A single S6III mutation uncouples the dual gating effects of Pc1a on Nav1.7
Abstract
Voltage-gated sodium (Nav) channels couple membrane depolarization to pore opening and fast inactivation. The bullet ant-derived δ-paraponeritoxin-Pc1a (Pc1a) is an unusual membrane-spanning Nav agonist that facilitates activation while suppressing fast inactivation. It remains unclear if and how these two effects are coupled. We previously identified a Pc1a-binding site between voltage-sensing domain in repeat II (VSDII) and pore domain III (PDIII) in Nav1.6 and found that mutation of an S6III residue could restore fast inactivation without abolishing the toxin-induced shift of activation. Here we use human Nav1.7 as a structurally tractable model to define this mechanism. A cryo-EM structure of Pc1a-bound Nav1.7 complex reveals a membrane-spanning binding pose nearly identical to that observed in Nav1.6. In the sensitized Nav1.7-M3 mutant (N1420K/Y1429D/S1430N), vetratridine (VTD) dose-dependently suppresses Pc1a-induced persistent current. Ensuing mutational analysis pinpointed residues that bidirectionally tune Pc1a-induced persistent current. We focused on L1449A in S6III, which nearly abolishes Pc1a-induced persistent current while preserving the hyperpolarizing shift of activation. In the absence of toxin, L1449A shifts activation toward more depolarized voltages and both fast and slow inactivation toward more hyperpolarized voltages, indicating a redistribution of channel gating toward inactivation-prone states. Together, these results separate the activation- and inactivation-modifying actions of Pc1a and identify Leu1449S6III,29 as a conserved determinant coupling Pc1a binding to fast inactivation.
References
1. M. de Lera Ruiz, R. L. Kraus, Voltage-Gated Sodium Channels: Structure, Function, Pharmacology, and Clinical Indications. J Med Chem 58, 7093–7118 (2015).
2. J. Huang, X. Pan, N. Yan, Structural biology and molecular pharmacology of voltage-gated ion channels. Nat Rev Mol Cell Biol 25, 904–925 (2024).
3. A. L. Hodgkin, A. F. Huxley, Resting and action potentials in single nerve fibres. J Physiol 104, 176–195 (1945).
4. A. L. Hodgkin, A. F. Huxley, A quantitative description of membrane current and its application to conduction and excitation in nerve. J Physiol 117, 500–544 (1952).
5. X. Pan et al., Structure of the human voltage-gated sodium channel Na(v)1.4 in complex with beta1. Science 362 (2018).
6. Z. Yan et al., Structure of the Na(v)1.4-beta1 Complex from Electric Eel. Cell 170, 470–482 e411 (2017).
7. X. Fan et al., Open-state structure of veratridine-activated human Nav1.7 reveals the molecular choreography of fast inactivation. Vita 1, 19–31 (2026).
8. H. Shen et al., Structural basis for the modulation of voltage-gated sodium channels by animal toxins. Science 362 (2018).
9. H. Shen, D. Liu, K. Wu, J. Lei, N. Yan, Structures of human Na(v)1.7 channel in complex with auxiliary subunits and animal toxins. Science 363, 1303–1308 (2019).
10. G. Huang et al., High-resolution structures of human Na(v)1.7 reveal gating modulation through alpha-pi helical transition of S6(IV). Cell Rep 39, 110735 (2022).
11. T. Piek et al., Poneratoxin, a novel peptide neurotoxin from the venom of the ant, Paraponera clavata. Comp Biochem Physiol C Comp Pharmacol Toxicol 99, 487–495 (1991).
12. A. Duval, C. O. Malecot, M. Pelhate, T. Piek, Poneratoxin, a new toxin from an ant venom, reveals an interconversion between two gating modes of the Na channels in frog skeletal muscle fibres. Pflugers Arch 420, 239–247 (1992).
13. S. D. Robinson et al., Ant venoms contain vertebrate-selective pain-causing sodium channel toxins. Nat Commun 14, 2977 (2023).
14. S. R. Johnson, H. G. Rikli, J. O. Schmidt, M. S. Evans, A reexamination of poneratoxin from the venom of the bullet ant Paraponera clavata. Peptides 98, 51–62 (2017).
15. X. Fan et al., Diverse binding poses of agonistic neurotoxins on human Na(v)1.6. Nature 656, 250–259 (2026).
16. X. Jin, J. Huang, H. Wang, K. Wang, N. Yan, A versatile residue numbering scheme for Na(v) and Ca(v) channels. Cell Chem Biol 31, 1394–1404 (2024).
17. Y. Liu, J. D. Galpin, C. A. Ahern, F. Bezanilla, Molecular basis of sodium channel inactivation. Nat Commun 16, 10565 (2025).
18. J. Huang et al., Cannabidiol inhibits Na(v) channels through two distinct binding sites. Nat Commun 14, 3613 (2023).
19. Q. Wu et al., Structural mapping of Na(v)1.7 antagonists. Nat Commun 14, 3224 (2023).
20. Z. Li et al., Structure of human Nav1.5 reveals the fast inactivation-related segments as a mutational hotspot for the long QT syndrome. Proc Natl Acad Sci U S A 118 (2021).
21. A. Goehring et al., Screening and large-scale expression of membrane proteins in mammalian cells for structural studies. Nat Protoc 9, 2574–2585 (2014).
22. T. Matsuda, C. L. Cepko, Electroporation and RNA interference in the rodent retina in vivo and in vitro. Proc Natl Acad Sci U S A 101, 16–22 (2004).
23. J. Song, C. Zhang, Y. Fu, H. Wang, N. Liu, The application of graphene grids in Cryo-EM sample preparation. Journal of Chinese Electron Microscopy Society 44, 106–114 (2025).
24. J. Lei, J. Frank, Automated acquisition of cryo-electron micrographs for single particle reconstruction on an FEI Tecnai electron microscope. J Struct Biol 150, 69–80 (2005).
25. T. Grant, N. Grigorieff, Measuring the optimal exposure for single particle cryo-EM using a 2.6 A reconstruction of rotavirus VP6. Elife 4, e06980 (2015).
26. K. Zhang, Gctf: Real-time CTF determination and correction. J Struct Biol 193, 1–12 (2016).
27. A. Punjani, J. L. Rubinstein, D. J. Fleet, M. A. Brubaker, cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat Methods 14, 290–296 (2017).
28. P. B. Rosenthal, R. Henderson, Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. J Mol Biol 333, 721–745 (2003).
29. S. Chen 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).
30. E. F. Pettersen et al., UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem 25, 1605–1612 (2004).
31. P. Emsley, B. Lohkamp, W. G. Scott, K. Cowtan, Features and development of Coot. Acta Crystallogr D Biol Crystallogr 66, 486–501 (2010).
32. P. D. Adams et al., PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr 66, 213–221 (2010).
33. A. Amunts et al., Structure of the yeast mitochondrial large ribosomal subunit. Science (New York, N.Y 343, 1485–1489 (2014).
34. Z. Li 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).
35. X. Huang 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).
36. X. Fan, J. Huang, X. Jin, N. Yan, Cryo-EM structure of human voltage-gated sodium channel Na(v)1.6. Proc Natl Acad Sci U S A 120, e2220578120 (2023).
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