TRPV1 structures in distinct conformations reveal activation mechanisms

Abstract

Transient receptor potential (TRP) channels are polymodal signal detectors that respond to a wide range of physical and chemical stimuli. Elucidating how these channels integrate and convert physiological signals into channel opening is essential to understanding how they regulate cell excitability under normal and pathophysiological conditions. Here we exploit pharmacological probes (a peptide toxin and small vanilloid agonists) to determine structures of two activated states of the capsaicin receptor, TRPV1. A domain (consisting of transmembrane segments 1–4) that moves during activation of voltage-gated channels remains stationary in TRPV1, highlighting differences in gating mechanisms for these structurally related channel superfamilies. TRPV1 opening is associated with major structural rearrangements in the outer pore, including the pore helix and selectivity filter, as well as pronounced dilation of a hydrophobic constriction at the lower gate, suggesting a dual gating mechanism. Allosteric coupling between upper and lower gates may account for rich physiological modulation exhibited by TRPV1 and other TRP channels.

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Figure 1: Structure of TRPV1 in complex with vanilloid ligand and spider toxin.
Figure 2: Binding sites for spider toxin and vanilloid agonists.
Figure 3: Comparison of ion permeation pathway in apo versus liganded channels.
Figure 4: Structural rearrangements in the outer pore region.
Figure 5: Opening of the lower gate.
Figure 6: Coupling of S4–S5 linker and S6 helix.

Accession codes

Accessions

Electron Microscopy Data Bank

Protein Data Bank

Data deposits

3D cryo-EM density maps of TRPV1 complexes without low-pass filter and amplitude modification have been deposited in the Electron Microscopy Data Bank under the accession numbers EMD-5776 (TRPV1–RTX/DkTx) and EMD-5777 (TRPV1–capsaicin). The coordinates of atomic models of TRPV1 in these two states have been deposited in the Protein Data Bank under the accession numbers 3J5Q and 3J5R.

References

  1. 1

    Basbaum, A. I., Bautista, D. M., Scherrer, G. & Julius, D. Cellular and molecular mechanisms of pain. Cell 139, 267–284 (2009)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  2. 2

    Baconguis, I. & Gouaux, E. Structural plasticity and dynamic selectivity of acid-sensing ion channel-spider toxin complexes. Nature 489, 400–405 (2012)

    CAS  Article  ADS  PubMed  PubMed Central  Google Scholar 

  3. 3

    Hansen, S. B., Tao, X. & MacKinnon, R. Structural basis of PIP2 activation of the classical inward rectifier K+ channel Kir2.2. Nature 477, 495–498 (2011)

    CAS  Article  ADS  PubMed  PubMed Central  Google Scholar 

  4. 4

    Hattori, M. & Gouaux, E. Molecular mechanism of ATP binding and ion channel activation in P2X receptors. Nature 485, 207–212 (2012)

    CAS  Article  ADS  PubMed  PubMed Central  Google Scholar 

  5. 5

    Whorton, M. R. & MacKinnon, R. X-ray structure of the mammalian GIRK2–βγ G-protein complex. Nature 498, 190–197 (2013)

    CAS  Article  ADS  PubMed  PubMed Central  Google Scholar 

  6. 6

    Bohlen, C. J. & Julius, D. Receptor-targeting mechanisms of pain-causing toxins: how ow? Toxicon 60, 254–264 (2012)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. 7

    Vriens, J., Appendino, G. & Nilius, B. Pharmacology of vanilloid transient receptor potential cation channels. Mol. Pharmacol. 75, 1262–1279 (2009)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  8. 8

    Liao, M., Cao, E., Julius, D. & Cheng, Y. Structure of the TRPV1 ion channel determined by electron cryo-microscopy. Nature http://dx.doi.org/10.1038/nature12823 (this issue)

  9. 9

    Catterall, W. A. Ion channel voltage sensors: structure, function, and pathophysiology. Neuron 67, 915–928 (2010)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  10. 10

    Long, S. B., Campbell, E. B. & Mackinnon, R. Voltage sensor of Kv1.2: structural basis of electromechanical coupling. Science 309, 903–908 (2005)

    CAS  Article  ADS  PubMed  PubMed Central  Google Scholar 

  11. 11

    Swartz, K. J. Sensing voltage across lipid membranes. Nature 456, 891–897 (2008)

    CAS  Article  ADS  PubMed  PubMed Central  Google Scholar 

  12. 12

    Zhu, S., Darbon, H., Dyason, K., Verdonck, F. & Tytgat, J. Evolutionary origin of inhibitor cystine knot peptides. FASEB J. 17, 1765–1767 (2003)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  13. 13

    Phillips, L. R. et al. Voltage-sensor activation with a tarantula toxin as cargo. Nature 436, 857–860 (2005)

    CAS  Article  ADS  PubMed  PubMed Central  Google Scholar 

  14. 14

    Swartz, K. J. & MacKinnon, R. Hanatoxin modifies the gating of a voltage-dependent K+ channel through multiple binding sites. Neuron 18, 665–673 (1997)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. 15

    Bohlen, C. J. et al. A bivalent tarantula toxin activates the capsaicin receptor, TRPV1, by targeting the outer pore domain. Cell 141, 834–845 (2010)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  16. 16

    Siemens, J. et al. Spider toxins activate the capsaicin receptor to produce inflammatory pain. Nature 444, 208–212 (2006)

    CAS  Article  ADS  PubMed  PubMed Central  Google Scholar 

  17. 17

    Grandl, J. et al. Temperature-induced opening of TRPV1 ion channel is stabilized by the pore domain. Nature Neurosci. 13, 708–714 (2010)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. 18

    Myers, B. R., Bohlen, C. J. & Julius, D. A yeast genetic screen reveals a critical role for the pore helix domain in TRP channel gating. Neuron 58, 362–373 (2008)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. 19

    Yang, F., Cui, Y., Wang, K. & Zheng, J. Thermosensitive TRP channel pore turret is part of the temperature activation pathway. Proc. Natl Acad. Sci. USA 107, 7083–7088 (2010)

    CAS  Article  ADS  PubMed  PubMed Central  Google Scholar 

  20. 20

    Zhou, Y., Morais-Cabral, J. H., Kaufman, A. & MacKinnon, R. Chemistry of ion coordination and hydration revealed by a K+ channel–Fab complex at 2.0 Å resolution. Nature 414, 43–48 (2001)

    CAS  Article  ADS  PubMed  PubMed Central  Google Scholar 

  21. 21

    Scheres, S. H. & Chen, S. Prevention of overfitting in cryo-EM structure determination. Nature Methods 9, 853–854 (2012)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  22. 22

    Chou, M. Z., Mtui, T., Gao, Y. D., Kohler, M. & Middleton, R. E. Resiniferatoxin binds to the capsaicin receptor (TRPV1) near the extracellular side of the S4 transmembrane domain. Biochemistry 43, 2501–2511 (2004)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  23. 23

    Gavva, N. R. et al. Molecular determinants of vanilloid sensitivity in TRPV1. J. Biol. Chem. 279, 20283–20295 (2004)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. 24

    Jordt, S. E. & Julius, D. Molecular basis for species-specific sensitivity to “hot” chili peppers. Cell 108, 421–430 (2002)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  25. 25

    Phillips, E., Reeve, A., Bevan, S. & McIntyre, P. Identification of species-specific determinants of the action of the antagonist capsazepine and the agonist PPAHV on TRPV1. J. Biol. Chem. 279, 17165–17172 (2004)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  26. 26

    Szallasi, A., Blumberg, P. M., Annicelli, L. L., Krause, J. E. & Cortright, D. N. The cloned rat vanilloid receptor VR1 mediates both R-type binding and C-type calcium response in dorsal root ganglion neurons. Mol. Pharmacol. 56, 581–587 (1999)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  27. 27

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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  28. 28

    Jordt, S. E., Tominaga, M. & Julius, D. Acid potentiation of the capsaicin receptor determined by a key extracellular site. Proc. Natl Acad. Sci. USA 97, 8134–8139 (2000)

    CAS  Article  ADS  PubMed  PubMed Central  Google Scholar 

  29. 29

    Kim, S. E., Patapoutian, A. & Grandl, J. Single residues in the outer pore of TRPV1 and TRPV3 have temperature-dependent conformations. PLoS ONE 8, e59593 (2013)

    CAS  Article  ADS  PubMed  PubMed Central  Google Scholar 

  30. 30

    Ryu, S., Liu, B., Yao, J., Fu, Q. & Qin, F. Uncoupling proton activation of vanilloid receptor TRPV1. J. Neurosci. 27, 12797–12807 (2007)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  31. 31

    Yeh, B. I., Kim, Y. K., Jabbar, W. & Huang, C. L. Conformational changes of pore helix coupled to gating of TRPV5 by protons. EMBO J. 24, 3224–3234 (2005)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  32. 32

    Bernèche, S. & Roux, B. A gate in the selectivity filter of potassium channels. Structure 13, 591–600 (2005)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. 33

    Cuello, L. G., Jogini, V., Cortes, D. M. & Perozo, E. Structural mechanism of C-type inactivation in K+ channels. Nature 466, 203–208 (2010)

    CAS  Article  ADS  PubMed  PubMed Central  Google Scholar 

  34. 34

    Hoshi, T. & Armstrong, C. M. C-type inactivation of voltage-gated K+ channels: pore constriction or dilation? J. Gen. Physiol. 141, 151–160 (2013)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  35. 35

    Hui, K., Liu, B. & Qin, F. Capsaicin activation of the pain receptor, VR1: multiple open states from both partial and full binding. Biophys. J. 84, 2957–2968 (2003)

    CAS  Article  ADS  PubMed  PubMed Central  Google Scholar 

  36. 36

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

    CAS  Article  ADS  PubMed  PubMed Central  Google Scholar 

  37. 37

    Tominaga, M. et al. The cloned capsaicin receptor integrates multiple pain-producing stimuli. Neuron 21, 531–543 (1998)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  38. 38

    Jiang, Y. et al. The open pore conformation of potassium channels. Nature 417, 523–526 (2002)

    CAS  Article  ADS  PubMed  PubMed Central  Google Scholar 

  39. 39

    Latorre, R., Zaelzer, C. & Brauchi, S. Structure–functional intimacies of transient receptor potential channels. Q. Rev. Biophys. 42, 201–246 (2009)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  40. 40

    Dai, J. et al. TRPV4-pathy, a novel channelopathy affecting diverse systems. J. Hum. Genet. 55, 400–402 (2010)

    Article  PubMed  PubMed Central  Google Scholar 

  41. 41

    Lin, Z. et al. Exome sequencing reveals mutations in TRPV3 as a cause of Olmsted syndrome. Am. J. Hum. Genet. 90, 558–564 (2012)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  42. 42

    Latorre, R., Vargas, G., Orta, G. & Brauchi, S. in TRP Ion Channel Function in Sensory Transduction and Cellular Signaling Cascades Frontiers in Neuroscience (eds Liedtke, W. B. & Heller, S. ). (2007)

  43. 43

    Matta, J. A. & Ahern, G. P. Voltage is a partial activator of rat thermosensitive TRP channels. J. Physiol. (Lond.) 585, 469–482 (2007)

    CAS  Article  Google Scholar 

  44. 44

    Nilius, B. et al. Gating of TRP channels: a voltage connection? J. Physiol. (Lond.) 567, 35–44 (2005)

    CAS  Article  Google Scholar 

  45. 45

    Loukin, S., Su, Z., Zhou, X. & Kung, C. Forward genetic analysis reveals multiple gating mechanisms of TRPV4. J. Biol. Chem. 285, 19884–19890 (2010)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  46. 46

    Cao, E., Cordero-Morales, J. F., Liu, B., Qin, F. & Julius, D. TRPV1 channels are intrinsically heat sensitive and negatively regulated by phosphoinositide lipids. Neuron 77, 667–679 (2013)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  47. 47

    van der Stelt, M. & Di Marzo, V. Endovanilloids. Putative endogenous ligands of transient receptor potential vanilloid 1 channels. Eur. J. Biochem. 271, 1827–1834 (2004)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  48. 48

    Brauchi, S., Orta, G., Salazar, M., Rosenmann, E. & Latorre, R. A hot-sensing cold receptor: C-terminal domain determines thermosensation in transient receptor potential channels. J. Neurosci. 26, 4835–4840 (2006)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  49. 49

    Papakosta, M. et al. The chimeric approach reveals that differences in the TRPV1 pore domain determine species-specific sensitivity to block of heat activation. J. Biol. Chem. 286, 39663–39672 (2011)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  50. 50

    Yao, J., Liu, B. & Qin, F. Modular thermal sensors in temperature-gated transient receptor potential (TRP) channels. Proc. Natl Acad. Sci. USA 108, 11109–11114 (2011)

    CAS  Article  ADS  PubMed  PubMed Central  Google Scholar 

  51. 51

    Scheres, S. H. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  52. 52

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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  53. 53

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

    Google Scholar 

  54. 54

    Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  55. 55

    Smart, O. S., Neduvelil, J. G., Wang, X., Wallace, B. A. & Sansom, M. S. HOLE: a program for the analysis of the pore dimensions of ion channel structural models. J. Mol. Graph. 14, 354–360,–376 (1996)

    Article  Google Scholar 

  56. 56

    Takahashi, H. et al. Solution structure of hanatoxin1, a gating modifier of voltage-dependent K+ channels: common surface features of gating modifier toxins. J. Mol. Biol. 297, 771–780 (2000)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank X. Li for assistant in data acquisition using TF30 Polara and K2 Summit camera, and J.P Armache, C. Bohlen, J. Cordero-Morales and J. Osteen for discussion and reading of the manuscript. This work was supported by grants from the National Institutes of Health (R01GM098672 and S10RR026814 to Y.C. and R01NS065071 and R01NS047723 to D. J.), the National Science Foundation (DBI-0960271 to D. Agard and Y.C.) and the University of California, San Francisco Program for Breakthrough Biomedical Research (Y.C.). E.C. was a fellow of the Damon Runyon Cancer Research Foundation.

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Contributions

All authors designed experiments. E.C. expressed and purified all protein samples used in this work and performed all functional studies. M.L. carried out all cryo-EM experiments, including data acquisition and processing. E.C. built the atomic model on the basis of cryo-EM maps. All authors analysed data and wrote the manuscript.

Corresponding authors

Correspondence to Yifan Cheng or David Julius.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Cryo-EM of TRPV1 in complex with RTX and DkTx.

a, b, Fourier transform (a) of a representative image (b). c, Two dimensional (2D) class averages of cryo-EM particles. d, Enlarged view of three representative 2D class averages. Arrows indicate DkTx densities near the channel pore. e, Gold-standard FSC curve (red) of the final 3D reconstruction, marked with the resolutions that correspond to FSC = 0.5 and 0.143. The FSC curve between the final map and that calculated from the atomic model is shown in blue. f, Euler angle distribution of all particles used for calculating the final 3D reconstruction. The sizes of balls represent the number of particles. The accuracy of rotation is 5.213°, as reported by RELION.

Extended Data Figure 2 3D reconstruction of TRPV1–RTX/DkTx complexes filtered at 6 Å resolution.

ad, Four different views of the 3D reconstruction low-pass filtered at 6 Å and amplified by a temperature factor of −100 Å2, fitted with de novo atomic model of TRPV1–RTX/DkTx complex (toxin is shown in magenta) built as described in Methods. e, f, Two views of the 3D reconstruction displayed at two different isosurface levels (high in yellow and low in grey). At the low isosurface level, the belt-shaped density of amphipols is visible with a thickness of 30 Å. DkTx-related densities are also clearly visible, including the linker peptide that connects the toxin’s two inhibitor cysteine knot (ICK) moieties, as noted.

Extended Data Figure 3 Cryo-EM of TRPV1 in complex with capsaicin.

a, b, Fourier transform (a) of a representative image (b). c, 2D class averages of cryo-EM particles. d, Enlarged view of three representative 2D class averages. e, Gold-standard FSC curve (red) of the final 3D reconstruction, marked with the resolutions that correspond to FSC = 0.5 and 0.143. The FSC curve between the final map and that calculated from the atomic model is shown in blue. f, Euler angle distribution of all particles used for calculating the final 3D reconstruction. The sizes of balls represent the number of particles. The accuracy of rotation is 4.989°, as reported by RELION.

Extended Data Figure 4 3D reconstruction of TRPV1–capsaicin complex low-pass filtered at 6 Å resolution.

ad, Four different views of the 3D reconstruction low-pass filtered at 6 Å and amplified by a temperature factor of −100 Å2, fitted with de novo atomic model of TRPV1–capsaicin complex built as described in Methods. e, f, Two views of the 3D reconstruction displayed at two different isosurface levels (high in yellow and low in grey). At the low isosurface level, the belt-shaped density of amphipols is visible with a thickness of 30 Å.

Extended Data Figure 5 3D reconstruction of TRPV1–RTX/DkTx complex low-pass filtered at 3.8 Å resolution.

ad, Four different views of the 3D reconstruction low-pass filtered at 3.8 Å with a temperature factor of −100 Å2, fitted with de novo atomic model of TRPV1–RTX/DkTx complex (toxin is shown in magenta and indicated by arrows) built as described in Methods.

Extended Data Figure 6 3D reconstruction of TRPV1–capsaicin complex low-pass filtered at 4.2 Å resolution.

ad, Four different views of the 3D reconstruction low-pass filtered to 4.2 Å with a temperature factor of −150 Å2, fitted with de novo atomic model of TRPV1–capsaicin complex built as described in Methods.

Extended Data Figure 7 Observed densities in vanilloid pocket.

a, Non-protein associated densities in the region adjacent to S3–S4 transmembrane helices observed in 3D density maps of the apo TRPV1 structure (blue, 3.4 Å, −100 Å2) or TRPV1 in complex with RTX/DkTx (red, 3.8 Å, −150 Å2) or capsaicin (yellow, 3.9 Å, −150 Å2), as indicated. b, Density of bound capsaicin (blue) viewed from the side (left) and top-down (that is, from the extracellular face; right). Density is also observed in the apo-channel structure (purple), possibly representing an endogenous lipid or other small hydrophobic molecule. All maps were low-pass filtered to 4.5 Å with a temperature factor of −200 Å2, normalized and displayed at the same sigma level (8σ).

Extended Data Figure 8 Structural details of the TRPV1 pore with and without ligands.

ac, Density maps for pore region for two diagonally opposed monomers superimposed onto their atomic models (top). Distances between specific side-chain atoms along the pore are also indicated (bottom). d, Superimposed top-down view of apo and capsaicin-bound TRPV1 outer pore regions (orange and green, respectively). e, Density map of selectivity filter in the capsaicin-bound TRPV1 structure. The distance between carbonyl oxygens of diagonally opposed G643 residues (4.6 Å) does not differ from that of the apo structure (4.6 Å).

Extended Data Figure 9 S1–S4 as a stationary domain.

a, Superimposition of apo and RTX/DkTx-bound TRPV1 structures (orange and blue, respectively) from top-down view. S1–S4 domain (outlined in dashed box) shows near-complete overlap. b, Same comparison for apo and capsaicin-bound channel structures (orange and green, respectively). c, d, Superimposition of transmembrane core of apo versus RTX/DkTx- or capsaicin-bound TRPV1 structures (orange, blue and green, respectively). Dashed box denotes region highlighted in Fig. 6.

Extended Data Figure 10 Dual gate model for TRPV1 activation.

a, Pore helix and upper half of S5 helix are in close proximity and appear to be physically coupled, representing a potential mechanisms for allosteric coupling between upper and lower gates. Several residues on both helices are rendered as ball-and-stick to highlight close apposition. b, Downward tilt of pore helix away from the central pore is associated with movement of the S5 helix in RTX/DkTx structure (left). This structural arrangement is not observed in capsaicin-bound structure (right). c, Model depicting two gate mechanism of TRPV1 activation. Two main constriction points at the selectivity filter (1) and lower gate (2) block ion permeation in the apo, closed state (top left). Some pharmacological agents (for example, protons or spider toxins; gold hexagon) target the outer pore region of the channel to open or stabilize the conductive conformation of the selectivity filter (top right). Arrow denotes proposed coupling between the pore helix and S5. Small vanilloid ligands (for example, RTX and capsaicin; red ellipse) bind within a hydrophobic pocket formed by the S3–S4 helices, the S4–S5 linker and the pore module, eliciting conformational changes that expand the lower gate (bottom left). Arrows indicate proposed coupling between S4–S5 helix, S6 and TRP domain. Full expansion of the ion permeation pathway and ion conduction is achieved when both upper and lower gates are opened (bottom right). Pharmacological and mutagenesis data suggest that these gates are allosterically coupled.

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Cao, E., Liao, M., Cheng, Y. et al. TRPV1 structures in distinct conformations reveal activation mechanisms. Nature 504, 113–118 (2013). https://doi.org/10.1038/nature12823

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