Preprint / Version 2

Open-state structure of veratridine-activated human Nav1.7 reveals the molecular choreography of fast inactivation

This article is a preprint and has not been certified by peer review.
Now published in Vita. doi: 10.15302/vita.2026.01.0003

Authors

    XIAO FAN,  
    XIAO FAN
    • Shenzhen Medical Academy of Research and Translation
    Jiaofeng Chen,  
    Jiaofeng Chen
    Lingfeng Xue,  
    Lingfeng Xue
    Huan Wang,  
    Huan Wang
    Tong Wu,  
    Tong Wu
    Xiaoshuang Huang,  
    Xiaoshuang Huang
    • Shenzhen Medical Academy of Research and Translation
    Fangzhou Lu,  
    Fangzhou Lu
    • Shenzhen Medical Academy of Research and Translation
    Xueqin Jin,  
    Xueqin Jin
    Chen Song,  
    Chen Song
    Jian Huang,  
    Jian Huang
    • Shenzhen Medical Academy of Research and Translation
    Nieng Yan
    Nieng Yan
    • Shenzhen Medical Academy of Research and Translation
    • Tsinghua University image/svg+xml
Categories
Biochemistry & Biophysics
Keywords
Voltage gated sodium channel; Open-state; Fast inactivation; Veratridine

Abstract

Almost all the reported cryo-EM structures of eukaryotic voltage-gated sodium (Nav) channels, including those of human Nav1.1-Nav1.8, represent various inactivated states that are characteristic of a non-conductive pore domain (PD) surrounded by voltage-sensing domains (VSDs) in varying up conformations. To capture an open-state Navstructure, we treated purified human Nav1.7 with a natural neurotoxin veratridine (VTD) and solved its cryo-EM structures. Two VTD-bound Nav1.7 complexes were obtained. One, with VTD inserted in the IFM-binding corner (site I), resembles other inactivated structures. The other, wherein VTD traverses the central cavity (site C), represents an activated conformation with the constriction diameter of 8.2 Å at the intracellular gate. Structural analysis reveals the mechanism of action of VTD’s bimodal modulation of Nav channels. More importantly, structural comparison between the open and inactivated states provides advanced molecular insight into the fast inactivation process.

References

1. Hille, B. Ion channels of excitable membranes. 3rd edn, (Sinauer, 2001).

2. Hodgkin, A. L. & Huxley, A. F. Resting and action potentials in single nerve fibres. The Journal of physiology 104, 176-195 (1945).

3. Huang, J., Pan, X. & Yan, N. Structural biology and molecular pharmacology of voltage-gated ion channels. Nat Rev Mol Cell Biol 25, 904-925 (2024).

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

5. Kuhlbrandt, W. Biochemistry. The resolution revolution. Science 343, 1443-1444 (2014).

6. Shen, H. et al. Structure of a eukaryotic voltage-gated sodium channel at near-atomic resolution. Science 355, eaal4326 (2017).

7. Yan, Z. et al. Structure of the Nav1.4-beta1 Complex from Electric Eel. Cell 170, 470-482 e411 (2017).

8. Pan, X. et al. Structure of the human voltage-gated sodium channel Nav1.4 in complex with beta1. Science 362 (2018).

9. Jiang, D. et al. Structural basis for voltage-sensor trapping of the cardiac sodium channel by a deathstalker scorpion toxin. Nat Commun 12, 128 (2021).

10. Clairfeuille, T. et al. Structural basis of alpha-scorpion toxin action on Nav channels. Science 363 (2019).

11. Jiang, D. et al. Open-state structure and pore gating mechanism of the cardiac sodium channel. Cell 184, 5151-5162.e5111 (2021).

12. Huang, G. et al. Unwinding and spiral sliding of S4 and domain rotation of VSD during the electromechanical coupling in Na(v)1.7. Proc Natl Acad Sci U S A 119, e2209164119 (2022).

13. Li, Z. et al. Dissection of the structure-function relationship of Na(v) channels. Proc Natl Acad Sci U S A 121, e2322899121 (2024).

14. Cox, J. J. et al. An SCN9A channelopathy causes congenital inability to experience pain. Nature 444, 894-898 (2006).

15. Cummins, T. R., Dib-Hajj, S. D. & Waxman, S. G. Electrophysiological properties of mutant Nav1.7 sodium channels in a painful inherited neuropathy. J Neurosci 24, 8232-8236 (2004).

16. Nassar, M. A. et al. Nociceptor-specific gene deletion reveals a major role for Nav1.7 (PN1) in acute and inflammatory pain. Proc Natl Acad Sci U S A 101, 12706-12711 (2004).

17. Yang, Y. et al. Mutations in SCN9A, encoding a sodium channel alpha subunit, in patients with primary erythermalgia. Journal of medical genetics 41, 171-174 (2004).

18. Shen, H., Liu, D., Wu, K., Lei, J. & Yan, N. Structures of human Nav1.7 channel in complex with auxiliary subunits and animal toxins. Science 363, 1303-1308 (2019).

19. Huang, G. et al. High-resolution structures of human Na(v)1.7 reveal gating modulation through α-π helical transition of S6(IV). Cell Rep 39, 110735 (2022).

20. Zhang, J. et al. Structural basis for Na(V)1.7 inhibition by pore blockers. Nat Struct Mol Biol 29, 1208-1216 (2022).

21. Wu, Q. et al. Structural mapping of Na(v)1.7 antagonists. Nat Commun 14, 3224 (2023).

22. Huang, J. et al. Cannabidiol inhibits Na(v) channels through two distinct binding sites. Nat Commun 14, 3613 (2023).

23. Huang, J., Fan, X., Jin, X., Teng, L. & Yan, N. Dual-pocket inhibition of Na(v) channels by the antiepileptic drug lamotrigine. Proc Natl Acad Sci U S A 120, e2309773120 (2023).

24. Sutro, J. B. Kinetics of veratridine action on Na channels of skeletal muscle. J Gen Physiol 87, 1-24 (1986).

25. Leibowitz, M. D., Sutro, J. B. & Hille, B. Voltage-dependent gating of veratridine-modified Na channels. J Gen Physiol 87, 25-46 (1986).

26. Ulbricht, W. The effect of veratridine on excitable membranes of nerve and muscle. Ergeb Physiol 61, 18-71 (1969).

27. Zhang, X. Y., Bi, R. Y., Zhang, P. & Gan, Y. H. Veratridine modifies the gating of human voltage-gated sodium channel Nav1.7. Acta Pharmacol Sin 39, 1716-1724 (2018).

28. DeLano, W. L. The PyMOL Molecular Graphics System. on World Wide Web http://www.pymol.org (2002).

29. Fan, X. et al. Phrixotoxin-3 binds to three distinct antagonistic sites on human Na(v)1.6. Cell Res 35, 610-613 (2025).

30. Gao, S. et al. Structural basis for human Ca(v)1.2 inhibition by multiple drugs and the neurotoxin calciseptine. Cell 186, 5363-5374.e5316 (2023).

31. Jin, X., Huang, J., Wang, H., Wang, K. & Yan, N. A versatile residue numbering scheme for Nav and Cav channels. Cell Chemical Biology 31, 1394-1404 (2024).

32. Weiss, R. E. & Horn, R. Functional differences between two classes of sodium channels in developing rat skeletal muscle. Science 233, 361-364 (1986).

33. Liu, Y., Bassetto, C. A. Z., Jr., Pinto, B. I. & Bezanilla, F. A mechanistic reinterpretation of fast inactivation in voltage-gated Na(+) channels. Nat Commun 14, 5072 (2023).

34. Wang, G., Dugas, M., Armah, B. I. & Honerjager, P. Sodium channel comodification with full activator reveals veratridine reaction dynamics. Molecular pharmacology 37, 144-148 (1990).

35. Barnes, S. & Hille, B. Veratridine modifies open sodium channels. The Journal of general physiology 91, 421-443 (1988).

36. Shen, H. et al. Structural basis for the modulation of voltage-gated sodium channels by animal toxins. Science 362 (2018).

37. Armstrong, C. M., Bezanilla, F. & Rojas, E. Destruction of sodium conductance inactivation in squid axons perfused with pronase. The Journal of general physiology 62, 375-391 (1973).

38. West, J. W. et al. A cluster of hydrophobic amino acid residues required for fast Na(+)-channel inactivation. Proceedings of the National Academy of Sciences of the United States of America 89, 10910-10914 (1992).

39. Li, Z. et al. Structure of human Nav1.5 reveals the fast inactivation-related segments as a mutational hotspot for the long QT syndrome. Proceedings of the National Academy of Sciences 118 (2021).

40. Li, Z. et al. Quinidine-bound human Nav1.5 structure reveals a mutational hotspot for Long QT Syndrome mapped to inactivation-related segments Submitted (2020).

41. Hu, R.-M. et al. Mexiletine rescues a mixed biophysical phenotype of the cardiac sodium channel arising from the SCN5A mutation, N406K, found in LQT3 patients. Channels 12, 176-186 (2018).

42. Itoh, H., Shimizu, M., Takata, S., Mabuchi, H. & Imoto, K. A novel missense mutation in the SCN5A gene associated with Brugada syndrome bidirectionally affecting blocking actions of antiarrhythmic drugs. Journal of cardiovascular electrophysiology 16, 486-493 (2005).

43. Huang, H., Priori, S. G., Napolitano, C., O'Leary, M. E. & Chahine, M. Y1767C, a novel SCN5A mutation, induces a persistent Na+ current and potentates ranolazine inhibition of Nav1.5 channels. American Journal of Physiology-Heart and Circulatory Physiology 300, H288-H299 (2011).

44. Clancy, C. E., Tateyama, M., Liu, H., Wehrens, X. H. & Kass, R. S. Non-equilibrium gating in cardiac Na+ channels: an original mechanism of arrhythmia. Circulation 107, 2233-2237 (2003).

45. Meng, E. C., Pettersen, E. F., Couch, G. S., Huang, C. C. & Ferrin, T. E. Tools for integrated sequence-structure analysis with UCSF Chimera. BMC Bioinformatics 7, 339 (2006).

46. Smart, O. S., Neduveli, 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).

47. Li, Z. et al. Structure of human Na(v)1.5 reveals the fast inactivation-related segments as a mutational hotspot for the long QT syndrome. Proc Natl Acad Sci U S A 118 (2021).

48. Huang, X. et al. Structural basis for high-voltage activation and subtype-specific inhibition of human Na(v)1.8. Proc Natl Acad Sci U S A 119, e2208211119 (2022).

49. Fan, X., Huang, J., Jin, X. & Yan, N. Cryo-EM structure of human voltage-gated sodium channel Na(v)1.6. Proc Natl Acad Sci U S A 120, e2220578120 (2023).

50. Tegunov, D. & Cramer, P. Real-time cryo-electron microscopy data preprocessing with Warp. Nat Methods 16, 1146-1152 (2019).

51. 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).

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

53. Afonine, P. V. et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr D Biol Crystallogr 68, 352-367 (2012).

54. Croll, T. I. ISOLDE: a physically realistic environment for model building into low-resolution electron-density maps. Acta Crystallogr D Struct Biol 74, 519-530 (2018).

55. Jo, S., Kim, T., Iyer, V. G. & Im, W. CHARMM-GUI: A web-based graphical user interface for CHARMM. Journal of Computational Chemistry 29, 1859-1865 (2008).

56. Abraham, M. J. et al. GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX 1-2, 19-25 (2015).

57. Huang, J. et al. CHARMM36m: an improved force field for folded and intrinsically disordered proteins. Nature Methods 14, 71-73 (2017).

58. Bussi, G., Donadio, D. & Parrinello, M. Canonical sampling through velocity rescaling. The Journal of chemical physics 126, 014101 (2007).

59. Parrinello, M. & Rahman, A. Polymorphic transitions in single crystals: A new molecular dynamics method. Journal of Applied Physics 52, 7182-7190 (1981).

60. Darden, T., York, D. & Pedersen, L. Particle mesh Ewald: An N·log(N) method for Ewald sums in large systems. The Journal of chemical physics 98, 10089-10092 (1993).

61. Hess, B., Bekker, H., Berendsen, H. J. C. & Fraaije, J. G. E. M. LINCS: A linear constraint solver for molecular simulations. Journal of Computational Chemistry 18, 1463-1472 (1997).

62. Michaud-Agrawal, N., Denning, E. J., Woolf, T. B. & Beckstein, O. MDAnalysis: A toolkit for the analysis of molecular dynamics simulations. Journal of Computational Chemistry 32, 2319-2327 (2011).

Metrics

Favorites: 6
Views: 10438
Downloads: 2666

DOI:

https://doi.org/10.65215/pwqkq726

Submission ID:

1

Downloads

Additional Files

Supplemental File(s)

Posted

2025-12-04

Versions

How to Cite

FAN, X., Chen, J., Xue, L., Wang, H., Wu, T., Huang, X., Lu, F., Jin, X., Song, C., Huang, J., & Yan, N. (2025). Open-state structure of veratridine-activated human Nav1.7 reveals the molecular choreography of fast inactivation. LangTaoSha Preprint Server. https://doi.org/10.65215/pwqkq726

Declaration of Competing Interests

The authors declare no competing interests to disclose.

Copyright

The copyright holder for this preprint is the author/funder.

All rights reserved. This work is protected by copyright. No part of this work may be reproduced, distributed, or transmitted in any form or by any means without the prior written permission of the copyright holder.