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Mechanism of capsaicin entry into buried vanilloid sites in TRPV1

Nature Chemical Biology (2025)Cite this article

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Abstract

The transient receptor potential vanilloid 1 (TRPV1) receptor is a promising target for nonopioid analgesics, yet hyperthermic side effects have hindered drug development. The prevailing perspective maintains that extracellular hydrophobic vanilloid ligands, such as capsaicin, traverse the cell membrane to reach the buried vanilloid site during TRPV1 activation. Here, we present an alternative mechanism based on computational and experimental approaches, which suggests a distinct hydrophobic pathway at the TRPV1–cell membrane interface as the principal route for ligand entry to the vanilloid site, rather than direct membrane penetration. Modifications to residues within this pathway greatly delayed capsaicin entry without directly modulating TRPV1 channel gating. A compound designed to occupy this pathway’s entrance exhibited analgesic effects without inducing hyperthermia. Cryo-electron microscopy confirmed binding to TRPV1 and its role in perturbing capsaicin entry. Thus, our findings unveil a unique and targetable route for capsaicin access to the TRPV1 vanilloid site.

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Fig. 1: Potential pathways for capsaicin entry into buried vanilloid sites on TRPV1.
Fig. 2: Effects of amino acid substitutions at the TRPV1–membrane interface on capsaicin activation.
Fig. 3: Delayed capsaicin activation in the A539E mutant varies with the method of administration, independent of changes in TRPV1 gating.
Fig. 4: Covalent occupancy and photoswitching at A539C prolong topening following extracellular but not intracellular capsaicin application.
Fig. 5: Small molecules binding at the entrance of the binding pathway significantly delay the topening of capsaicin.
Fig. 6: Slowed TRPV1 activation enhances Ca2+ entry, expedites desensitization and strengthens analgesic outcomes.

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Data availability

All experimental data generated or analyzed in the course of this study are fully presented within the main text and the Supplementary Information. Atomic coordinates and cryo-EM density maps for the rTRPV1 channel in complex with PSFL426-S5 (6) were deposited to the PDB and EM Data Bank under accession codes 9ISG and EMD-60835, respectively. Previously resolved TRPV1 structures were retrieved from the PDB and EM Data Bank under accession codes 7L2H and EMD-23128 and 3J5P and EMD-5778, respectively. 1H-NMR spectra of PSFL426-S1 (2)–PSFL426-S21 (22), corresponding to Extended Data Fig. 7, are provided in Supplementary Data 1. Computational data presented in Fig. 2 and Extended Data Figs. 3, 6 and 9 are available in Supplementary Data 2–4. All additional datasets supporting the conclusions of this study are available from the corresponding author upon reasonable request. Source data are provided with this paper.

References

  1. Kraut, J. How do enzymes work? Science 242, 533–540 (1988).

    Article  CAS  PubMed  Google Scholar 

  2. Consalvi, S., Biava, M. & Poce, G. COX inhibitors: a patent review (2011–2014). Expert Opin. Ther. Pat. 25, 1357–1371 (2015).

    Article  CAS  PubMed  Google Scholar 

  3. Kawate, T. et al. Crystal structure of the ATP-gated P2X4 ion channel in the closed state. Nature 460, 592–598 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Yu, Y. et al. A nonproton ligand sensor in the acid-sensing ion channel. Neuron 68, 61–72 (2010).

    Article  CAS  PubMed  Google Scholar 

  5. Basak, S. et al. Cryo-EM reveals two distinct serotonin-bound conformations of full-length 5-HT3A receptor. Nature 563, 270–274 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Jasti, J. et al. Structure of acid-sensing ion channel 1 at 1.9 Å resolution and low pH. Nature 449, 316–323 (2007).

    Article  CAS  PubMed  Google Scholar 

  7. Polovinkin, L. et al. Conformational transitions of the serotonin 5-HT3 receptor. Nature 563, 275–279 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Huang, Y. et al. Ligand recognition and gating mechanism through three ligand-binding sites of human TRPM2 channel. eLife 8, e50175 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  9. Chun, L., Zhang, W. H. & Liu, J. F. Structure and ligand recognition of class C GPCRs. Acta Pharm. Sin. 33, 312–323 (2012).

    Article  CAS  Google Scholar 

  10. Koehl, A. et al. Structure of the μ-opioid receptor–Gi protein complex. Nature 558, 547–552 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Zhang, X. et al. Dynamic recognition of naloxone, morphine and endomorphin1 in the same pocket of micro-opioid receptors. Front. Mol. Biosci. 9, 925404 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Yang, P. L. et al. GSK1702934A and M085 directly activate TRPC6 via a mechanism of stimulating the extracellular cavity formed by the pore helix and transmembrane helix S6. J. Biol. Chem. 297, 101125 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Liao, M. et al. Structure of the TRPV1 ion channel determined by electron cryo-microscopy. Nature 504, 107–112 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Domene, C. et al. A potential route of capsaicin to its binding site in the TRPV1 ion channel. J. Chem. Inf. Model. 62, 2481–2489 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Hanson, S. M. et al. Capsaicin interaction with TRPV1 channels in a lipid bilayer: molecular dynamics simulation. Biophys. J. 108, 1425–1434 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. 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).

    Article  PubMed  Google Scholar 

  18. Roberts, J. C., Davis, J. B. & Benham, C. D. [3H]Resiniferatoxin autoradiography in the CNS of wild-type and TRPV1 null mice defines TRPV1 (VR-1) protein distribution. Brain Res. 995, 176–183 (2004).

    Article  CAS  PubMed  Google Scholar 

  19. Nilius, B. & Flockerzi, V. (eds) Mammalian Transient Receptor Potential (TRP) Cation Channels (Springer, 2014).

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

    Article  CAS  PubMed  Google Scholar 

  21. Bevan, S. & Yeats, J. Protons activate a cation conductance in a sub-population of rat dorsal root ganglion neurones. J. Physiol. 433, 145–161 (1991).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Clapham, D. E. TRP channels as cellular sensors. Nature 426, 517 (2003).

    Article  CAS  PubMed  Google Scholar 

  23. Benitez-Angeles, M. et al. TRPV1: structure, endogenous agonists, and mechanisms. Int. J. Mol. Sci. 21, 3421 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Iftinca, M., Defaye, M. & Altier, C. TRPV1-targeted drugs in development for human pain conditions. Drugs 81, 7–27 (2021).

    Article  CAS  PubMed  Google Scholar 

  25. Gouin, O. et al. TRPV1 and TRPA1 in cutaneous neurogenic and chronic inflammation: proinflammatory response induced by their activation and their sensitization. Protein Cell 8, 644–661 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Chung, M.-K. & Campbell, J. N.Use of capsaicin to treat pain: mechanistic and therapeutic considerations. Pharmaceuticals (Basel) 9, 66 (2016).

    Article  PubMed  Google Scholar 

  27. Moran, M. M. et al. Transient receptor potential channels as therapeutic targets. Nat. Rev. Drug Discov. 10, 601–620 (2011).

    Article  CAS  PubMed  Google Scholar 

  28. Iadarola, M. J. & Gonnella, G. L. Resiniferatoxin for pain treatment: an interventional approach to personalized pain medicine. Open Pain. J. 6, 95–107 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Knotkova, H., Pappagallo, M. & Szallasi, A. Capsaicin (TRPV1 agonist) therapy for pain relief: farewell or revival? Clin. J. Pain 24, 142–154 (2008).

    Article  PubMed  Google Scholar 

  30. Diaz-Franulic, I. et al. Allosterism and structure in thermally activated transient receptor potential channels. Annu. Rev. Biophys. 45, 371–398 (2016).

    Article  CAS  PubMed  Google Scholar 

  31. Gavva, N. R. Body-temperature maintenance as the predominant function of the vanilloid receptor TRPV1. Trends Pharm. Sci. 29, 550–557 (2008).

    Article  CAS  PubMed  Google Scholar 

  32. Yonghak, P., Miyata, S. & Kurganov, E. TRPV1 is crucial for thermal homeostasis in the mouse by heat loss behaviors under warm ambient temperature. Sci. Rep. 10, 8799 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Chovancova, E. et al. CAVER 3.0: a tool for the analysis of transport pathways in dynamic protein structures. PLoS Comput. Biol. 8, e1002708 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Wang, Y. et al. Single molecule FRET reveals pore size and opening mechanism of a mechano-sensitive ion channel. eLife 3, e01834 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  35. van den Bogaart, G., Krasnikov, V. & Poolman, B. Dual-color fluorescence-burst analysis to probe protein efflux through the mechanosensitive channel MscL. Biophys. J. 92, 1233–1240 (2007).

    Article  PubMed  Google Scholar 

  36. Li, J. et al. Mechanical coupling of the multiple structural elements of the large-conductance mechanosensitive channel during expansion. Proc. Natl Acad. Sci. USA 112, 10726–10731 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Laio, A. & Gervasio, F. L. Metadynamics: a method to simulate rare events and reconstruct the free energy in biophysics, chemistry and material science. Rep. Prog. Phys. 71, 126601 (2008).

    Article  Google Scholar 

  38. Wu, X. et al. Application of molecular dynamics simulation in biomedicine. Chem. Biol. Drug Des. 99, 789–800 (2022).

    Article  CAS  PubMed  Google Scholar 

  39. Barducci, A., Bussi, G. & Parrinello, M. Well-tempered metadynamics: a smoothly converging and tunable free-energy method. Phys. Rev. Lett. 100, 020603 (2008).

    Article  PubMed  Google Scholar 

  40. Leech, J., Prins, J. F. & Hermans, J.SMD: visual steering of molecular dynamics for protein design. IEEE Comput. Sci. Eng. 3, 38–45 (1996).

    Article  CAS  Google Scholar 

  41. Amann, R. & Maggi, C. A. Ruthenium red as a capsaicin antagonist. Life Sci. 49, 849–856 (1991).

    Article  CAS  PubMed  Google Scholar 

  42. Lee, B. H. & Zheng, J. Proton block of proton-activated TRPV1 current. J. Gen. Physiol. 146, 147–159 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Winter, Z. et al. Functionally important amino acid residues in the transient receptor potential vanilloid 1 (TRPV1) ion channel—an overview of the current mutational data. Mol. Pain 9, 30 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Yang, F. et al. The conformational wave in capsaicin activation of transient receptor potential vanilloid 1 ion channel. Nat. Commun. 9, 2879 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  45. Lee, H. S. et al. Genetic incorporation of a small, environmentally sensitive, fluorescent probe into proteins in Saccharomyces cerevisiae. J. Am. Chem. Soc. 131, 12921–12923 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Chatterjee, A. et al. A genetically encoded fluorescent probe in mammalian cells. J. Am. Chem. Soc. 135, 12540–12543 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Sun, M. Y. et al. Vanilloid agonist-mediated activation of TRPV1 channels requires coordinated movement of the S1–S4 bundle rather than a quiescent state. Sci. Bull. (Beijing) 67, 1062–1076 (2022).

    Article  CAS  PubMed  Google Scholar 

  48. Browne, L. E. et al. Optical control of trimeric P2X receptors and acid-sensing ion channels. Proc. Natl Acad. Sci. USA 111, 521–526 (2014).

    Article  CAS  PubMed  Google Scholar 

  49. Fattori, V. et al. Capsaicin: current understanding of its mechanisms and therapy of pain and other pre-clinical and clinical uses. Molecules 21, 844 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  50. Szallasi, A. & Sheta, M. Targeting TRPV1 for pain relief: limits, losers and laurels. Expert Opin. Investig. Drugs 21, 1351–1369 (2012).

    Article  CAS  PubMed  Google Scholar 

  51. Liu, B., Zhang, C. & Qin, F. Functional recovery from desensitization of vanilloid receptor TRPV1 requires resynthesis of phosphatidylinositol 4,5-bisphosphate. J. Neurosci. 25, 4835–4843 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Starowicz, K., Nigam, S. & Di Marzo, V. Biochemistry and pharmacology of endovanilloids. Pharm. Ther. 114, 13–33 (2007).

    Article  CAS  Google Scholar 

  53. Zhang, H. et al. Structure-guided peptide engineering of a positive allosteric modulator targeting the outer pore of TRPV1 for long-lasting analgesia. Nat. Commun. 14, 4 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Garami, A. et al. Hyperthermia induced by transient receptor potential vanilloid-1 (TRPV1) antagonists in human clinical trials: insights from mathematical modeling and meta-analysis. Pharm. Ther. 208, 107474 (2020).

    Article  CAS  Google Scholar 

  55. He, S. et al. A human TRPV1 genetic variant within the channel gating domain regulates pain sensitivity in rodents. J. Clin. Invest. 133, e163735 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Wong, G. Y. & Gavva, N. R. Therapeutic potential of vanilloid receptor TRPV1 agonists and antagonists as analgesics: recent advances and setbacks. Brain Res. Rev. 60, 267–277 (2009).

    Article  CAS  PubMed  Google Scholar 

  57. Cao, E. et al. TRPV1 structures in distinct conformations reveal activation mechanisms. Nature 504, 113–118 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Gao, Y. et al. TRPV1 structures in nanodiscs reveal mechanisms of ligand and lipid action. Nature 534, 347–351 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Arnold, W. R. et al. Structural basis of TRPV1 modulation by endogenous bioactive lipids. Nat. Struct. Mol. Biol. 31, 1377–1385 (2024).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Sharma, S. K., Vij, A. S. & Sharma, M. Mechanisms and clinical uses of capsaicin. Eur. J. Pharm. 720, 55–62 (2013).

    Article  CAS  Google Scholar 

  61. Arora, V., Campbell, J. N. & Chung, M. K. Fight fire with fire: neurobiology of capsaicin-induced analgesia for chronic pain. Pharm. Ther. 220, 107743 (2021).

    Article  CAS  Google Scholar 

  62. Rahaman, O. & Ganguly, D. Endocannabinoids in immune regulation and immunopathologies. Immunology 164, 242–252 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Di Marzo, V., Blumberg, P. M. & Szallasi, A. Endovanilloid signaling in pain. Curr. Opin. Neurobiol. 12, 372–379 (2002).

    Article  PubMed  Google Scholar 

  64. Dib, B. Effects of intracerebroventricular capsaicin on thermoregulatory behavior in the rat. Pharm. Biochem Behav. 16, 23–27 (1982).

    Article  CAS  Google Scholar 

  65. Phan, T. X. et al. TRPV1 expressed throughout the arterial circulation regulates vasoconstriction and blood pressure. J. Physiol. 598, 5639–5659 (2020).

    Article  CAS  PubMed  Google Scholar 

  66. Yue, W. W. S., Yuan, L., Braz, J. M., Basbaum, A. I. & Julius, D.TRPV1 drugs alter core body temperature via central projections of primary afferent sensory neurons. eLife 11, e80139 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Huang, Y.-Z. et al. TRPV1 analgesics disturb core body temperature via a biased allosteric mechanism involving conformations distinct from that for nociception. Neuron 112, 1815–1831 (2024).

    Article  CAS  PubMed  Google Scholar 

  68. Jordan, M. & Wurm, F. Transfection of adherent and suspended cells by calcium phosphate. Methods 33, 136–143 (2004).

    Article  CAS  PubMed  Google Scholar 

  69. Schink, M. et al. Reactive species modify NaV1.8 channels and affect action potentials in murine dorsal root ganglion neurons. Pflug. Arch. 468, 99–110 (2016).

    Article  CAS  Google Scholar 

  70. Malin, S. A., Davis, B. M. & Molliver, D. C. Production of dissociated sensory neuron cultures and considerations for their use in studying neuronal function and plasticity. Nat. Protoc. 2, 152–160 (2007).

    Article  CAS  PubMed  Google Scholar 

  71. Yao, J., Liu, B. & Qin, F. Rapid temperature jump by infrared diode laser irradiation for patch-clamp studies. Biophys. J. 96, 3611–3619 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Yao, J., Liu, B. & Qin, F. Kinetic and energetic analysis of thermally activated TRPV1 channels. Biophys. J. 99, 1743–1753 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. Yang, S. et al. A pain-inducing centipede toxin targets the heat activation machinery of nociceptor TRPV1. Nat. Commun. 6, 8297 (2015).

    Article  CAS  PubMed  Google Scholar 

  74. 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 

  75. Punjani, A. et al. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).

    Article  CAS  PubMed  Google Scholar 

  76. Emsley, P. et al. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  77. Liebschner, D. et al. Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in PHENIX. Acta Crystallogr. D Struct. Biol. 75, 861–877 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  79. Pettersen, E. F. et al. UCSF ChimeraX: structure visualization for researchers, educators, and developers. Protein Sci. 30, 70–82 (2021).

    Article  CAS  PubMed  Google Scholar 

  80. Mansoor, S. E. et al. X-ray structures define human P2X3 receptor gating cycle and antagonist action. Nature 538, 66–71 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  81. Laskowski, R. A. et al. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26, 283–291 (1993).

    Article  CAS  Google Scholar 

  82. Wang, J. et al. Druggable negative allosteric site of P2X3 receptors. Proc. Natl Acad. Sci. USA 115, 4939–4944 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  83. Laio, A. & Parrinello, M. Escaping free-energy minima. Proc. Natl Acad. Sci. USA 99, 12562–12566 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  84. Shaw, D. E. A fast, scalable method for the parallel evaluation of distance-limited pairwise particle interactions. J. Comput. Chem. 26, 1318–1328 (2005).

    Article  CAS  PubMed  Google Scholar 

  85. Orgensen, W. L., Maxwell, D. S. & Tirado-Rives, J. Development and testing of the OPLS all-atom force field on conformational energetics and properties of organic liquids. J. Am. Chem. Soc. 118, 11225–11236 (1996).

    Article  Google Scholar 

  86. Banks, J. L. et al. Integrated Modeling Program, Applied Chemical Theory (IMPACT). J. Comput Chem. 26, 1752–1780 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  87. Phillips, J. C. et al. Scalable molecular dynamics with NAMD. J. Comput. Chem. 26, 1781–1802 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  88. Feng, S. et al. CHARMM-GUI Membrane Builder: past, current, and future developments and applications. J. Chem. Theory Comput. 19, 2161–2185 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  89. Vanommeslaeghe, K. & MacKerell, A. D. Jr. Automation of the CHARMM General Force Field (CGenFF) I: bond perception and atom typing. J. Chem. Inf. Model. 52, 3144–3154 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  90. Zhu, S. Validation of the generalized force fields GAFF, CGenFF, OPLS-AA, and PRODRGFF by testing against experimental osmotic coefficient data for small drug-like molecules. J. Chem. Inf. Model. 59, 4239–4247 (2019).

    Article  CAS  PubMed  Google Scholar 

  91. Wijtmans, M. et al. Optical control of class A G protein-coupled receptors with photoswitchable ligands. Curr. Opin. Pharm. 63, 102192 (2022).

    Article  CAS  Google Scholar 

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Acknowledgements

This study was supported with funds from the National Natural Science Foundation of China (32371289 to Y.Y., 32401027 to S.M.Y. and 32421001, 32122040 and 31971040 to F.Y.), Changsha ‘Jie Bang Gua Shuai’ Major Science and Technology Program (KQ2301004), Innovation and Entrepreneurship (Shuangchuang) Program of Jiangsu Province (JSSCTD202350 to Y.Y.), Zhejiang Provincial Natural Science Foundation of China (LRG25C050001 to F.Y.), National Key R&D Program of China (2023YFC2308200 to P.C.), Natural Science Foundation of Jiangsu Province (BK20202002 to Y.Y.), ‘Xing Yao’ Leading Scholars of China Pharmaceutical University (2021 to Y.Y.), China Postdoctoral Science Foundation (2023M733896 to S.M.Y. and 2023M743097 to X.C.), CAMS Innovation Fund for Medical Sciences (2019-I2M-5-074 to Y.Y.) and Medical Innovation and Development Project of Lanzhou University (lzuyxcx-2022-156 to Y.Y.).

Author information

Author notes

  1. These authors contributed equally: Meng-Yang Sun, Yu-Jing Bian, Xiao-Ying Chen, Xue Zhang.

Authors and Affiliations

  1. Schools of Basic Medicine and Clinical Pharmacy, State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, China

    Meng-Yang Sun, Yu-Jing Bian, Xue Zhang, Ming Li, Bo-Ying Zhou, Yi-Zhe Huang, Rui Yang, Yu-Hao Gao, Si-Jia Zhu, Yun-Tao Lei & Ye Yu

  2. Key Laboratory of Preclinical Study for New Drugs of Gansu Province, School of Basic Medical Sciences, Lanzhou University, Lanzhou, China

    Meng-Yang Sun, Wen-Wen Cui & Ye Yu

  3. Department of Biophysics and Kidney Disease Center of the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China

    Xiao-Ying Chen & Fan Yang

  4. Liang zhu Laboratory, Zhejiang University Medical Center, Hangzhou, China

    Xiao-Ying Chen & Fan Yang

  5. Department of Pharmacology and Chemical Biology, Institute of Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai, China

    Yang Yang & Ye Yu

  6. School of Basic Medical Sciences and Forensic Medicine, Hangzhou Medical College, Hangzhou, China

    Ya-Qi Wang

  7. Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing, China

    Peng Cao

  8. State Key Laboratory of Utilization of Woody Oil Resource, Hunan Academy of Forestry, Changsha, China

    Chang-Zhu Li

  9. Department of Integrative Biology and Pharmacology, McGovern Medical School, The University of Texas Health Science Center at Houston, Houston, TX, USA

    Michael X. Zhu

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Contributions

Y. Yu designed the project. M.Y.S., Y.J.B., X.Z., S.J.Z. and Y. Yang performed the cell culture, patch-clamp recording and VCF. Y.J.B. and X.Z. conducted the animal experiments. Y. Yu, Y.T.L, M.L. and B.Y.Z. designed and synthesized the compounds. X.Y.C., Y.Q.W. and F.Y. obtained the cryo-EM structure of the PSFL426-S5 (6)-bound TRPV1 complex. R.Y. and W.W.C. performed the mutations. Y. Yu, Y.Z.H., Y.H.G. and P.C. conducted the MetaD, CMD and SMD simulations. Y. Yu and M.Y.S. analyzed the data. Y. Yu, M.X.Z., M.Y.S. and C.Z.L. wrote the manuscript. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Yun-Tao Lei, Fan Yang or Ye Yu.

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The authors declare no competing interests.

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Nature Chemical Biology thanks Jianhan Chen, Vladimir Chubanov, Daohua Jiang and the other, anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Differential localization of ligand binding sites and activation kinetics highlight distinctions between P2X3 receptors and TRPV1 channels.

(a) Crystal structure of P2X3 receptor bound with ATP, with subunits colored green, cyan, and magenta. (b) Representative current trace of ATP in HEK-293 cells expressing rP2X3 receptors. (c) Cryo-EM structure of TRPV1 bound with capsaicin, integrated into the plasma membrane, with subunits colored individually (green, yellow, cyan, and magenta). (d) Representative current trace of capsaicin in cells expressing rTRPV1. Individual sample sizes (n) are indicated in panels b and d.

Source data

Extended Data Fig. 2 TRPV1 activation by capsaicin under distinct electrophysiological configurations.

(a, b) Representative current traces demonstrating TRPV1 activation by capsaicin in whole-cell (a) and cell-attached (b) configurations. (c) Schematic representation of TRPV1 activation induced by capsaicin contained in the patch-pipette solution under cell-attached patch-clamp conditions. (d, e) Representative current traces recorded from HEK293 cells expressing TRPV1 or non-expressing control cells, illustrating TRPV1 activation by 300 nM capsaicin in the patch-pipette solution, in the presence or absence of 100 nM AMG517, or in an extracellular Ca2+/Mg2+-free solution. The red-dotted line indicates the baseline current (0 pA). (f) Representative current traces depicting TRPV1 activation in response to 100 nM capsaicin, with or without 100 nM AMG517, in the patch-pipette solution during cell-attached patch-clamp recordings. The baseline is indicated by a red-dotted line. (g) Summary of inward current amplitudes recorded under the above conditions. Data are presented as individual values (circles), each representing a distinct HEK293 cell. Statistical significance was determined by one-way ANOVA followed by Bonferroni’s multiple comparisons test (F(5,14) = 18.15; 95% confidence interval (CI)). n values are noted in the bar graphs. (h) TRPV1 activation by capsaicin under a cell-attached patch-clamp configuration in HEK293 cells co-expressing Mscl-G26C, treated with 1 mM MTSET.

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Extended Data Fig. 3 Metadynamics (MetaD) and steered molecular dynamics (SMD) simulations of the TRPV1-capsaicin complex.

(a) Definition of the collective variables (CVs) employed in MetaD simulations to explore capsaicin binding and unbinding. (b) Root-mean-square deviation (RMSD) values of the protein Cα atoms (rat TRPV1, rTRPV1, green) and capsaicin atoms (purple) over time in MetaD simulations. (c) Free energy landscapes describing capsaicin’s binding and unbinding pathways within rTRPV1, derived from MetaD simulations. (d) Torsion plots of capsaicin illustrating conformational changes in its rotatable bonds throughout the simulation, with a 2D schematic showing color-coded rotatable bonds (upper right). Radial plots depict torsional conformations over time, while bar plots show probability density, elucidating the ligand’s conformational dynamics stabilizing the rTRPV1-bound state. (e) Statistical analysis of contacts between capsaicin and amino acids along the binding pathway, highlighting key interacting residues at different stages of binding. Orange lines indicate the extent of interaction compactness. (f) Comparative analysis of binding pathway reproducibility using well-tempered MetaD simulations with varied CVs and parameters. Top panels illustrate reconstructed 3D free energy surfaces (FES), highlighting low-energy pathways. Bottom panels show representative snapshots of capsaicin traversing entry points near the TRPV1–lipid hydrophobic interface, engaging residues A539, V658, and F655. (g, h) Side-view (g) and top-view (h) snapshots of the rTRPV1/capsaicin complex during SMD simulations. The green arrow indicates the direction of applied force. Zoomed-in views depict capsaicin’s movement pathways during SMD simulations, with residue A539 highlighted as spheres. (i) Force profile as a function of the center-of-mass (COM) distance between capsaicin and residues at the base of the vanilloid pocket, such as R557. Zoomed-in views detail force fluctuations relative to capsaicin’s progression along the entry route.

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Extended Data Fig. 4 Critical role of A539 in the capsaicin binding pathway of TRPV1.

(a)Top-view of the TRPV1-capsaicin complex. Capsaicin is represented as a sphere, situated within the TRPV1-cell membrane interface. Zooming into the binding pathway entrance highlights specific key amino acids (depicted as orange sticks). (b) Summary of capsaicin-induced topening values during two successive activations with 1 μM capsaicin for WT and A539 mutant channels. Data are shown as individual cells (circles); bars represent mean ± S.E. For the first activation: F(10, 65) = 8.906, P < 0.0001; for the second: F(10, 65) = 13.00, P < 0.0001 (Dunnett’s multiple comparisons test; 95% CI). (c) Representative current traces in HEK-293 cells expressing TRPV1 WT or TRPV1A539E in response to saturating capsaicin (10 μM). (d-e) Summary of capsaicin-induced activation (topening) and the residual current ratio (Iwashout, after 5-min washing capsaicin out) to the maximal current (Imax) compared between TRPV1 WT and A539 mutants. Data points represent individual HEK293 cells (circles); bars indicate mean ± S.E. Statistical analysis was performed using one-way ANOVA with Bonferroni’s multiple comparisons test; D, F(8, 51) = 14.06, P < 0.0001; E, F(8, 43) = 11.56, P < 0.0001; 95% CI. (f) Representative single-channel recordings of TRPV1 and A539E induced by saturating capsaicin (10 μM). (g) The concentration-response curve of capsaicin, with and without PSFL426-S5(6), for both TRPV1 and A539E is depicted. The data, expressed as means ± S.E. from independent experiments, were fitted to the Hill1 equation to illustrate the capsaicin-dependent activation profiles. The EC50 values for the WT TRPV1, as previously shown in panel e of Fig. 1, are included here as a reference control. Individual sample sizes (n) are noted in the graphs of each panel.

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Extended Data Fig. 5 Covalent modification and photoswitching analysis in rTRPV1-4CG-A539C, and responses of TRPV1 mutants with delayed capsaicin activation to alternative stimuli.

(a) Zoom-in view of the introduction of DTNB (yellow sticks) into rTRPV1-4CG-A539C. Capsaicin and linked TNB group are shown as grey and peach sticks, respectively. (b) Representative current traces illustrating the impact of DTNB (4 mM) on capsaicin-induced opening in the TRPV14CG mutant. (c) Zoom-in view of the introduction of BMA (trans state, magenta sticks) into rTRPV1-4CG-A539C. (d) Typical current traces demonstrating the modulation of capsaicin-induced opening by various-wavelength lights following BMA (25 μM) application in the TRPV1-4CG mutants. (e) Pooled data of capsaicin-induced topening of rTRPV1, rTRPV1-A539C and rTRPV1-4CG-A539C. Each data point represents a distinct HEK293 cell; bars denote mean ± S.E. Statistical analysis via one-way ANOVA with Dunnett’s multiple comparisons test: F(2,13) = 2.546, P = 0.1167; 95% CI. (f) Inside-out patch-clamp recordings of rTRPV1-4CG-A539C activated by 100 nM capsaicin with DTNB (4 mM) or BMA (25 μM) applied intracellularly. (g) Quantification of current opening following DTNB or BMA application, normalized to pre-treatment values. Each circle represents one HEK293 cell; bars show mean ± S.E.; No significant difference was detected (two-sided paired t-test, 95% CI). (h) Representative current traces of rTRPV1-WT and rTRPV1-A539E in response to acidosis (pH 4.0), elevated temperature (heat), and 2-APB (3 mM). (i-k) Summary of the topening for 2-APB, acidosis, and heat-induced activation of TRPV1-WT mutant channels. Data shown as circles from different experimental units (HEK293 cells); Data are shown as individual cells (circles); bars represent mean ± S.E. Statistical testing via one-way ANOVA with Bonferroni correction: 2-APB: F(7,56) = 1.373, P = 0.2348; pH 4.0: F(6,35) = 1.619, P = 0.1712; heat: F(6,30) = 1.893, P = 0.1147; all analyses conducted with 95% CI. Individual sample sizes (n) are noted in the graphs of each panel.

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Extended Data Fig. 6 Extracellular capsaicin accumulation facilitated by hydrophobic residues at the entrance of the access tunnel.

(a) Side view (left) and top view (right) of the unbiased conventional molecular dynamics (CMD) simulation setup. Here, 20 capsaicin molecules (green spheres) were initially distributed randomly above TRPV1 receptors and allowed to move freely during CMD simulations. (b) Several free capsaicin molecules (green, molecules 2, 3, and 4) accumulated and became trapped at the entrance of the binding pathway, while a minority (molecules 1 and 5) contacts with the cell membrane independently. (c) Temporal changes in the distance between capsaicin and residues at the entrance of the binding pathway during CMD simulations. (d) The 3D reconstructions of the FES of capsaicin’s binding pathway reproducibility through extended conventional MetaD (80 ns), highlighting the lowest free-energy pathways for capsaicin passage based on selected CVs. (e) The snapshot of capsaicin at specific channel entry points along the TRPV1-lipid hydrophobic interface, interacting with residues A539, V658, and F655, as inferred from the FES. (f) The snapshot illustrating capsaicin’s transient interaction with TRPV1 surface residues R455, K535, and D652 before entering the TRPV1-lipid hydrophobic interface. This configuration is consistent with the observation that PSFL426-S5 (6) delays capsaicin binding by engaging in interactions with these interfacial residues.

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Extended Data Fig. 7 Discovery, design, and characterization of PSFL426 (1) and analogues targeting the vanilloid ligand entry site of TRPV1.

(a) Chemical structures of PSFL426 (1) analogues on activation by 100 nM capsaicin. Data are presented as the ratio of current opening (topening) after compound application to that before treatment. (b-c) Proposed binding modes of PSFL426 (1) and PSFL457 (32) obtained by virtual screening (b), and their quantified delay effects (100–500 μM) on capsaicin-induced TRPV1 currents (c). Each point represents an independent HEK293 cell; bars indicate mean ± S.E.; two-sided paired t-test; 95% CI. (d-e) Representative current traces (d) and summary data (e) showing the effect of PSFL426-S5 (6) on TRPV1 responses to 2-APB (3 mM), pH 4.0, and noxious heat. Current opening after compound application is normalized to pre-application values. P > 0.05, two-sided paired t-test; 95% CI. (f) Effects of PSFL426-S5 (6) on other TRP channel subfamilies. Left panel: ratio of current opening post- vs. pre-treatment. Right panel: current opening comparison with or without 200 μM PSFL426-S5 (6). Bars represent mean ± S.E.M.; left: paired t-test; right: unpaired t-test; P > 0.05; 95% CI (g) Functional impact of mutations at TRPV1 residues interacting with PSFL426-S5(6), based on structural analysis of the resolved complex. Data represent the ratio of capsaicin-evoked current opening post- vs. pre-treatment with PSFL426-S5 (6); one-way ANOVA with Dunnett’s post hoc test: F(11,51) = 8.350, P < 0.0001, 95% CI. Data for K535L, E600D, and V538T are reproduced from Fig. 5h (200 μM PSFL426-S5(6)). (h) Inhibition of endogenously expressed TRPV1 in DRG neurons by PSFL426-S5 (6), assessed in response to 1 μM capsaicin. Data are shown as mean ± S.E.; two-sided unpaired t-test; 95% CI. Individual sample sizes (n) are noted in the graphs of each panel.

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Extended Data Fig. 8 Single-particle cryo-EM data processing of PSFL426-S5 (6) bound rTRPV1 and cryo-EM density mapping of ligands and key domains in the TRPV1/PSFL426-S5 (6) complex.

(a) Size-exclusion chromatography of rat TRPV1 on Superose 6 (GE Healthcare) and SDS-PAGE analysis of the final sample. (b) Representative cryo-EM micrograph of the TRPV1/PSFL426-S5 (6) complex. (c) Flowchart of image processing for PSFL426-S5 (6) bound TRPV1. (d) Density map of the TRPV1/ PSFL426-S5 (6) complex colored by local resolution. (e) Distribution of particle orientations in azimuth and elevation angles for particles used in the final map calculation. (f) Gold standard Fourier Shell Correlation (FSC) curve of the final 3D reconstruction of the TRPV1/PSFL426-S5 (6) complex, and FSC curve for cross-validation between the map and model of the TRPV1/PSFL426-S5 (6) complex. (g) Sample maps in TRPV1/PSFL426-S5 (6) complex. The density is shown as a mask at the level of 0.08 in UCSF Chimera.

Extended Data Fig. 9 CMD simulations of the refined cryo-EM structure of the rTRPV1-PSFL426-S5 (6) complex and elucidation of receptor-ligand interaction dynamics.

(a) Statistical analysis of contacts between PSFL426-S5 (6) and amino acids at or near the capsaicin-entry pathway entrance during binding. Highlighted orange lines indicate key interacting amino acids with PSFL426-S5 (6), illustrating interaction density. (b) Potential interactions between TRPV1 and PSFL426-S5 (6) observed throughout CMD simulations. (c) Conformational dynamics of PSFL426-S5 (6) throughout the simulation trajectory. Backbone atoms of PSFL426-S5(6) are labeled (upper left). RMSF analysis of PSFL426-S5(6) binding to TRPV1 during CMD simulation (upper right). Rotatable bonds of PSFL426-S5(6) are shown in light blue, summarizing conformational changes in radial and bar plots throughout the simulation trajectory. (d) Cα-Cα distances between F655 and A539 with/without PSFL426-S5(6) binding and detailed view of residues (sticks) at the capsaicin-entry pathway entrance. Time-dependent change of Cα –Cα distance between F655 and A539 are shown with (red) or without (blue) PSFL426-S5(6) binding. (e) Torsion angles of dihedral angles for key residues (sticks) at the capsaicin-entry pathway entrance during CMD simulations. Atoms forming dihedral angles for each residue are numbered. Radial plots illustrate dihedral angles with (red) and without (blue) PSFL426-S5(6) for each residue throughout the simulation trajectory.

Extended Data Fig. 10 Delayed capsaicin activation reduces DRG neuron excitability by enhancing extracellular Ca2+ influx, promoting TRPV1 desensitization and analgesia.

(a-b) Representative Ca2+ imaging (a) and quantification (b) of HEK293 cells expressing WT or rTRPV1A539E, loaded with Fura-4, in response to 1 μM capsaicin and 10 μM ATP. The integral area under the curve (AUC) of the calcium signal reflects total Ca2+ influx. Data shown as individual cell responses (circles); mean ± S.E., two-sided unpaired t-test, 95% CI. (c) Quantification of peak TRPV1 currents in HEK293 cells expressing WT or A539E in response to 1 μM capsaicin under standard extracellular conditions (2 mM Ca2+, 1.5 mM Mg2+). two-sided unpaired t-test, 95% CI. (d) Comparison of peak capsaicin-induced TRPV1 currents in DRG neurons, with or without PSFL426-S5(6) (200 μM), recorded in standard saline (2 mM Ca2+, 1.5 mM Mg2+); two-sided unpaired t-test, 95% CI. (e) Action potential trains recorded from mouse DRG neurons following 1 μM capsaicin or co-application with PSFL426-S5(6); stimulus protocol indicated above. (f) Behavioral quantification of paw-licking responses in WT mice injected with PSFL426-S5(6) or vehicle control. Data represent mean ± S.E.; two-way ANOVA with Sidak’s multiple comparisons test; F(3, 21) = 0.94; P = 0.439; 95% CI. (g) Changes in CBT in Trpv1+/+ mice following injection of capsaicin (0.2 mg/kg), with or without PSFL426-S5(6) (4.0 mg/kg), or vehicle. Statistical comparisons: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. vehicle; #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001 vs. capsaicin alone. Two-way ANOVA with Tukey’s post hoc test; F(14, 56) = 21.57. (h-i) Effect of PSFL426-S5(6) (4.0 mg/kg) on CFA-induced thermal hyperalgesia in Trpv1+/+ (h) and Trpv1−/− (i) mice. Data shown as mean ± S.E.; two-way ANOVA with Sidak’s multiple comparisons test; F(7, 42) = 9.777 for Trpv1+/+, F(7, 42) = 2.101 for Trpv1−/−; *P < 0.05, **P < 0.01, ***P < 0.001. Individual sample sizes (n) are noted in the graphs of each panel.

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Supplementary information

Supplementary Information

Supplementary Tables 1 and 2, Protocols and Notes.

Reporting Summary

Supplementary Video 1

MetaD simulation illustrating the capsaicin-binding pathway.

Supplementary Video 2

SMD simulation demonstrating the capsaicin-binding pathway.

Supplementary Video 3

CMD simulation showing spontaneous capsaicin binding at the entrance of the binding pathway.

Supplementary Data 1

The 1H-NMR of PSFL426-S1 (2)–PSFL426-S21 (22).

Supplementary Data 2

The SMD trajectory, well-tempered MetaD and MetaD of capsaicin pullout from VBP.

Supplementary Data 3

Raw data and trajectory of CMD and conventional MetaD of capsaicin contracting with the entrance of the binding pathway.

Supplementary Data 4

Raw data of CMD simulations of the refined cryo-EM structure of the rTRPV1–PSFL426-S5 (6) complex.

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Sun, MY., Bian, YJ., Chen, XY. et al. Mechanism of capsaicin entry into buried vanilloid sites in TRPV1. Nat Chem Biol (2025). https://doi.org/10.1038/s41589-025-01966-5

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