Structure of human NaV1.6 channel reveals Na+ selectivity and pore blockade by 4,9-anhydro-tetrodotoxin

Li, Yue; Yuan, Tian; Huang, Bo; Zhou, Feng; Peng, Chao; Li, Xiaojing; Qiu, Yunlong; Yang, Bei; Zhao, Yan; Huang, Zhuo; Jiang, Daohua

doi:10.1038/s41467-023-36766-9

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Published: 23 February 2023

Structure of human Na_V1.6 channel reveals Na⁺ selectivity and pore blockade by 4,9-anhydro-tetrodotoxin

Yue Li ORCID: orcid.org/0000-0001-6387-6032^1,2,3^na1,
Tian Yuan^4,5^na1,
Bo Huang ORCID: orcid.org/0000-0003-3822-9110⁶^na1,
Feng Zhou⁶,
Chao Peng^4,5,
Xiaojing Li^1,3,
Yunlong Qiu^1,3,
Bei Yang¹,
Yan Zhao ORCID: orcid.org/0000-0002-4348-256X^1,3,
Zhuo Huang ORCID: orcid.org/0000-0001-9198-4778^4,5 &
…
Daohua Jiang ORCID: orcid.org/0000-0002-4397-5807^2,3

Nature Communications volume 14, Article number: 1030 (2023) Cite this article

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Abstract

The sodium channel Na_V1.6 is widely expressed in neurons of the central and peripheral nervous systems, which plays a critical role in regulating neuronal excitability. Dysfunction of Na_V1.6 has been linked to epileptic encephalopathy, intellectual disability and movement disorders. Here we present cryo-EM structures of human Na_V1.6/β1/β2 alone and complexed with a guanidinium neurotoxin 4,9-anhydro-tetrodotoxin (4,9-ah-TTX), revealing molecular mechanism of Na_V1.6 inhibition by the blocker. The apo-form structure reveals two potential Na⁺ binding sites within the selectivity filter, suggesting a possible mechanism for Na⁺ selectivity and conductance. In the 4,9-ah-TTX bound structure, 4,9-ah-TTX binds to a pocket similar to the tetrodotoxin (TTX) binding site, which occupies the Na⁺ binding sites and completely blocks the channel. Molecular dynamics simulation results show that subtle conformational differences in the selectivity filter affect the affinity of TTX analogues. Taken together, our results provide important insights into Na_V1.6 structure, ion conductance, and inhibition.

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Introduction

Voltage-gated sodium (Na_V) channels mediate the generation and propagation of action potentials in excitable cells^1,2. In humans, nine Na_V channel subtypes (Na_V1.1–1.9) had been identified, which are involved in a broad range of physiological processes due to their tissue-specific distributions in various excitable tissues^3,4. Subtype Na_V1.6, encoded by the gene SCN8A, is ubiquitously expressed in neurons of both the central nervous system (CNS) and the peripheral nervous system (PNS), especially enriched in the distal end of axon initial segment (AIS) and in the node of Ranvier of myelinated excitatory neurons. The Na_V1.6 channel is believed to play a primary role in the initiation and propagation of action potentials in those neurons by lowering the threshold voltage^{5,6,7,8,9,10,11}. Emerging evidence suggests that Na_V1.6 is also expressed in some inhibitory interneurons and plays a role in establishing synaptic inhibition in the thalamic networks^12,13,14. Compared with other Na_V channel subtypes, Na_V1.6 possesses unique biophysical properties including activation at more hyperpolarized voltage, higher levels of persistent current and resurgent current, and higher frequency of repetitive neuronal firing in neurons such as cerebellar Purkinje cells^{15,16,17,18,19,20,21,22,23}. These features make Na_V1.6 a critical and favorable mediator in regulating neuronal excitability in those neurons. Meanwhile, dozens of mutations in Na_V1.6 have been linked to human diseases, most of which exhibit gain-of-function phenotypes, increase neuronal excitability, and cause different types of epileptic encephalopathy^{24,25,26,27,28}; whereas loss-of-function mutations are often associated with later onset seizures, intellectual disability, isolated cognitive impairment and movement disorders^29,30,31. Thus, Na_V1.6 is an important drug target; effective and subtype-selective therapeutics are eagerly awaited for the treatment of Na_V1.6-related epilepsy and other neurological diseases.

Eukaryotic Na_V channels are composed of a pore-forming α subunit and auxiliary β subunits³². The four-domain α subunit exerts voltage sensing, gate opening, ion permeation, and inactivation^4,33. Meanwhile, one or two β subunits bind to the α subunit to regulate Na_V channel kinetics and trafficking. Among the four types of β subunits^34,35,36,37, β1 and β3 subunits non-covalently bind to the α subunit, while β2 and β4 subunits are covalently linked to the α subunit via a disulfide bond^32,38. To date, high-resolution cryo-electron microscopy (cryo-EM) structures of seven mammalian Na_V channels (Na_V1.1–1.5, Na_V1.7–1.8) have been reported^{39,40,41,42,43,44,45}. Together with the resting-state⁴⁶, open-state⁴⁷, and multiple ligand-bound Na_V channel structures^48,49,50, these structures revealed the general molecular mechanisms of voltage-sensing, electromechanically coupling, fast inactivation, sodium permeation, and ligand modulation. Among those Na_V channel modulators, the guanidinium neurotoxin tetrodotoxin (TTX) has long been used as a useful tool to study Na_V channels, which can potently inhibit Na_V1.1–1.4 and Na_V1.6–1.7 at nanomolar level (TTX-sensitive Na_V channels), and less potently inhibit Na_V1.5, Na_V1.8, and Na_V1.9 at a micromolar concentration (TTX-insensitive Na_V channels). The detailed binding mode of TTX had been revealed in the Na_V channel-TTX complex structures^44,51. Furthermore, two guanidinium neurotoxin derivatives, ST-2262 and ST-2530, were reported as potent and selective inhibitors for Na_V1.7, indicating that TTX analogs could potentially be developed as selective therapeutics^52,53. Interestingly, 4,9-anhydro-tetrodotoxin (4,9-ah-TTX), a metabolite of TTX, has been reported to selectively block Na_V1.6 with a blocking efficacy of 40- to 160-fold higher than other TTX-sensitive Na_V channels⁵⁴. However, the structure of Na_V1.6 and how 4,9-ah-TTX blocks Na_V1.6 remain elusive.

In this work, we show a fully-functional shorter-form construct of human Na_V1.6 suitable for structural studies, and present cryo-EM structures of Na_V1.6/β1/β2 apo-form and in complex with 4,9-ah-TTX. Complemented with electrophysiological results and molecular dynamics (MD) simulations, our structures reveal Na_V1.6 structural features, sodium conductance, and pore-blockade by 4,9-ah-TTX.

Results

Construct optimization of Na_V1.6 for cryo-EM study

To conduct structural studies of Na_V1.6, human wide-type Na_V1.6 (named Na_V1.6^WT) was co-expressed with human β1 and β2 subunits in HEK293F cells and was purified similarly to previously reported Na_V channels^41,44. Although the amino acid sequence of Na_V1.6 is highly conserved with other Na_V channel subtypes (e.g., 70% identity with Na_V1.7); however, the purified Na_V1.6^WT sample exhibited poor quality and did not permit high-resolution structural analysis (Supplementary Fig. 1a, b). Construct optimization had been proven to be successful in improving the sample quality of Na_V1.7 and Na_V1.5^55,56, we therefore carried out construct screening of human Na_V1.6 by removing unstructured intracellular loops and C-terminus. We found that deletion of S478-G692 between D_I and D_II (Na_V1.6^ΔDI-DII), S1115-L1180 between D_II and D_III (Na_V1.6^ΔDII-DIII), or R1932-C1980 of the C-terminus (Na_V1.6^ΔCter) showed improved sample homogeneity based on the size-exclusion chromatography (SEC) profiles (Supplementary Fig. 1a). Strikingly, when we combined these modifications and deleted all of the three unstructured regions, it displayed a sharp mono-disperse SEC profile, which is much better than that of Na_V1.6^WT and any of the single-deletion constructs (Fig. 1a, b, Supplementary Fig. 1a). We next examined the functional characteristics of the triple-deletion construct by whole-cell voltage-clamp recording of Na_V1.6-expressing HEK293T cells. The candidate construct exhibits typical voltage-dependent activation and inactivation (Fig. 1c). The resulting V_1/2 values of the voltage-dependence of activation and steady-state fast inactivation are −31.3 ± 0.3 mV (n = 15) and −77.3 ± 0.2 mV (n = 15), respectively, which are close to the reported V_1/2 values of human wide-type Na_V1.6^57,58. These results confirmed that the triple-deletion construct fulfills similar electrophysiological functions to the Na_V1.6^WT. The preliminary cryo-EM analysis of this triple-deletion construct showed that the micrograph contains a rich distribution of mono-disperse particles, which gave rise to much better 2D class averages with well-resolved features than the Na_V1.6^WT (Supplementary Fig. 1b, c). Thus, this triple-deletion construct (named Na_V1.6^EM) was selected for further structural studies.

**Fig. 1: Topology and functional characterization of the Na_V1.6^EM/β1/β2 complex.**

The overall structure of human Na_V1.6

The purified Na_V1.6^EM/β1/β2 sample was frozen in vitreous ice for cryo-EM data collection (Supplementary Fig. 2). After processing, the final reconstruction map from the best class of ~41 k particles was refined to an overall resolution of 3.4 Å (Fig. 2a, Supplementary Figs. 3–5). As expected, the resulting Na_V1.6^EM/β1/β2 structure closely resembles the reported structures of human Na_V channels due to high sequence similarity (Fig. 2b). For example, the binding modes of the β subunits are consistent with the structures of human Na_V1.7/β1/β2 and Na_V1.3/β1/β2^41,44; the pore-forming α-subunit of Na_V1.6^EM can be well superimposed with Na_V1.7 with a backbone (1107 Cα) root mean square deviation (RMSD) of 1.4 Å (Fig. 2c). However, marked local conformational differences were observed between the two structures, especially in the extracellular loops (ECLs) (Fig. 2c, d). The ECLs are less conserved regions among the nine Na_V channel subtypes (Supplementary Fig. 6a), which form the outer mouth of the selectivity filters (SFs) and contribute to the binding of β subunits. Superposition of the Domain I ECLs of Na_V1.6^EM and Na_V1.7 shows that the ECL_I of Na_V1.6^EM lacks the short α2 helix, but instead forms an extended hairpin-like turn (Fig. 2d). Importantly, the ECL_I of Na_V1.6^EM exhibits more N-linked glycosylation modification sites than Na_V1.7; N308-linked glycosylation site appears to be unique for Na_V1.6 based on the sequence alignment (Supplementary Fig. 6a). Although these structural differences in the ECLs do not affect the binding of β subunits to Na_V1.6 (Fig. 2a), the glycosylation and other modifications shape the surface properties of Na_V1.6, which play important roles in its trafficking, localization, and pathology^59,60. For instance, a unique glycosylation site in the ECL_I of Na_V1.5 blocks the binding of the β1 subunit to Na_V1.5⁴³.

**Fig. 2: Cryo-EM structure of the Na_V1.6^EM/β1/β2 complex.**

We next compared the fast inactivation gate and intracellular activation gate between Na_V1.6^EM and Na_V1.7, which only display subtle conformational shifts (Fig. 2e, f), indicating that those key structural elements are highly conserved to fulfill their similar biological roles. Consistently, the signature fast inactivation gate, Ile-Phe-Met motif (IFM-motif), binds tightly to its receptor site adjacent to the intracellular activation gate (Fig. 2e), resulting in a non-conductive activation gate constricted by A411, L977, I1464, and I1765 from the four S6 helices, respectively (Fig. 2f). The van der Waals diameter of the activation gate is less than 6 Å, suggesting that the gate is functionally closed (Fig. 3a, b).

**Fig. 3: Potential Na⁺ binding sites in the SF of Na_V1.6^EM.**

Potential Na⁺ sites in the SF

The ion path of Na_V1.6 has two constriction sites, the extracellular SF and intracellular activation gate, respectively (Fig. 3a, b). The sodium selectivity of mammalian Na_V channels is determined by the extracellular SF^61,62, which is composed of an Asp from D_I, Glu from D_II, Lys from D_III, and Ala from D_IV, known as the DEKA-locus^63,64. Based on structural analysis, the acidic residues of the DEKA-locus are believed to act as a high-field strength site, which attracts and coordinates Na⁺; and the Lys in D_III was proposed as a favorable binding ligand for Na⁺ which facilitates the ions passing through the SF^43,65. In coincidence with other mammalian Na_V channels^43,44, the SF of Na_V1.6^EM adopts an asymmetric conformation composed of the DEKA-locus (Fig. 3b, c). No oblivious Na⁺ binding site had been identified in previous structures of mammalian Na_V channels. In contrast, densities for Ca²⁺ were consistently reported in the structures of bacterial Ca_VAb channel and mammalian Ca_V1.1, Ca_V2.2, and Ca_V3.1 channels^66,67,68,69. Interestingly, two strong blobs of EM densities were observed in the SF of Na_V1.6^EM (Fig. 3d, e), which are deduced as potential Na⁺ binding sites because Na⁺ ions are the only major cations in the solutions throughout the purification processes. The upper site (namely Na1) closely engages E936 of the DEKA-locus and an additional acidic residue E939 (Fig. 3c). The distances of this Na1 to the E936 and E939 are at ~3.5 Å, suggesting that Na⁺ in Na1 site may still be hydrated. Meanwhile, D370 of the DEKA-locus contributes minorly to this Na⁺ binding site at a distance of ~7.5 Å (Fig. 3c). This observation is in line with previous studies showing that E936/K1413 of the DEKA-locus are the most prominent residues for Na⁺ permeation and selectivity, while D370 of the DEKA-locus is not absolutely required⁶³. This potential Na1 site may represent the first step for Na⁺ conductance, that is, E936 of the DEKA-locus attracts and captures one hydrated Na⁺ from the extracellular solution with the assistance of E939. The second blob of density is located inside the SF, namely the Na2 site, which is about ~5.3 Å away from the Na1 site (Fig. 3d, e). Interestingly, the Na2 is close to the short side-chain residue A1705 of the DEKA-locus and is coordinated with the strictly conserved E373 at a distance of ~3.3 Å (Fig. 3c, d). We also noticed that D370/E936 of the DEKA-locus contribute negligibly to the Na2 at distances of 5.6–6.6 Å (Fig. 3c). Thus, we hypothesize that the Na2 may represent the second step for sodium conductance, that is, after captured and partially dehydrated in Na1 site, at least partially-dehydrated Na⁺ can fit into the Na2 site which is going to enter the narrowest asymmetric constriction site of the SF. The possible partial dehydration of Na⁺ in the Na2 site is reflected by its relatively weaker density compared to the Na1 (Fig. 3d, e). Furthermore, the K1413 points its long side-chain deep into the SF, forming the narrowest part of the SF. It has been proposed that this residue serves as a key coordination ligand in favor of Na⁺ or Li⁺ but is unfavorable for other cations⁴³. In line with this hypothesis, Na⁺ from the Na2 site can quickly pass through the SF and enter the central cavity accelerated by the amino group of the K1413. We found additional elongated density below the K1413 at a distance of ~3.5 Å, which may represent a third Na⁺ site (namely Na3) (Fig. 3d, e). Consistently, previous MD simulations studies suggested that two Na⁺ ions spontaneously occupy the symmetric SF of the bacterial Na_V channels, and three Na⁺ sites were proposed in the asymmetric SF of the eukaryotic Na_V channel^70,71,72, which are similar to the Na2, Na3 sites and Na1-3 sites of our Na_V1.6 structure, respectively.

In Ca_V channels, the Ca²⁺ binding sites were revealed in the SFs^66,67,68,73, suggesting a possible step-wise “knock-off” mechanism for Ca²⁺ conducntance⁶⁶. Superposition of the SFs of the Na_V1.6^EM and the Ca_VAb shows that the Na1 and Na2 sites are roughly at the same height levels as Ca1 and Ca2 sites in Ca_VAb, respectively (Fig. 3d, e). However, the two Na⁺ sites are off the central axis of the SF, while the Ca²⁺ sites are in the center (Supplementary Fig. 7). This difference is in agreement with the asymmetric characteristics of the SFs of mammalian Na_V channels. As shown in the Na_V1.6^EM structure, similar to the Ca_V channels, two or more potential Na⁺ sites exist in the SFs of Na_V channels. In fact, the SFs of Na_V and Ca_V channels are closely related, point-mutations in the SF of the Na_V channel can convert it into a highly Ca²⁺ favorable channel^66,74. Nevertheless, these subtle compositional and conformational differences at the SFs determine the ion selectivity and conductance.

Blockade of Na_V1.6 by 4,9-ah-TTX

The guanidinium neurotoxin TTX and its derivatives can potently inhibit eukaryotic Na_V channels⁷⁵. TTX was reported to be more potent in inhibiting Na_V1.6 than other TTX-sensitive Na_V channels⁷⁶. Interestingly, one of the TTX metabolites, 4,9-ah-TTX, has been reported to preferentially block Na_V1.6 over the other eight Na_V channel subtypes⁵⁴. We first examined the TTX sensitivity of Na_V1.6^EM, Na_V1.2, and Na_V1.7, yielding IC₅₀ values of 1.9 nM (n = 5), 4.9 nM (n = 5), and 16.7 nM (n = 4), respectively. Consistent with previous reports, TTX indeed favors Na_V1.6 (Supplementary Fig. 6c). Then we tested the inhibitory effects of 4,9-ah-TTX on Na_V1.2, Na_V1.7, and Na_V1.6^EM. As illustrated in Fig. 4a–c, 4,9-ah-TTX gradually inhibits both Na_V1.7 and Na_V1.6^EM in a concentration-dependent manner. However, the resulting IC₅₀ values of 4,9-ah-TTX are significantly different, which are 257.9 nM (n = 6) for Na_V1.2, 1340 nM (n = 6) for Na_V1.7 and 52.0 nM (n = 5) for Na_V1.6^EM, respectively (Fig. 4c). Those results confirmed that the potency of 4,9-ah-TTX is ~27-fold weaker than TTX in inhibiting Na_V1.6^EM, and 4,9-ah-TTX is indeed a Na_V1.6 preferred blocker.

**Fig. 4: Blockade of the Na_V1.6^EM by 4,9-ah-TTX.**

To better understand the underlying mechanism of Na_V1.6 modulation by 4,9-ah-TTX, we solved the cryo-EM structure of Na_V1.6^EM/β1/β2 in complex with 4,9-ah-TTX (named Na_V1.6^4,9-ahTTX) at a resolution of 3.3 Å (Supplementary Fig. 4). The overall structure of Na_V1.6^4,9-ahTTX is indistinguishable to the Na_V1.6^EM (RMSD at 0.2 Å). However, unambiguous EM density located above the SF of Na_V1.6^4,9-ahTTX was observed, which fits a 4,9-ah-TTX molecule very well (Fig. 4d, e, Supplementary Fig. 4b). A closer look shows that the 4,9-ah-TTX occupies the Na⁺ binding sites and sticks into the SF of Na_V1.6 via extensive interactions (Fig. 4f). D370 and E373 from D_I, E936, and E939 from D_II, and D1708 from D_IV form electrostatic interactions with the 4,9-ah-TTX, Y371, and K1413 also contribute to stabilizing the blocker by forming van der Waals interactions (Fig. 4f). Superposition of the Na_V1.6^4,9-ahTTX and the TTX bound Na_V1.7 (Na_V1.7^TTX) show a very similar binding mode for the two blockers (Fig. 4f–h). This similar binding mode is reasonable because the chemical structures of TTX and 4,9-ah-TTX are very similar; secondly, these key interacting residues are identical among the TTX-sensitive Na_V channels (Supplementary Fig. 6b). However, subtle conformational differences were observed. The 4,9-ah-TTX binds ~1.4 Å deeper in the pocket of Na_V1.6 than TTX in Na_V1.7 (Fig. 4h). In addition, the 4,9-ah-TTX lacks two hydroxyl groups at the 4 and 9 positions of TTX, which form two more hydrogen-bonds with E364 and G1407 of Na_V1.7, respectively (Fig. 4g). TTX should form the same interactions with Na_V1.6 as found in Na_V1.7. Thus, the binding of TTX to Na_V1.6 is stronger than the binding of 4,9-ah-TTX, which agrees with the higher potency of TTX in inhibiting Na_V1.6 than 4,9-ah-TTX (Fig. 4c and Supplementary Fig. 6c).

Then how does 4,9-ah-TTX preferentially inhibit Na_V1.6 over Na_V1.7 in a nearly identical pocket? By carefully checking the pore-loop sequences of Na_V1.6, we found that L1712 in the D_IV P-loop of Na_V1.6 is a major different residue in the P-loop regions not similar to other Na_V channels (Supplementary Fig. 6b). We tested the effect of 4,9-ah-TTX on L1712A mutant of Na_V1.6 (Na_V1.6^L1712A), the resulting IC₅₀ value of 4,9-ah-TTX for Na_V1.6^L1712A is 61.1 nM (n = 4), which is close to that of the Na_V1.6^EM (Supplementary Fig. 6d). This result suggests that L1712 is not relevant to the binding of 4,9-ah-TTX. To test whether the accessibility affects the binding of 4,9-ah-TTX, we substituted the ECL_I of Na_V1.6 (F273-F356) with that of Na_V1.7 (F267-F347) or the ECL_III (F1349-V1399) with that of Na_V1.7 (F1343-V1392), namely Na_V1.6^ECL1 and Na_V1.6^ECL3, respectively. Surprisingly, the substitution of the ECL_I dramatically drops the IC₅₀ values of the 4,9-ah-TTX and TTX by 149-fold and 86-fold, respectively; in contrast, ECL_III substitution only decreases the IC₅₀ values of the 4,9-ah-TTX and TTX by 2.6-fold and 1.1-fold, respectively (Supplementary Fig. 6d, e). These results show that the ECL substitutions especially ECL_I do affect the potency of TTX analogs, but do not discriminate them.

To further dissect the preferential inhibition of Na_V1.6 by 4,9-ah-TTX, we carried out MD simulations of TTX binding to Na_V1.6 or Na_V1.7, and 4,9-ah-TTX binding to Na_V1.6 or Na_V1.7. Six independent 100 ns MD simulations were performed for each complex and the trajectories were used for binding affinity calculations using the method of Molecular Mechanics with Generalized Born and Surface Area solvation (MM/GBSA)⁷⁷. The simulation results show that the binding affinity of TTX to Na_V1.6 is significantly higher than that of 4,9-ah-TTX to Na_V1.6, and the affinity of 4,9-ah-TTX to Na_V1.6 is greater than 4,9-ah-TTX to Na_V1.7 (Supplementary Fig. 8a). These MD binding affinity results fairly agree with our electrophysiological results (Fig. 4c, Supplementary Fig. 6c). The simulations also show that there is only one predominant conformation for 4,9-ah-TTX binding to Na_V1.6; while there are four major conformations for 4,9-ah-TTX binding to Na_V1.7 (Fig. 5a, Supplementary Fig. 8b–f). More specifically, E373, E936, and E939 mainly contributed to the binding of 4,9-ah-TTX to Na_V1.6, consistent with our structural observation (Fig. 4f, Supplementary Fig. 8f); however, E930 and E927 of Na_V1.7, the counterparts of E939 and E936 in Na_V1.6, appeared to be very dynamic and contributed less stably to the binding of 4,9-ah-TTX (Fig. 5b). A contact analysis (Supplementary Fig. 9) was conducted to provide more details to understand the dynamics of the ligands (Supplementary Fig. 10). Specifically, E930 and E927 in Na_V1.7 interact with 4,9-ah-TTX with a frequency ranging from 21% to 87% for the most populated conformation cluster, whereas the frequency is over 90% for the interactions between such ligand and E939 and E936 in Na_V1.6. Superposition of the two representative conformations provides us an assumption that R922 of P1_II helix is more flexible in Na_V1.7 than the equivalent R931 in Na_V1.6 because of the small side-chain T1409 on P2_III helix, which in turn increases the flexibility of E930 and E927 and thereby negatively affects the binding of 4,9-ah-TTX to Na_V1.7 (Fig. 5c). To validate this assumption, we tested the potency of 4,9-ah-TTX on Na_V1.6 with double-mutations of M1416T/D1417I (Na_V1.6^{M1416T/D1417I}) using whole-cell voltage-clamp recordings. The resulting IC₅₀ value is 257 nM (n = 5), which is 5-fold less potent than that of Na_V1.6^EM, in coincidence with the findings by MD simulations (Fig. 5d). Taken together, our results confirmed that TTX has the highest affinity to Na_V1.6 among the TTX-sensitive Na_V channels; the TTX analog 4,9-ah-TTX is less potent than TTX in inhibiting Na_V1.6, but does exhibit preferential inhibition of Na_V1.6 over Na_V1.7.

**Fig. 5: MD simulations of 4,9-ah-TTX binding to Na_V1.6 and Na_V1.7.**

Pathogenic mutation map of Na_V1.6

The Na_V1.6 channels are abundantly distributed in neurons of both the CNS and the PNS. Compared to other Na_V channel subtypes, the Na_V1.6 channel has unique properties including activation at more hyperpolarized potential and generating a large proportion of resurgent current and persistent current, which plays important roles in regulating neuronal excitability and repetitive firing^17,19. To date, at least 16 gain-of-function mutations in Na_V1.6 causing hyperactivity are linked to Developmental and Epileptic Encephalopathy (DEE)⁷⁸; meanwhile, 9 loss-of-function mutations in Na_V1.6 causing reduced neuronal excitability are associated with intellectual disability and movement disorders. We highlighted 14 gain-of-function and 7 loss-of-function mutations in our Na_V1.6^EM structure (Fig. 6). The 14 gain-of-function mutations are mainly distributed in the VSDs, fast inactivation gate, and activation gate. In particular, mutations G1475R, E1483K, M1492V, and A1650V/T target the fast inactivation gate, presumably causing overactivity of the Na_V1.6 variants by impairing the binding of the IFM-motif to its receptor site. Mutation N1768D, located at the end of the DIV-S6 helix, was reported to generate elevated persistent current and resurgent current^24,79, which may cause improper gate closing to generate these aberrant currents. Meanwhile, two loss-of-function variants, G964R and E1218K cause intellectual disability without seizure³⁰. G964 is located in the middle of S6_II, which is believed to serve as a hinge in the pore-lining S6 helix during gating⁸⁰. A G964R mutation can certainly impair the flexibility of the S6_II helix; in addition, the additional long side-chain of the mutant can cause clashes with neighboring residues. E1218 belongs to the extracellular negatively-charged clusters (ENCs) of VSD_III, which play an important role in interacting with the positively-charged gating-charges. The E1218K mutation provides an opposite charge which can disrupt the voltage sensing. This mutant may also destabilize the variant, reflected by its significantly reduced express level³⁰.

**Fig. 6: Mapping the pathogenic mutations on the Na_V1.6^EM.**

Discussion

In this study, we presented cryo-EM structures of human Na_V1.6/β1/β2 apo-form and complexed with the Na_V1.6 preferred blocker 4,9-ah-TTX. To facilitate the structural studies, we obtained the core construct of Na_V1.6^EM which displayed improved sample quality. This construct and the structures can be a useful tool for future Na_V1.6-related structural and biochemical studies. The apo-form Na_V1.6 structure reveals three potential Na⁺ sites, which are coordinated by the important residues in the SF, suggesting a possible mechanism for Na⁺ recognition, selection, and conductance. By comparison with the Ca²⁺ sites in the bacterial and mammalian Ca_V channels^66,67,68,69, the unique asymmetric SF of mammalian Na_V channels provides a precise tunnel to separate Na⁺ from other cations. However, the exact hydration state of those potential Na⁺ sites cannot be identified here due to the resolution limit. Future high-resolution structure of Na_V1.6 would be required to investigate more detailed mechanisms of Na⁺ conductance. The 4,9-anhydro-TTX bound Na_V1.6 structure demonstrated that 4,9-anhydro-TTX and its closely-related analog TTX share a similar binding pocket, which is composed of nearly identical residues above the SFs. However, TTX has greater potency than 4,9-anhydro-TTX in inhibiting Na_V1.6 very likely due to TTX can form two additional hydrogen bonds with Na_V1.6. Our MD simulations show that 4,9-anhydro-TTX exhibits a more stable binding mode and greater binding energy with Na_V1.6 than Na_V1.7. Specifically, the increased flexibility of E930 and E927 may cause the loose binding of 4,9-anhydro-TTX to Na_V1.7. Those results potentially explain the higher potency of TTX to Na_V1.6 than other TTX-sensitive Na_V channels and the favorable inhibition of Na_V1.6 by 4,9-anhydro-TTX. In addition, an interesting observation needed to be mentioned here is the existence of some differences between the binding poses of 4,9-anhydro-TTX in the Na_V1.6^4,9-ahTTX EM structure and our MD simulation models (Fig. 4f, Supplementary Fig. 8f). The MD study was conducted with the assumption that the NH group of guanidine in 4,9-anhydro-TTX is fully protonated into NH₂⁺. However, since such NH in the EM structure is only ~3 Å from the amine group of Y371, it implies an uncertainty of the protonation state of the guanidine of 4,9-anhydro-TTX. When we performed another MD study using unprotonated 4,9-anhydro-TTX and found that the ligand adopts a similar binding pose as observed in the EM structure. Our findings on the protonation state of 4,9-anhydro-TTX binding with Na_V1.6 requires further systemic investigation. Taken together, our results provide important insights into Na_V channel structure, Na⁺ selectivity and conductance, and modulation by TTX and its analog 4,9-anhydro-TTX.

Methods

Whole-cell recordings

HEK293T cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, USA) supplemented with 15% Fetal Bovine Serum (FBS, PAN-Biotech, Germany) at 37 °C and 5% CO₂. The P2 viruses of Na_V1.6^EM and Na_V1.6 variants were obtained using Sf9 insect cells and used to infect HEK293T cells for 9 h. The plasmids expressing Na_V1.2^WT or Na_V1.7^WT were transfected into HEK293T cells using lipofectamine 2000 (Thermo Fisher Scientific, USA). 12–24 h after transfection or infection, whole-cell recordings were obtained using a HEKA EPC-10 patch-clamp amplifier (HEKA Electronic, Germany) and PatchMaster software (HEKA Electronic, Germany). The extracellular recording solution contained (in mM): 140 NaCl, 3 KCl, 1 CaCl₂, 1 MgCl₂, 10 Glucose, and 10 HEPES (310 mOsm/L, pH 7.30 with NaOH). The recording pipette intracellular solution contained (in mM): 140 CsF, 10 NaCl, 1 EGTA, and 10 HEPES (300 mOsm/L, pH 7.30 with CsOH). The pipettes were fabricated by a DMZ Universal Electrode puller (Zeitz Instruments, Germany) using borosilicate glass, with a resistance of 1.5–2.5 MΩ. The currents were acquired at a 50 kHz sample rate and series resistance (R_s) compensation was set to 70–90%. All experiments were performed at room temperature.

Data analyses were performed using Origin 2020b (OriginLab, USA), Excel 2016 (Microsoft, USA), and GraphPad Prism 9.1.1 (GraphPad Software, USA). Steady-state fast inactivation (I–V) and conductance-voltage (G–V) relationships were fitted to Boltzmann equations:

$${{{{{\rm{I}}}}}}/{{{{{{\rm{I}}}}}}}_{{{{{{\rm{max }}}}}}}=1/(1+\exp (({{{{{{\rm{V}}}}}}}_{{{{{{\rm{m}}}}}}}-{{{{{{\rm{V}}}}}}}_{1/2})/{{{{{\rm{k}}}}}}))$$

(1)

$${{{{{\rm{G}}}}}}/{{{{{{\rm{G}}}}}}}_{{{\max }}}=1/(1+\exp (({{{{{\rm{Vm}}}}}}-{{{{{{\rm{V}}}}}}}_{1/2})/{{{{{\rm{k}}}}}}))$$

(2)

$${{{{{\rm{G}}}}}}={{{{{\rm{I}}}}}}/({{{{{{\rm{V}}}}}}}_{{{{{{\rm{m}}}}}}}-{{{{{{\rm{E}}}}}}}_{{{{{{\rm{Na}}}}}}})$$

(3)

where I is the peak current, G is conductance, V_m is the stimulus potential, V_1/2 is the half-maximal activation potential, E_Na is the equilibrium potential, and k is the slope factor.

To assess the potency of 4,9-anhydro-TTX and TTX on Na_V channels, HEK293T cells were held at −120 mV and the inward sodium currents were elicited by a 50-ms step to −10 mV with a low frequency of 1/15 Hz. The concentration-response curves were fitted to a four-parameter Hill equation with constraints of Bottom = 0 and Top = 1:

$${{{{{\rm{Y}}}}}}={{{{{\rm{Bottom}}}}}}+({{{{{\rm{Top}}}}}}-{{{{{\rm{Bottom}}}}}})\big/\left(1+{10}^{\left({{{{{\rm{X}}}}}}-{{{{{\rm{lgIC50}}}}}}\right)}\right)$$

(4)

where Y is the value of I_Drug/I_Control, Top is the maximum response, Bottom is the minimum response, X is the lg of drug concentration, and IC₅₀ is the drug concentration producing the half-maximum response. The significance of fitted IC₅₀ values compared to the control was analyzed using the extra sum-of-squares F test. The drug inhibition statistics are presented in Supplementary Table 1.

Na_V1.6/β1/β2 cloning and expression

The DNA fragments encoding human Na_V1.6 (UniProt ID: Q9UQD0), β1 (Uniprot ID: Q07699), and β2 (Uniprot ID: O60939) were amplified from a HEK293 cDNA library. The full-length or truncated Na_V1.6, β1, and β2 genes were cloned into the pEG BacMam vector, respectively. For Na_V1.6^EM, residues of inter-domain linkers 478–692, 1115–1180, and 1932 to the last residue were deleted by PCR to optimize the biochemical properties of the purified protein sample. Specifically, Na_V1.6^EM was fused before a PreScission Protease recognition site, which is succeeded by a mCherry fluorescent protein and a Twin-Strep II tag at the C terminus. A superfolder green fluorescent protein (sfGFP) and His10 tag were introduced at the C terminus of β1. The sequences of all primers used in this study are provided in Supplementary Table 2. For protein expression, recombinant baculoviruses were generated in Sf9 cells using the Bac-to-Bac baculovirus expression system (Invitrogen, 11496015). HEK293F (Gibco, 11625019) cells were cultured under 5% CO₂ at 37 °C and were used for transfection at a density of 2.5 × 10⁶ cells/ml. The Na_V1.6^EM, β1, and β2 viruses were co-transfected into HEK 293F cells at a ratio of 1% (v/v) supplemented with 1% (v/v) FBS. After 8–12 h, sodium butyrate was added into the culture at a final concentration of 10 mM, and the cell was incubated for another 48 h under 30 °C. Cells were then harvested by centrifugation at 1640 × g for 5 min, and finally stored at −80 °C after freezing in liquid nitrogen.

Purification of human Na_V1.6/β1/β2 complex

The Na_V1.6/β1/β2 complex was purified following a protocol as was applied in the purification of the Na_V1.3/β1/β2 complex⁴¹. Cells expressing Na_V1.6^EM complex were resuspended in buffer A (20 mM HEPES pH 7.5, 150 mM NaCl, 2 mM β-mercaptoethanol (β-ME), aprotinin (2 μg/mL), leupeptin (1.4 μg/mL), pepstatin A (0.5 μg/mL)) using a Dounce homogenizer and centrifuged at 100,000 × g for 1 h. After resuspension in buffer B (buffer A supplemented with 1% (w/v) n-Dodecyl-β-D-maltoside (DDM, Anatrace), 0.15% (w/v) cholesteryl hemisuccinate (CHS, Anatrace), 5 mM MgCl₂ and 5 mM ATP), the suspension was agitated at 4 °C for 2 h and the insoluble fraction was removed by centrifugation again at 100,000 × g for 1 h. The supernatant containing solubilized Na_V1.6^EM was then passed through Streptactin Beads (Smart-Lifesciences, China) via gravity flow at 4 °C to enrich the protein complex. The resin was subsequently washed with buffer C (buffer A supplemented with 0.03% (w/v) glycol-diosgenin (GDN, Anatrace)) for 10 column volumes. The purified Na_V1.6^EM complex was eluted with buffer D (buffer C plus 5 mM desthiobiotin (Sigma, USA)) and was subsequently concentrated to 1 mL using a 100 kDa cut-off Amicon ultra centrifugal filter (Merck Millipore, Germany). The concentrated protein sample was further purified by size exclusion chromatography (SEC) using a Superose 6 Increase 10/300 GL (GE Healthcare) column pre-equilibrated with the buffer E (20 mM HEPES pH 7.5, 150 mM NaCl, 2 mM β-ME, 0.007% GDN). Finally, the fractions containing homogeneous-distributed protein particles were collected and concentrated to ~4 mg/mL for cryo-EM sample preparation.

Cryo-EM sample preparation and data acquisition

For the preparation of cryo-EM grids, 300-mesh Cu R1.2/1.3 grids (Quantifoil Micro Tools, Germany) were glow-discharged under H2-O2 condition for 60 s. A droplet of 2.5 μL of purified Na_V1.6^EM complex was applied to the grid followed by blotting for 4–5 s at 4 °C under 100% humidity using a Vitrobot Mark IV (Thermo Fisher Scientific, USA). In the case of the preparation of Na_V1.6^EM complex with 4,9-anhydro-TTX, 50 μM 4,9-anhydro-TTX (Tocris, UK) was added to the sample before vitrification. Cryo-EM data were collected on a 300-kV Titan Krios transmission electron microscope (Thermo Fisher Scientific, USA) equipped with a Gatan K2 Summit Direct Electron Detector (Gatan, USA) located behind the GIF quantum energy filter (20 e^-V). SerialEM⁸¹ was used to collect movie stacks at a magnification of ×130,000 (1.04 Å pixel size) with a nominal defocus range from –1.2 to –2.2 μm. A total dose of 50–60 e⁻/Å² was acquired for each movie stack under a dose rate of ~9.2 e-/(Å²s) and dose-fractionated into 32 frames. A total of 3,985 and 2929 movie stacks were collected for the apo- and 4,9-anhydro-TTX-bound Na_V1.6 complex, respectively.

Data processing

For the data processing of apo and 4,9-anhydro-TTX-bound Na_V1.6 complex, a similar procedure was performed and a detailed diagram was presented in Supplementary Figs. 3 and 4. All the data were processed in RELION3.0⁸² or cryoSPARC⁸³. Movies were motion-corrected and dose-weighted using MotionCor2. Contrast transfer function (CTF) estimation was performed with GCTF⁸⁴. Particles were picked using the AutoPick tool in RELION with templates and extracted into 256 × 256-pixel boxes. Several rounds of 2D and 3D classifications were performed to remove junk particles, followed by 3D autorefine, Bayesian polish, and CTF refinement to improve the map quality. The final EM density maps were generated by the non-uniform (NU) refinement in cryoSPARC and reported at 3.4 Å and 3.3 Å, respectively, according to the golden standard Fourier shell correlation (GSFSC) criterion.

Model building

The sequence of human Na_V1.6 and Na_V1.7 were aligned using Jalview⁸⁵, and a homology model of Na_V1.6 was generated using the molecular replacement tool in PHENIX⁸⁶. The atomic models of β1 and β2 subunits were extracted from the structure of Na_V1.7 (PDB ID: 6J8I). All of the models were fitted into the cryo-EM map as rigid bodies using the UCSF Chimera⁸⁷. Restraints for 4,9-anhydro-TTX were derived by eLBOW in PHENIX and examined in Coot⁸⁸. All residues were manually checked and adjusted to fit the map in Coot and were subsequently subjected to rounds of real-space refinement in PHENIX. Model validation was performed using the comprehensive validation (cryo-EM) in PHENIX. The cryo-EM data collection, refinement, and model validation statistics are presented in Supplementary Table 3.

All figures were prepared with UCSF ChimeraX⁸⁹ or PyMOL (Schrödinger, USA)⁹⁰.

Molecular dynamics simulations

The structures and force fields for protein, DMPC lipids, and ligands were prepared using the CHARMM-GUI website. The Amber ff14SB force field was used for both protein and lipids with the TIP3P model for water molecules⁹¹. The GAFF2 force field parameters were used for the ligands⁹². The simulated systems were solvated in water with 150 mM NaCl. The energy minimization was performed using the steepest descent method, followed by six equilibrium steps. During the 2 ns equilibrium steps, the protein backbone atoms were restrained to their initial positions using a harmonic potential with a force constant of 1 kcal mol⁻¹ Å⁻² and the restraints were subsequently removed. Berendsen’s coupling scheme was used for both temperature and pressure⁹³. Water molecules and all bond lengths to hydrogen atoms were constrained using LINCS⁹⁴. Finally, six independent production runs were performed for 100 ns. The overall temperature of the system was kept constant, coupling independently for protein, lipids, and solvents at 303.15 K with a Nose-Hoover thermostat⁹⁵. A constant pressure of 1 bar was maintained using a Parrinello–Rahman barostat in a semi-isotropic coupling type for x/y, and z directions, respectively⁹⁶. The temperature and pressure time constants of the coupling were 1 and 5 ps, and the compressibility was 4.5 × 10⁻⁵ bar⁻¹ for pressure. The integration of the equations of motion was performed by using a leapfrog algorithm with a time step of 2 fs. Periodic boundary conditions were implemented in all systems. A cutoff of 0.9 nm was implemented for the Lennard–Jones and the direct space part of the Ewald sum for Coulombic interactions. The Fourier space part of the Ewald splitting was computed by using the particle-mesh-Ewald method⁹⁷, with a grid length of 0.12 nm on the side and a cubic spline interpolation.

The binding affinities were calculated by MM/GBSA method^{98,99,100,101}. The MM part consists of the bonded (bond, angle, and dihedral), electrostatic, and van der Waals interactions. The solvation free energies were obtained by using the generalized Born model (GB part), and the non-polar term is obtained from a linear relation to the solvent-accessible surface area (SA part). For each independent trajectory, the first 20 ns trajectory was discarded and 800 frames from 20–100 ns were used for MM/GBSA calculations. The final binding affinity for each ligand-protein complex was obtained by taking the average of the six independent trajectories. Regarding the clustering analysis, structure alignment was first performed for each two of the structures in the trajectory by using Least Squares algorithm which aligns two sets of structure by rotating and translating one of the structures so that the RMSD between matching atoms of the two structures is minimal. Then the clustering analysis was performed by using GROMOS¹⁰² with a RMSD cut-off of 1.5 Å to determine the structurally similar clusters. All the simulations were performed using the GROMACS 2021 suite of programs¹⁰³.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The data that support this study are available from the corresponding authors upon reasonable request. The three-dimensional cryo-EM density maps have been deposited in the Electron Microscopy Data Bank (EMDB) under accession codes EMD-34387 (Na_V1.6/β1/β2) and EMD-34388 (Na_V1.6/β1/β2-4,9-anhydro-TTX). The atomic coordinates have been deposited in the Protein Data Bank (PDB) under accession codes 8GZ1 (Na_V1.6/β1/β2) and 8GZ2 (Na_V1.6/β1/β2-4,9-anhydro-TTX). The UniProt accession codes for the sequences of human Na_V1.6, β1, and β2 are Q9UQD0, Q07699, and O60939, respectively. The accession codes for the coordinates of Na_V1.7, Ca_VAb, and Ca_V3.1 used in this study are 6J8J (Na_V1.7), 4MS2 (Ca_VAb), and 6KZO (Ca_V3.1). Source data are provided as a Source data file. Source data are provided with this paper.

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Acknowledgements

We thank X. Huang, B. Zhu, X. Li, L. Chen, and other staff members at the Center for Biological Imaging (CBI), Core Facilities for Protein Science at the Institute of Biophysics, Chinese Academy of Science (IBP, CAS), and D. Sun at the SM10 Cryo-EM Facility at the Institute of Physics, Chinese Academy of Sciences (IOP, CAS) for the support in cryo-EM data collection. We thank Prof. Xuejun Cai Zhang for his helpful discussions, and Yan Wu and Wei Fan for their research assistance service. This work is funded by the Institute of Physics, Chinese Academy of Sciences (E0VK101 and E2V4101 to D.J.), the National Natural Science Foundation of China (T2221001 and 32271272 to D.J., 92157102 to Y.Z., 31871083 and 82271498 to Z.H.), Chinese Academy of Sciences Strategic Priority Research Program (Grant XDB37030304 to Y.Z.), the National Natural Science Foundation of China (Grant 92157102 to Y.Z.), the Chinese National Programs for Brain Science and Brain-like intelligence technology (2021ZD0202102 to Z.H.).

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These authors contributed equally: Yue Li, Tian Yuan, Bo Huang.

Authors and Affiliations

National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China
Yue Li, Xiaojing Li, Yunlong Qiu, Bei Yang & Yan Zhao
Laboratory of Soft Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing, 100190, China
Yue Li & Daohua Jiang
University of Chinese Academy of Sciences, Beijing, 100049, China
Yue Li, Xiaojing Li, Yunlong Qiu, Yan Zhao & Daohua Jiang
State Key Laboratory of Natural and Biomimetic Drugs, Department of Molecular and Cellular Pharmacology, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing, 100191, China
Tian Yuan, Chao Peng & Zhuo Huang
IDG/McGovern Institute for Brain Research, Peking University, Beijing, 100871, China
Tian Yuan, Chao Peng & Zhuo Huang
Beijing StoneWise Technology Co Ltd., 15 Haidian Street, Haidian District, Beijing, China
Bo Huang & Feng Zhou

Authors

Yue Li
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Tian Yuan
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Bo Huang
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Feng Zhou
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Chao Peng
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Xiaojing Li
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Yunlong Qiu
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Bei Yang
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Yan Zhao
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Zhuo Huang
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Daohua Jiang
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Contributions

D.J., Z.H., and Y.Z. conceived and designed the experiments. Y.L. and X.L. prepared samples for the cryo-EM study and made all the constructs. Y.Q. and B.Y. prepared cells for protein expression. Y.L. collected cryo-EM data. Y.L. and D.J. processed the data, and built and refined the models. Y.L. and T.Y. prepared figures. T.Y. collected the electrophysiology data. B.H., F.Z., and C.P. performed MD studies. Y.L., T.Y., B.H., Y.Z., Z.H., and D.J. analyzed and interpreted the results. L.Y., T.Y., B.H., and D.J. wrote the paper, and all authors reviewed and revised the paper.

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Correspondence to Yan Zhao, Zhuo Huang or Daohua Jiang.

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Li, Y., Yuan, T., Huang, B. et al. Structure of human Na_V1.6 channel reveals Na⁺ selectivity and pore blockade by 4,9-anhydro-tetrodotoxin. Nat Commun 14, 1030 (2023). https://doi.org/10.1038/s41467-023-36766-9

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Received: 03 October 2022
Accepted: 15 February 2023
Published: 23 February 2023
DOI: https://doi.org/10.1038/s41467-023-36766-9

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