Characterizing fenestration size in sodium channel subtypes and their accessibility to inhibitors

Voltage-gated sodium channels (Nav) underlie the electrical activity of nerve and muscle cells. Humans have nine different subtypes of these channels, which are the target of small-molecule inhibitors commonly used to treat a range of conditions. Structural studies have identified four lateral fenestrations within the Nav pore module that have been shown to influence Nav pore blocker access during resting-state inhibition. However, the structural differences among the nine subtypes are still unclear. In particular, the dimensions of the four individual fenestrations across the Nav subtypes and their differential accessibility to pore blockers is yet to be characterized. To address this, we applied classical molecular dynamics simulations to study the recently published structures of Nav1.1, Nav1.2, Nav1.4, Nav1.5, and Nav1.7. Although there is significant variability in the bottleneck sizes of the Nav fenestrations, the subtypes follow a common pattern, with wider DI-II and DIII-IV fenestrations, a more restricted DII-III fenestration, and the most restricted DI-IV fenestration. We further identify the key bottleneck residues in each fenestration and show that the motions of aromatic residue sidechains govern the bottleneck radii. Well-tempered metadynamics simulations of Nav1.4 and Nav1.5 in the presence of the pore blocker lidocaine also support the DI-II fenestration being the most likely access route for drugs. Our computational results provide a foundation for future in vitro experiments examining the route of drug access to sodium channels. Understanding the fenestrations and their accessibility to drugs is critical for future analyses of diseases mutations across different sodium channel subtypes, with the potential to inform pharmacological development of resting-state inhibitors and subtype-selective drug design.     

Folding similarity of the outer pore region in prokaryotic and eukaryotic sodium channels revealed by docking of conotoxins GIIIA,PIIIA, and KIIIA in a NavAb-based model of Nav1.4

Voltage-gated sodium channels are targets for many drugs and toxins. However, the rational design of medically relevant channel modulators is hampered by the lack of x-ray structures of eukaryotic channels. Here, we used a homology model based on the x-ray structure of the NavAb prokaryotic sodium channel together with published experimental data to analyze interactions of the μ-conotoxins GIIIA, PIIIA, and KIIIA with the Nav1.4 eukaryotic channel. Using Monte Carlo energy minimizations and published experimentally defined pairwise contacts as distance constraints, we developed a model in which specific contacts between GIIIA and Nav1.4 were readily reproduced without deformation of the channel or toxin backbones. Computed energies of specific interactions between individual residues of GIIIA and the channel correlated with experimental estimates. The predicted complexes of PIIIA and KIIIA with Nav1.4 are consistent with a large body of experimental data. In particular, a model of Nav1.4 interactions with KIIIA and tetrodotoxin (TTX) indicated that TTX can pass between Nav1.4 and channel-bound KIIIA to reach its binding site at the selectivity filter. Our models also allowed us to explain experimental data that currently lack structural interpretations. For instance, consistent with the incomplete block observed with KIIIA and some GIIIA and PIIIA mutants, our computations predict an uninterrupted pathway for sodium ions between the extracellular space and the selectivity filter if at least one of the four outer carboxylates is not bound to the toxin. We found a good correlation between computational and experimental data on complete and incomplete channel block by native and mutant toxins. Thus, our study suggests similar folding of the outer pore region in eukaryotic and prokaryotic sodium channels.     

Use-dependent potentiation of the Nav1.6 sodium channel

Nav1.2 and Nav1.6 are two voltage-gated sodium channel isoforms that are abundant in the adult central nervous system. These channels are expressed in different cells and localized in different neuronal regions, which may reflect functional specialization. To examine this possibility, we compared the properties of Nav1.2 and Nav1.6 in response to a rapid series of repetitive depolarizations. Currents through Nav1.6 coexpressed with beta1 demonstrated use-dependent potentiation during a rapid train of depolarizations. This potentiation was in contrast to the use-dependent decrease in current for Nav1.2 with beta1. The voltage dependence of potentiation correlated with the voltage dependence of activation, and it still occurred when fast inactivation was removed by mutation. Rapid stimulation accelerated a slow phase of activation in the Nav1.6 channel that had fast inactivation removed, resulting in faster channel activation. Although the Nav1.2 channel with fast inactivation removed also demonstrated slightly faster activation, that channel showed very pronounced slow inactivation compared to Nav1.6. These results indicate that potentiation of Nav1.6 sodium currents results from faster channel activation, and that this effect is masked by slow inactivation in Nav1.2. The data suggest that Nav1.6 might be more resistant to inactivation, which might be helpful for high-frequency firing at nodes of Ranvier compared to Nav1.2.     

Calmodulin mediates Ca2+ sensitivity of sodium channels

Ca2+ has been proposed to regulate Na+ channels through the action of calmodulin (CaM) bound to an IQ motif or through direct binding to a paired EF hand motif in the Nav1 C terminus. Mutations within these sites cause cardiac arrhythmias or autism, but details about how Ca2+ confers sensitivity are poorly understood. Studies on the homologous Cav1.2 channel revealed non-canonical CaM interactions, providing a framework for exploring Na+ channels. In contrast to previous reports, we found that Ca2+ does not bind directly to Na+ channel C termini. Rather, Ca2+ sensitivity appears to be mediated by CaM bound to the C termini in a manner that differs significantly from CaM regulation of Cav1.2. In Nav1.2 or Nav1.5, CaM bound to a localized region containing the IQ motif and did not support the large Ca(2+)-dependent conformational change seen in the Cav1.2.CaM complex. Furthermore, CaM binding to Nav1 C termini lowered Ca2+ binding affinity and cooperativity among the CaM-binding sites compared with CaM alone. Nonetheless, we found suggestive evidence for Ca2+/CaM-dependent effects upon Nav1 channels. The R1902C autism mutation conferred a Ca(2+)-dependent conformational change in Nav1.2 C terminus.CaM complex that was absent in the wild-type complex. In Nav1.5, CaM modulates the Cterminal interaction with the III-IV linker, which has been suggested as necessary to stabilize the inactivation gate, to minimize sustained channel activity during depolarization, and to prevent cardiac arrhythmias that lead to sudden death. Together, these data offer new biochemical evidence for Ca2+/CaM modulation of Na+ channel function.     

Isoform-specific effects of the beta2 subunit on voltage-gated sodium channel gating

Voltage-gated sodium channels (Nav) are complex glycoproteins comprised of an alpha subunit and often one to several beta subunits. We have shown that sialic acid residues linked to Nav alpha and beta1 subunits alter channel gating. To determine whether beta2-linked sialic acids similarly impact Nav gating, we co-expressed beta2 with Nav1.5 or Nav1.2 in Pro5 (complete sialylation) and in Lec2 (essentially no sialylation) cells. Beta2 sialic acids caused a significant hyperpolarizing shift in Nav1.5 voltage-dependent gating, thus describing for the first time an effect of beta2 on Nav1.5 gating. In contrast, beta2 caused a sialic acid-independent depolarizing shift in Nav1.2 gating. A deglycosylated mutant, beta(2-DeltaN), had no effect on Nav1.5 gating, indicating further the impact of beta2 N-linked sialic acids on Nav1.5 gating. Conversely, beta(2-DeltaN) modulated Nav1.2 gating virtually identically to beta2, confirming that beta2 N-linked sugars have no impact on Nav1.2 gating. Thus, beta2 modulates Nav gating through multiple mechanisms possibly determined by the associated alpha subunit. Beta1 and beta2 were expressed together with Nav1.5 or Nav1.2 in Pro5 and Lec2 cells. Together beta1 and beta2 produced a significantly larger sialic acid-dependent hyperpolarizing shift in Nav1.5 gating. Under fully sialylating conditions, the Nav1.2.beta1.beta2 complex behaved like Nav1.2 alone. When sialylation was reduced, only the sialic acid-independent depolarizing effects of beta2 on Nav1.2 gating were apparent. Thus, the varied effects of beta1 and beta2 on Nav1.5 and Nav1.2 gating are apparently synergistic and highlight the complex manner, through subunit- and sugar-dependent mechanisms, by which Nav activity is modulated.     

Dynamic compartmentalization of the voltage-gated sodium channels in axons

One of the major physiological roles of the neuronal voltage-gated sodium channel is to generate action potentials at the axon hillock/initial segment and to ensure propagation along myelinated or unmyelinated fibers to nerve terminal. These processes require a precise distribution of sodium channels accumulated at high density in discrete subdomains of the nerve membrane. In neurons, information relevant to ion channel trafficking and compartmentalization into sub-domains of the plasma membrane is far from being elucidated. Besides, whereas information on dendritic targeting is beginning to emerge, less is known about the mechanisms leading to the polarized distribution of proteins in axon. To obtain a better understanding of how neurons selectively target sodium channels to discrete subdomains of the nerve, we addressed the question as to whether any of the large intracellular regions of Nav1.2 contain axonal sorting and/or clustering signals. We first obtained evidence showing that addition of the cytoplasmic carboxy-terminal region of Nav1.2 restricted the distribution of a dendritic-axonal reporter protein to axons of hippocampal neurons. The analysis of mutants revealed that a di-leucine-based motif mediates chimera compartmentalization in axons and its elimination in soma and dendrites by endocytosis. The analysis of the others generated chimeras showed that the determinant conferring sodium channel clustering at the axonal initial segment is contained within the cytoplasmic loop connecting domains II-III of Nav1.2. Expression of a soluble Nav1.2 II-III linker protein led to the disorganization of endogenous sodium channels. The motif was sufficient to redirect a somatodendritic potassium channel to the axonal initial segment, a process involving association with ankyrin G. Thus, it is conceivable that concerted action of the two determinants is required for sodium channel compartmentalization in axons.     

Functional properties and toxin pharmacology of a dorsal root ganglion sodium channel viewed through its voltage sensors

The voltage-activated sodium (Nav) channel Nav1.9 is expressed in dorsal root ganglion (DRG) neurons where it is believed to play an important role in nociception. Progress in revealing the functional properties and pharmacological sensitivities of this non-canonical Nav channel has been slow because attempts to express this channel in a heterologous expression system have been unsuccessful. Here, we use a protein engineering approach to dissect the contributions of the four Nav1.9 voltage sensors to channel function and pharmacology. We define individual S3b-S4 paddle motifs within each voltage sensor, and show that they can sense changes in membrane voltage and drive voltage sensor activation when transplanted into voltage-activated potassium channels. We also find that the paddle motifs in Nav1.9 are targeted by animal toxins, and that these toxins alter Nav1.9-mediated currents in DRG neurons. Our results demonstrate that slowly activating and inactivating Nav1.9 channels have functional and pharmacological properties in common with canonical Nav channels, but also show distinctive pharmacological sensitivities that can potentially be exploited for developing novel treatments for pain.     

Characterization of a new class of potent inhibitors of the voltage-gated sodium channel Nav1.7

Voltage-gated sodium channels (Nav1) transmit pain signals from peripheral nociceptive neurons, and blockers of these channels have been shown to ameliorate a number of pain conditions. Because these drugs can have adverse effects that limit their efficacy, more potent and selective Nav1 inhibitors are being pursued. Recent human genetic data have provided strong evidence for the involvement of the peripheral nerve sodium channel subtype, Nav1.7, in the signaling of nociceptive information, highlighting the importance of identifying selective Nav1.7 blockers for the treatment of chronic pain. Using a high-throughput functional assay, novel Nav1.7 blockers, namely, the 1-benzazepin-2-one series, have recently been identified. Further characterization of these agents indicates that, in addition to high-affinity inhibition of Nav1.7 channels, selectivity against the Nav1.5 and Nav1.8 subtypes can also be achieved within this structural class. The most potent, nonselective member of this class of Nav1.7 blockers has been radiolabeled with tritium. [3H]BNZA binds with high affinity to rat brain synaptosomal membranes (Kd = 1.5 nM) and to membranes prepared from HEK293 cells stably transfected with hNav1.5 (Kd = 0.97 nM). In addition, and for the first time, high-affinity binding of a radioligand to hNav1.7 channels (Kd = 1.6 nM) was achieved with [3H]BNZA, providing an additional means for identifying selective Nav1.7 channel inhibitors. Taken together, these data suggest that members of the novel 1-benzazepin-2-one structural class of Nav1 blockers can display selectivity toward the peripheral nerve Nav1.7 channel subtype, and with appropriate pharmacokinetic and drug metabolism properties, these compounds could be developed as analgesic agents.     

铝暴露对大鼠海马神经元长时程增强和Na+通道表达的影响

海马神经元长时程增强(LTP) 被认为与学习和记忆的形成有关.Na⁺在诱导 LTP产生的过程中十分重要.实验发现,慢性铝暴露可以影响大鼠海马神经元LTP的产生,随着铝暴露浓度的增加,LTP 的幅值逐渐降低.RT-PCR 法对大鼠海马神经元 9 种类型Na⁺ 通道（即 Nav1.1～Nav1.9）的 mRNA 进行检测发现,除 Nav1.4 和 Nav1.8 Na⁺通道 mRNA 在大鼠海马神经元中未见表达外,慢性染铝组大鼠海马神经元7种Na⁺ 通道 mRNA 表达均明显增高（P<0.05）.蛋白印迹法对一种脑型 Na⁺通道 (Nav 1.2) 蛋白检测证明, Na⁺通道蛋白表达亦明显升高.结果提示,铝进入神经元后,可能通过影响 Na⁺ 通道蛋白的表达而影响了突触后神经细胞的去极化,进而影响了LTP的诱导过程,从而预示铝的暴露可能损害大鼠学习和记忆能力.     

Reciprocal Changes in Phosphorylation and Methylation of Mammalian Brain Sodium Channels in Response to Seizures

Voltage-gated sodium (Nav) channels initiate action potentials in brain neurons and are primary therapeutic targets for anti-epileptic drugs controlling neuronal hyperexcitability in epilepsy. The molecular mechanisms underlying abnormal Nav channel expression, localization, and function during development of epilepsy are poorly understood but can potentially result from altered posttranslational modifications (PTMs). For example, phosphorylation regulates Nav channel gating, and has been proposed to contribute to acquired insensitivity to anti-epileptic drugs exhibited by Nav channels in epileptic neurons. However, whether changes in specific brain Nav channel PTMs occur acutely in response to seizures has not been established. Here, we show changes in PTMs of the major brain Nav channel, Nav1.2, after acute kainate-induced seizures. Mass spectrometry-based proteomic analyses of Nav1.2 purified from the brains of control and seizure animals revealed a significant down-regulation of phosphorylation at nine sites, primarily located in the interdomain I-II linker, the region of Nav1.2 crucial for phosphorylation-dependent regulation of activity. Interestingly, Nav1.2 in the seizure samples contained methylated arginine (MeArg) at three sites. These MeArgs were adjacent to down-regulated sites of phosphorylation, and Nav1.2 methylation increased after seizure. Phosphorylation and MeArg were not found together on the same tryptic peptide, suggesting reciprocal regulation of these two PTMs. Coexpression of Nav1.2 with the primary brain arginine methyltransferase PRMT8 led to a surprising 3-fold increase in Nav1.2 current. Reciprocal regulation of phosphorylation and MeArg of Nav1.2 may underlie changes in neuronal Nav channel function in response to seizures and also contribute to physiological modulation of neuronal excitability.     

Resting potential-dependent regulation of the voltage sensitivity of sodium channel gating in rat skeletal muscle in vivo

Normal muscle has a resting potential of -85 mV, but in a number of situations there is depolarization of the resting potential that alters excitability. To better understand the effect of resting potential on muscle excitability we attempted to accurately simulate excitability at both normal and depolarized resting potentials. To accurately simulate excitability we found that it was necessary to include a resting potential-dependent shift in the voltage dependence of sodium channel activation and fast inactivation. We recorded sodium currents from muscle fibers in vivo and found that prolonged changes in holding potential cause shifts in the voltage dependence of both activation and fast inactivation of sodium currents. We also found that altering the amplitude of the prepulse or test pulse produced differences in the voltage dependence of activation and inactivation respectively. Since only the Nav1.4 sodium channel isoform is present in significant quantity in adult skeletal muscle, this suggests that either there are multiple states of Nav1.4 that differ in their voltage dependence of gating or there is a distribution in the voltage dependence of gating of Nav1.4. Taken together, our data suggest that changes in resting potential toward more positive potentials favor states of Nav1.4 with depolarized voltage dependence of gating and thus shift voltage dependence of the sodium current. We propose that resting potential-induced shifts in the voltage dependence of sodium channel gating are essential to properly regulate muscle excitability in vivo.     

Determinants of voltage-gated sodium channel clustering in neurons

In mammalian neurons, the generation and propagation of the action potential result from the presence of dense clusters of voltage-gated sodium channels (Nav) at the axonal initial segment (AIS) and nodes of Ranvier. In these two structures, the assembly of specific supra-molecular complexes composed of numerous partners, such as cytoskeletal scaffold proteins and signaling proteins ensures the high concentration of Nav channels. Understanding how neurons regulate the expression and discrete localization of Nav channels is critical to understanding the diversity of normal neuronal function as well as neuronal dysfunction caused by defects in these processes. Here, we review the mechanisms establishing the clustering of Nav channels at the AIS and in the node and discuss how the alterations of Nav channel clustering can lead to certain pathophysiologies.     

Tonic and phasic guanidinium toxin-block of skeletal muscle Na channels expressed in Mammalian cells

The blockage of skeletal muscle sodium channels by tetrodotoxin (TTX) and saxitoxin (STX) have been studied in CHO cells permanently expressing rat Nav1.4 channels. Tonic and use-dependent blockage were analyzed in the framework of the ion-trapped model. The tonic affinity (26.6 nM) and the maximum affinity (7.7 nM) of TTX, as well as the "on" and "off" rate constants measured in this preparation, are in remarkably good agreement with those measured for Nav1.2 expressed in frog oocytes, indicating that the structure of the toxin receptor of Nav1.4 and Nav1.2 channels are very similar and that the expression method does not have any influence on the pore properties of the sodium channel. The higher affinity of STX for the sodium channels (tonic and maximum affinity of 1.8 nM and 0.74 nM respectively) is explained as an increase on the "on" rate constant (approximately 0.03 s(-1) nM(-1)), compared to that of TTX (approximately 0.003 s(-1) nM(-1)), while the "off" rate constant is the same for both toxins (approximately 0.02 s(-1)). Estimations of the free-energy differences of the toxin-channel interaction indicate that STX is bound in a more external position than TTX. Similarly, the comparison of the toxins free energy of binding to a ion-free, Na(+)- and Ca(2+)-occupied channel, is consistent with a binding site in the selectivity filter for Ca(2+) more external than for Na(+). This data may be useful in further attempts at sodium-channel pore modeling.     

Sodium channel beta1 subunit-mediated modulation of Nav1.2 currents and cell surface density is dependent on interactions with contactin and ankyrin

Voltage-gated sodium channels are composed of a pore-forming alpha subunit and at least one auxiliary beta subunit. Both beta1 and beta2 are cell adhesion molecules that interact homophilically, resulting in ankyrin recruitment. In contrast, beta1, but not beta2, interacts heterophilically with contactin, resulting in increased levels of cell surface sodium channels. We took advantage of these results to investigate the molecular basis of beta1-mediated enhancement of sodium channel cell surface density, including elucidating structure-function relationships for beta1 association with contactin, ankyrin, and Nav1.2. beta1/beta2 subunit chimeras were used to assign putative sites of contactin interaction to two regions of the beta1 Ig loop. Recent studies have shown that glutathione S-transferase fusion proteins containing portions of Nav1.2 intracellular domains interact directly with ankyrinG. We show that native Nav1.2 associates with ankyrinG in cells in the absence of beta subunits and that this interaction is enhanced in the presence of beta1 but not beta1Y181E, a mutant that does not interact with ankyrinG. beta1Y181E does not modulate Nav1.2 channel function despite efficient association with Nav1.2 and contactin. beta1Y181E increases Nav1.2 cell surface expression, but not as efficiently as wild type beta1. beta1/beta2 chimeras exchanging various regions of the beta1 Ig loop were all ineffective in increasing Nav1.2 cell surface density. Our results demonstrate that full-length beta1 is required for channel modulation and enhancement of sodium channel cell surface expression.     

Animal toxins and ion channels

Animal venoms contain various toxins which act on ion-channels, responsible for either sodium, potassium, calcium or chloride permeation. Structure determination of these toxins demonstrate that they are organised around two different structural motifs: potassium and sodium channel effectors are organised around an alpha-helix connected by two disulfide bridges to a two- or three-stranded beta sheet whereas calcium channels effectors are structured around an "Inhibitory Cystine Knot" motif made of a dense disulfide-rich core from which emerge several loops. Analysis of local structural modifications allows us to understand the structural basis of the selectivity of these effectors towards the various ion channels. This is the first step in the design of new synthetic molecules which are potent therapeutic drugs for diseases involving ion channel dysfunctioning.     

Cannabidiol inhibits the skeletal muscle Nav1.4 by blocking its pore and by altering membrane elasticity

Cannabidiol (CBD) is the primary nonpsychotropic phytocannabinoid found in Cannabis sativa, which has been proposed to be therapeutic against many conditions, including muscle spasms. Among its putative targets are voltage-gated sodium channels (Navs), which have been implicated in many conditions. We investigated the effects of CBD on Nav1.4, the skeletal muscle Nav subtype. We explored direct effects, involving physical block of the Nav pore, as well as indirect effects, involving modulation of membrane elasticity that contributes to Nav inhibition. MD simulations revealed CBD’s localization inside the membrane and effects on bilayer properties. Nuclear magnetic resonance (NMR) confirmed these results, showing CBD localizing below membrane headgroups. To determine the functional implications of these findings, we used a gramicidin-based fluorescence assay to show that CBD alters membrane elasticity or thickness, which could alter Nav function through bilayer-mediated regulation. Site-directed mutagenesis in the vicinity of the Nav1.4 pore revealed that removing the local anesthetic binding site with F1586A reduces the block of I_Na by CBD. Altering the fenestrations in the bilayer-spanning domain with Nav1.4-WWWW blocked CBD access from the membrane into the Nav1.4 pore (as judged by MD). The stabilization of inactivation, however, persisted in WWWW, which we ascribe to CBD-induced changes in membrane elasticity. To investigate the potential therapeutic value of CBD against Nav1.4 channelopathies, we used a pathogenic Nav1.4 variant, P1158S, which causes myotonia and periodic paralysis. CBD reduces excitability in both wild-type and the P1158S variant. Our in vitro and in silico results suggest that CBD may have therapeutic value against Nav1.4 hyperexcitability.     

Structural Model of the CaV1.2 Pore

Understanding the structure and functional mechanisms of voltage-gated calcium channels remains a major task in membrane biophysics. In the absence of three dimensional structures, homology modelling techniques are the method of choice, to address questions concerning the structure of these channels. We have developed models of the open Cav1.2 pore, based on the crystal structure of the mammalian voltage-gated potassium channel Kv1.2 and a model of the bacterial sodium channel NaChBac. Our models are developed to be consistent with experimental data and modelling criteria. The models highlight major differences between voltage-gated potassium and calcium channels, in the P segments, as well as the inner pore helices. Molecular dynamics simulations support the hypothesis of a clockwise domain arrangement and experimental observations of asymmetric calcium channel behaviour. In the accompanying paper these models were used to study structural effects of a channelopathy mutation.     

A naturally occurring amino acid substitution in the voltage-dependent sodium channel selectivity filter affects channel gating

The pore of sodium channels contains a selectivity filter made of 4 amino acids, D/E/K/A. In voltage sensitive sodium channel (Nav) channels from jellyfish to human the fourth amino acid is Ala. This Ala, when mutated to Asp, promotes slow inactivation. In some Nav channels of pufferfishes, the Ala is replaced with Gly. We studied the biophysical properties of an Ala-to-Gly substitution (A1529G) in rat Nav1.4 channel expressed in Xenopus oocytes alone or with a β1 subunit. The Ala-to-Gly substitution does not affect monovalent cation selectivity and positively shifts the voltage-dependent inactivation curve, although co-expression with a β1 subunit eliminates the difference between A1529G and WT. There is almost no difference in channel fast inactivation, but the β1 subunit accelerates WT current inactivation significantly more than it does the A1529G channels. The Ala-to-Gly substitution mainly influences the rate of recovery from slow inactivation. Again, the β1 subunit is less effective on speeding recovery of A1529G than the WT. We searched Nav channels in numerous databases and noted at least four other independent Ala-to-Gly substitutions in Nav channels in teleost fishes. Thus, the Ala-to-Gly substitution occurs more frequently than previously realized, possibly under selection for alterations of channel gating.     

Two Alternative Conformations of a Voltage-Gated Sodium Channel

Activation and inactivation of voltage-gated sodium channels (Navs) are well studied, yet the molecular mechanisms governing channel gating in the membrane remain unknown. We present two conformations of a Nav from Caldalkalibacillus thermarum reconstituted into lipid bilayers in one crystal at 9 Å resolution based on electron crystallography. Despite a voltage sensor arrangement identical with that in the activated form, we observed two distinct pore domain structures: a prominent form with a relatively open inner gate and a closed inner-gate conformation similar to the first prokaryotic Nav structure. Structural differences, together with mutational and electrophysiological analyses, indicated that widening of the inner gate was dependent on interactions among the S4–S5 linker, the N-terminal part of S5 and its adjoining part in S6, and on interhelical repulsion by a negatively charged C-terminal region subsequent to S6. Our findings suggest that these specific interactions result in two conformational structures.