Cryo-EM (electron cryo-microscopy) structures of human NaV1.6 published in 2023 (apo at 3.4 Å and bound to the guanidinium neurotoxin 4,9-anhydro-tetrodotoxin, 4,9-ah-TTX, at 3.3 Å) left two questions about the pore unresolved: the hydration states of the putative Na+ binding sites in the selectivity filter, which underlie the detailed mechanism of Na+ selectivity and conductance, and the protonation state of the 4,9-ah-TTX guanidinium group in its binding pocket, where the cryo-EM pose and the initial molecular-dynamics pose disagree unless that guanidine nitrogen is left unprotonated (the usual monocationic guanidinium, +1). No deposited NaV1.6 structure better than 2.9 Å resolves the selectivity-filter waters or the toxin protonation state, so both questions remain open. Determining this ion- and ligand-coordination chemistry would complete the mechanistic picture of Na+ conductance and guanidinium-toxin blockade at the mammalian sodium-channel pore.

Background

Voltage-gated sodium channels (NaV) initiate and propagate action potentials in excitable cells. The human subtype NaV1.6, encoded by the SCN8A gene, is concentrated at the axon initial segment and in Purkinje neurons, and its dysfunction causes epileptic encephalopathy, intellectual disability, and movement disorders. Eukaryotic NaV channels are large membrane proteins with four homologous repeats forming the ion-conducting pore; the narrowest part, the selectivity filter, is built from the signature DEKA motif (one Asp, Glu, Lys, Ala from the four repeats) plus an outer acidic ring, and this asymmetric arrangement — unlike the symmetric filters of potassium channels — selects Na+ over other cations. Eukaryotic NaV channels are blocked from the extracellular side by guanidinium neurotoxins such as tetrodotoxin (TTX, pufferfish poison) and its analogue 4,9-anhydro-TTX (4,9-ah-TTX), which plug the pore directly above the selectivity filter. In 2023, Li and colleagues reported cryo-EM structures of human NaV1.6 (in complex with its auxiliary β1 and β2 subunits) alone and in complex with 4,9-ah-TTX (PDB 8GZ1 at 3.4 Å and 8GZ2 at 3.3 Å; DOI 10.1038/s41467-023-36766-9). The apo structure revealed three putative Na+ binding sites coordinated by selectivity-filter residues, suggesting a possible Na+ recognition and conductance mechanism, and the toxin-bound structure showed 4,9-ah-TTX occupying those sites in a pocket shared with TTX, with TTX being more potent, plausibly because it forms two additional hydrogen bonds with the channel. Two issues were explicitly left open in that paper's Discussion. First, at 3.3–3.4 Å resolution, ordered water molecules cannot be placed, so the hydration states of the putative Na+ sites — whether Na+ is fully or partially hydrated (partially dehydrated, water-bridged) at each site during permeation — could not be determined; distinguishing a Na+ ion from a water oxygen is in any case difficult because they scatter a similar number of electrons. Since hydration underlies the debated permeation mechanism (knock-on between hydrated ions versus stepwise partial dehydration), the authors state that a future higher-resolution NaV1.6 structure is required to investigate the more detailed mechanism of Na+ conductance. Second, the molecular-dynamics (MD) study of the bound toxin was run assuming that the guanidine nitrogen of 4,9-ah-TTX carries an extra proton (NH2+, i.e. the toxin modeled as doubly protonated, net +2, rather than in its usual monocationic guanidinium state, net +1), but in the EM structure that NH sits only about 3 Å from Y371, an electrostatically disfavored contact for the doubly protonated state; repeating the MD with that nitrogen unprotonated (the usual +1 toxin) reproduced the EM binding pose. The protonation state of the bound toxin, and how it shapes the binding pose and affinity, therefore remained unsettled and, in the authors' assessment, requires further systematic investigation. A contemporaneous independent NaV1.6 structure (Fan et al., PNAS 2023, 3.1 Å, DOI 10.1073/pnas.2220578120) did not resolve either issue, and as of August 2026 no NaV1.6 structure better than 2.9 Å (an unpublished gating-modifier-toxin complex, PDB 9DK5) has been deposited — a resolution at which neither selectivity-filter waters nor ion-versus-water discrimination is possible. Recent MD work on the homologous cardiac channel NaV1.5 (Anguita-Ortiz and Nogueira, 2025, DOI 10.1021/acs.biochem.4c00664) explicitly concludes that current force fields misdescribe the ion/protein/solvation energy balance governing Na+ versus K+ selectivity, so computation alone has not settled the hydration question either, and no systematic study of TTX-family protonation states in the channel pore has appeared.

Problem Statement

Determine the ion- and ligand-coordination chemistry of the human NaV1.6 pore at the selectivity filter, as left unresolved by Li et al. (2023), namely: (a) the hydration states of the putative Na+ binding sites in the NaV1.6 selectivity filter — which sites bind fully versus partially hydrated Na+ during permeation — and the resulting detailed mechanism of Na+ selectivity and conductance; and (b) the protonation state of the guanidinium group of 4,9-anhydro-tetrodotoxin in its NaV1.6 binding pocket, and how that protonation state shapes the toxin's binding pose and affinity.

References

  1. Li, Yue; Yuan, Tian; Huang, Bo; Zhou, Feng; Peng, Chao; Li, Xiaojing; Qiu, Yunlong; Yang, Bei; Zhao, Yan G.; Huang, Zhuo; Jiang, D. 'Structure of human NaV1.6 channel reveals Na+ selectivity and pore blockade by 4,9-anhydro-tetrodotoxin', Nature Communications 14, 1030 (2023). doi:10.1038/s41467-023-36766-9, https://www.nature.com/articles/s41467-023-36766-9 (source paper; full text at PMC9950489)
  2. Fan, Xiao; Huang, Jian; Jin, Xueqin; Yan, Nieng. 'Cryo-EM structure of human voltage-gated sodium channel NaV1.6', Proceedings of the National Academy of Sciences USA 120(5), e2220578120 (2023). doi:10.1073/pnas.2220578120, https://doi.org/10.1073/pnas.2220578120 (PDB 8FHD, 3.1 Å)
  3. Fan, Xiao; Huang, Jian; Yang, Lin; Chen, Jiaofeng; Wang, Huan; Huang, Xiaoshuang; Geng, Jinli; Wu, Qinglin; Xie, Yuzhen; Lu, Fangzhou; Guo, Qinmeng; Shen, Zilin; Jin, Xueqin; Yan, Nieng. 'Diverse binding poses of agonistic neurotoxins on human Nav1.6', Nature 656, 250-259 (2026). doi:10.1038/s41586-026-10661-x, https://www.nature.com/articles/s41586-026-10661-x
  4. Wu, T.; Yang, X.; Jin, X.; Yan, N.; Li, Z. 'Critical role of extracellular loops in differential modulations of TTX-sensitive and TTX-resistant Nav channels', Proceedings of the National Academy of Sciences USA 122(32), e2510355122 (2025). doi:10.1073/pnas.2510355122, https://pmc.ncbi.nlm.nih.gov/articles/PMC12358880/ (PMID 40768348)
  5. Anguita-Ortiz, N.; Nogueira, J. J. 'Role of Hydration and Amino Acid Interactions on the Ion Permeation Mechanism in the hNaV1.5 Channel', Biochemistry 64(1), 47-56 (2025). doi:10.1021/acs.biochem.4c00664, https://doi.org/10.1021/acs.biochem.4c00664
  6. Maeda, Y. K.; Kojima, K.; Nakamura, T. Y.; Nakatsu, T.; Irie, K. 'Structure-function analysis of the lithium-ion selectivity of the voltage-gated sodium channel', Journal of General Physiology 158(3) (2026). doi:10.1085/jgp.202513855, https://doi.org/10.1085/jgp.202513855
  7. Chen, Huiwen; Xia, Zhanyi; Dong, Jie; Huang, Bo; Zhang, Jiangtao; Zhou, Feng; Yan, Rui; Shi, Yiqiang; Gong, Jianke; Jiang, Juquan; Huang, Zhuo; Jiang, Daohua. 'Structural mechanism of voltage-gated sodium channel slow inactivation', Nature Communications 15, 3691 (2024). doi:10.1038/s41467-024-48125-3, https://www.nature.com/articles/s41467-024-48125-3 (bacterial NaVEh channel, not NaV1.6)
  8. Wang, H.; Wei, Y.; Wang, J.; Liu, J.; Ou, S.; Wang, J. 'Structure and function of voltage-gated sodium channel Nav1.6: Involvement in the pathological process of neural injury', Neural Regeneration Research 21(8), 3352-3362 (2026). doi:10.4103/NRR.NRR-D-25-00354, https://doi.org/10.4103/NRR.NRR-D-25-00354
  9. PDB entry 9DK5 (Fan, X. et al., to be published). 'Cryo-EM structure of human Nav1.6 in complex with PaurTx3', 2.9 Å. doi:10.2210/pdb9DK5/pdb, https://doi.org/10.2210/pdb9DK5/pdb