixel sampling, 0.40 nm per pixel.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Lansky, S., Betancourt, J.M., Zhang, J. et al. A pentameric TRPV3 channel with a dilated pore. Nature 621, 206–214 (2023). https://doi.org/10.1038/s41586-023-06470-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41586-023-06470-1

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

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