Pathogenic gating pore current conducted by autism-related mutations in the NaV1.2 brain sodium channel.

Autism spectrum disorder (ASD) is a complex neurodevelopmental condition characterized by social and communication deficits and repetitive behaviors. The genetic heterogeneity of ASD presents a challenge to the development of an effective treatment targeting the underlying molecular defects. ASD gating charge mutations in the KCNQ/KV7 potassium channel cause gating pore currents (Igp) and impair action potential (AP) firing of dopaminergic neurons in brain slices. Here, we investigated ASD gating charge mutations of the voltage-gated SCN2A/NaV1.2 brain sodium channel, which ranked high among the ion channel genes with mutations in individuals with ASD. Our results show that ASD mutations in the gating charges R2 in Domain-II (R853Q), and R1 (R1626Q) and R2 (R1629H) in Domain-IV of NaV1.2 caused Igp in the resting state of ~0.1% of the amplitude of central pore current. The R1626Q mutant also caused significant changes in the voltage dependence of fast inactivation, and the R1629H mutant conducted proton-selective Igp. These potentially pathogenic Igp were exacerbated by the absence of the extracellular Mg2+ and Ca2+. In silico simulation of the effects of these mutations in a conductance-based single-compartment cortical neuron model suggests that the inward Igp reduces the time to peak for the first AP in a train, increases AP rates during a train of stimuli, and reduces the interstimulus interval between consecutive APs, consistent with increased neural excitability and altered input/output relationships. Understanding this common pathophysiological mechanism among different voltage-gated ion channels at the circuit level will give insights into the underlying mechanisms of ASD.

Dual receptor-sites reveal the structural basis for hyperactivation of sodium channels by poison-dart toxin batrachotoxin.

The poison dart toxin batrachotoxin is exceptional for its high potency and toxicity, and for its multifaceted modification of the function of voltage-gated sodium channels. By using cryogenic electron microscopy, we identify two homologous, but nonidentical receptor sites that simultaneously bind two molecules of toxin, one at the interface between Domains I and IV, and the other at the interface between Domains III and IV of the cardiac sodium channel. Together, these two bound toxin molecules stabilize α/π helical conformation in the S6 segments that gate the pore, and one of the bound BTX-B molecules interacts with the crucial Lys1421 residue that is essential for sodium conductance and selectivity via an apparent water-bridged hydrogen bond. Overall, our structure provides insight into batrachotoxin's potency, efficacy, and multifaceted functional effects on voltage-gated sodium channels via a dual receptor site mechanism.

Structural basis for severe pain caused by mutations in the voltage sensors of sodium channel NaV1.7.

Voltage-gated sodium channels in peripheral nerves conduct nociceptive signals from nerve endings to the spinal cord. Mutations in voltage-gated sodium channel NaV1.7 are responsible for a number of severe inherited pain syndromes, including inherited erythromelalgia (IEM). Here, we describe the negative shifts in the voltage dependence of activation in the bacterial sodium channel NaVAb as a result of the incorporation of four different IEM mutations in the voltage sensor, which recapitulate the gain-of-function effects observed with these mutations in human NaV1.7. Crystal structures of NaVAb with these IEM mutations revealed that a mutation in the S1 segment of the voltage sensor facilitated the outward movement of S4 gating charges by widening the pathway for gating charge translocation. In contrast, mutations in the S4 segments modified hydrophobic interactions with surrounding amino acid side chains or membrane phospholipids that would enhance the outward movement of the gating charges. These results provide key structural insights into the mechanisms by which these IEM mutations in the voltage sensors can facilitate outward movements of the gating charges in the S4 segment and cause hyperexcitability and severe pain in IEM. Our work gives new insights into IEM pathogenesis at the near-atomic level and provides a molecular model for mutation-specific therapy of this debilitating disease.

Cell-cycle arrest at the G1/S boundary enhances transient voltage-gated ion channel expression in human and insect cells.

Heterologous expression of recombinant ion channel subunits in cell lines is often limited by the presence of a low number of channels at the cell surface level. Here, we introduce a combination of two techniques: viral expression using the baculovirus system plus cell-cycle arrest at the G1/S boundary using either thymidine or hydroxyurea. This method achieved a manifold increase in the peak current density of expressed ion channels compared with the classical liposome-mediated transfection methods. The enhanced ionic current was accompanied by an increase in the density of gating charges, confirming that the increased yield of protein and ionic current reflects the functional localization of channels in the plasma membrane. This modified method of viral expression coordinated with the cell cycle arrest will pave the way to better decipher the structure and function of ion channels and their association with ion channelopathies.

Structural basis for severe pain caused by mutations in the S4-S5 linkers of voltage-gated sodium channel NaV1.7.

Gain-of-function mutations in voltage-gated sodium channel NaV1.7 cause severe inherited pain syndromes, including inherited erythromelalgia (IEM). The structural basis of these disease mutations, however, remains elusive. Here, we focused on three mutations that all substitute threonine residues in the alpha-helical S4-S5 intracellular linker that connects the voltage sensor to the pore: NaV1.7/I234T, NaV1.7/I848T, and NaV1.7/S241T in order of their positions in the amino acid sequence within the S4-S5 linkers. Introduction of these IEM mutations into the ancestral bacterial sodium channel NaVAb recapitulated the pathogenic gain-of-function of these mutants by inducing a negative shift in the voltage dependence of activation and slowing the kinetics of inactivation. Remarkably, our structural analysis reveals a common mechanism of action among the three mutations, in which the mutant threonine residues create new hydrogen bonds between the S4-S5 linker and the pore-lining S5 or S6 segment in the pore module. Because the S4-S5 linkers couple voltage sensor movements to pore opening, these newly formed hydrogen bonds would stabilize the activated state substantially and thereby promote the 8 to 18 mV negative shift in the voltage dependence of activation that is characteristic of the NaV1.7 IEM mutants. Our results provide key structural insights into how IEM mutations in the S4-S5 linkers may cause hyperexcitability of NaV1.7 and lead to severe pain in this debilitating disease.

When the Gates Swing Open Only: Arrhythmia Mutations That Target the Fast Inactivation Gate of Nav1.5.

Nav1.5 is the main voltage-gated sodium channel found in cardiac muscle, where it facilitates the fast influx of Na+ ions across the cell membrane, resulting in the fast depolarization phase-phase 0 of the cardiac action potential. As a result, it plays a major role in determining the amplitude and the upstroke velocity of the cardiac impulse. Quantitively, cardiac sodium channel activates in less than a millisecond to trigger the cardiac action potential and inactivates within 2-3 ms to facilitate repolarization and return to the resting state in preparation for firing the next action potential. Missense mutations in the gene that encodes Nav1.5 (SCN5A), change these time constants which leads to a wide spectrum of cardiac diseases ranging from long QT syndrome type 3 (LQT3) to sudden cardiac death. In this mini-review I will focus on the missense mutations in the inactivation gate of Nav1.5 that results in arrhythmia, attempting to correlate the location of the missense mutation to their specific phenotype.

Convergent regulation of CaV1.2 channels by direct phosphorylation and by the small GTPase RAD in the cardiac fight-or-flight response.

The L-type calcium currents conducted by the cardiac CaV1.2 calcium channel initiate excitation-contraction coupling and serve as a key regulator of heart rate, rhythm, and force of contraction. CaV1.2 is regulated by ß-adrenergic/protein kinase A (PKA)-mediated protein phosphorylation, proteolytic processing, and autoinhibition by its carboxyl-terminal domain (CT). The small guanosine triphosphatase (GTPase) RAD (Ras associated with diabetes) has emerged as a potent inhibitor of CaV1.2, and accumulating evidence suggests a key role for RAD in mediating ß-adrenergic/PKA upregulation of channel activity. However, the relative roles of direct phosphorylation of CaV1.2 channels and phosphorylation of RAD in channel regulation remain uncertain. Here, we investigated the hypothesis that these two mechanisms converge to regulate CaV1.2 channels. Both RAD and the proteolytically processed distal CT (dCT) strongly reduced CaV1.2 activity. PKA phosphorylation of RAD and phosphorylation of Ser-1700 in the proximal CT (pCT) synergistically reversed this inhibition and increased CaV1.2 currents. Our findings reveal that the proteolytically processed form of CaV1.2 undergoes convergent regulation by direct phosphorylation of the CT and by phosphorylation of RAD. These parallel regulatory pathways provide a flexible mechanism for upregulation of the activity of CaV1.2 channels in the fight-or-flight response.

Druggability of Voltage-Gated Sodium Channels-Exploring Old and New Drug Receptor Sites.

Voltage-gated ion channels are important drug targets because they play crucial physiological roles in both excitable and non-excitable cells. About 15% of clinical drugs used for treating human diseases target ion channels. However, most of these drugs do not provide sufficient specificity to a single subtype of the channels and their off-target side effects can be serious and sometimes fatal. Recent advancements in imaging techniques have enabled us for the first time to visualize unique and hidden parts of voltage-gated sodium channels in different structural conformations, and to develop drugs that further target a selected functional state in each channel subtype with the potential for high precision and low toxicity. In this review we describe the druggability of voltage-gated sodium channels in distinct functional states, which could potentially be used to selectively target the channels. We review classical drug receptors in the channels that have recently been structurally characterized by cryo-electron microscopy with natural neurotoxins and clinical drugs. We further examine recent drug discoveries for voltage-gated sodium channels and discuss opportunities to use distinct, state-dependent receptor sites in the voltage sensors as unique drug targets. Finally, we explore potential new receptor sites that are currently unknown for sodium channels but may be valuable for future drug discovery. The advancement presented here will help pave the way for drug development that selectively targets voltage-gated sodium channels.

Fenestropathy of Voltage-Gated Sodium Channels.

Voltage-gated sodium channels (Nav) are responsible for the initiation and propagation of action potentials in excitable cells. From pain to heartbeat, these integral membrane proteins are the ignition stations for every sensation and action in human bodies. They are large (>200 kDa, 24 transmembrane helices) multi-domain proteins that couple changes in membrane voltage to the gating cycle of the sodium-selective pore. Nav mutations lead to a multitude of diseases - including chronic pain, cardiac arrhythmia, muscle illnesses, and seizure disorders - and a wide variety of currently used therapeutics block Nav. Despite this, the mechanisms of action of Nav blocking drugs are only modestly understood at this time and many questions remain to be answered regarding their state- and voltage-dependence, as well as the role of the hydrophobic membrane access pathways, or fenestrations, in drug ingress or egress. Nav fenestrations, which are pathways that connect the plasma membrane to the central cavity in the pore domain, were discovered through functional studies more than 40 years ago and once thought to be simple pathways. A variety of recent genetic, structural, and pharmacological data, however, shows that these fenestrations are actually key functional regions of Nav that modulate drug binding, lipid binding, and influence gating behaviors. We discovered that some of the disease mutations that cause arrhythmias alter amino acid residues that line the fenestrations of Nav1.5. This indicates that fenestrations may play a critical role in channel's gating, and that individual genetic variation may also influence drug access through the fenestrations for resting/inactivated state block. In this review, we will discuss the channelopathies associated with these fenestrations, which we collectively name "Fenestropathy," and how changes in the fenestrations associated with the opening of the intracellular gate could modulate the state-dependent ingress and egress of drugs binding in the central cavity of voltage gated sodium channels.

Autism-associated mutations in KV7 channels induce gating pore current.

Autism spectrum disorder (ASD) adversely impacts >1% of children in the United States, causing social interaction deficits, repetitive behaviors, and communication disorders. Genetic analysis of ASD has advanced dramatically through genome sequencing, which has identified >500 genes with mutations in ASD. Mutations that alter arginine gating charges in the voltage sensor of the voltage-gated potassium (KV) channel KV7 (KCNQ) are among those frequently associated with ASD. We hypothesized that these gating charge mutations would induce gating pore current (also termed ω-current) by causing an ionic leak through the mutant voltage sensor. Unexpectedly, we found that wild-type KV7 conducts outward gating pore current through its native voltage sensor at positive membrane potentials, owing to a glutamine in the third gating charge position. In bacterial and human KV7 channels, gating charge mutations at the R1 and R2 positions cause inward gating pore current through the resting voltage sensor at negative membrane potentials, whereas mutation at R4 causes outward gating pore current through the activated voltage sensor at positive potentials. Remarkably, expression of the KV7.3/R2C ASD-associated mutation in vivo in midbrain dopamine neurons of mice disrupts action potential generation and repetitive firing. Overall, our results reveal native and mutant gating pore current in KV7 channels and implicate altered control of action potential generation by gating pore current through mutant KV7 channels as a potential pathogenic mechanism in autism.

Open-state structure and pore gating mechanism of the cardiac sodium channel.

The heartbeat is initiated by voltage-gated sodium channel NaV1.5, which opens rapidly and triggers the cardiac action potential; however, the structural basis for pore opening remains unknown. Here, we blocked fast inactivation with a mutation and captured the elusive open-state structure. The fast inactivation gate moves away from its receptor, allowing asymmetric opening of pore-lining S6 segments, which bend and rotate at their intracellular ends to dilate the activation gate to ∼10 Å diameter. Molecular dynamics analyses predict physiological rates of Na+ conductance. The open-state pore blocker propafenone binds in a high-affinity pose, and drug-access pathways are revealed through the open activation gate and fenestrations. Comparison with mutagenesis results provides a structural map of arrhythmia mutations that target the activation and fast inactivation gates. These results give atomic-level insights into molecular events that underlie generation of the action potential, open-state drug block, and fast inactivation of cardiac sodium channels, which initiate the heartbeat.

Novel G1481V and Q1491H SCN5A Mutations Linked to Long QT Syndrome Destabilize the Nav1.5 Inactivation State.

BACKGROUND: Nav1.5, which is encoded by the SCN5A gene, is the predominant voltage-gated Na+ channel in the heart. Several mutations of this gene have been identified and reported to be involved in several cardiac rhythm disorders, including type 3 long QT interval syndrome, that can cause sudden cardiac death. We analyzed the biophysical properties of 2 novel variants of the Nav1.5 channel (Q1491H and G1481V) detected in 5- and 12-week-old infants diagnosed with a prolonged QT interval. METHODS: The Nav1.5 wild-type and the Q1491H and G1481V mutant channels were reproduced in vi tr o. Wild-type or mutant channels were cotransfected in human embryonic kidney (HEK) 293 cells with the beta 1 regulatory subunit. Na+ currents were recorded using the whole-cell configuration of the patch-clamp technique. RESULTS: The Q1491H mutant channel exhibited a lower current density, a persistent Na+ current, an enhanced window current due to a +20-mV shift of steady-state inactivation, a +10-mV shift of steady-state activation, a faster onset of slow inactivation, and a recovery from fast inactivation with fast and slow time constants of recovery. The G1481V mutant channel exhibited an increase in current density and a +7-mV shift of steady-state inactivation. The observed defects are characteristic of gain-of-function mutations typical of type 3 long QT interval syndrome. CONCLUSIONS: The 5- and 12-week-old infants displayed prolonged QT intervals. Our analyses of the Q1491H and G1481V mutations correlated with the clinical diagnosis. The observed biophysical dysfunctions associated with both mutations were most likely responsible for the sudden deaths of the 2 infants.

INTRODUCTION: Le canal Nav1.5, codé par le gène SCN5A, est le canal Na+ dépendant du voltage prédominant dans le cœur. Plusieurs mutations de ce gène sont impliquées dans plusieurs anomalies du rythme cardiaque, dont le syndrome du QT long de type 3, qui peut provoquer la mort subite d'origine cardiaque. Nous avons analysé les propriétés biophysiques de deux nouveaux variants du canal Nav1.5 (Q1491H et G1481V) détectés chez deux bébés âgés respectivement de 5 et 12 semaines qui avaient une prolongation de l'intervalle QT. MÉTHODES: Le canal Nav1.5 de type sauvage et les canaux mutants Q1491H et G1481V ont été reproduits in vi tr o. Les canaux de type sauvage ou mutants ont été co-transfectés dans les cellules des reins embryonnaires humains (REH) 293 avec la sous-unité régulatrice bêta 1. Les courants Na+ ont été enregistrés à partir de la configuration en cellule entière via la technique de patch-clamp. RÉSULTATS: Le canal mutant Q1491H montre une densité de courant plus faible, un courant Na+ persistant, un courant fenêtre augmenté en raison d'un changement dép de +20 mV de l'inactivation à l'état stable, un changement de +10 mV de l'activation à l'état stable, une entrée plus rapide de l'inactivation lente et une récupération de l'inactivation rapide avec des constantes de temps rapides et lentes. Le canal mutant G1481V montre une augmentation de la densité de courant et un changement de +7 mV de l'inactivation à l'état stable. Les anomalies observées sont caractéristiques des mutations avec gain de fonction typiques du syndrome du QT long de type 3. CONCLUSIONS: Les deux bébés âgés respectivement de cinq 5 et 12 semaines montraient une prolongation des intervalles QT. Nos analyses des mutations Q1491H et G1481V montrent une corrélation avec le diagnostic clinique. Les dysfonctions biophysiques observées qui sont associées aux deux mutations étaient fort probablement responsables des morts subites des deux bébés.

Structural basis for voltage-sensor trapping of the cardiac sodium channel by a deathstalker scorpion toxin.

Voltage-gated sodium (NaV) channels initiate action potentials in excitable cells, and their function is altered by potent gating-modifier toxins. The α-toxin LqhIII from the deathstalker scorpion inhibits fast inactivation of cardiac NaV1.5 channels with IC50 = 11.4 nM. Here we reveal the structure of LqhIII bound to NaV1.5 at 3.3 Å resolution by cryo-EM. LqhIII anchors on top of voltage-sensing domain IV, wedged between the S1-S2 and S3-S4 linkers, which traps the gating charges of the S4 segment in a unique intermediate-activated state stabilized by four ion-pairs. This conformational change is propagated inward to weaken binding of the fast inactivation gate and favor opening the activation gate. However, these changes do not permit Na+ permeation, revealing why LqhIII slows inactivation of NaV channels but does not open them. Our results provide important insights into the structural basis for gating-modifier toxin binding, voltage-sensor trapping, and fast inactivation of NaV channels.

Structural Basis for High-Affinity Trapping of the NaV1.7 Channel in Its Resting State by Tarantula Toxin.

Voltage-gated sodium channels initiate electrical signals and are frequently targeted by deadly gating-modifier neurotoxins, including tarantula toxins, which trap the voltage sensor in its resting state. The structural basis for tarantula-toxin action remains elusive because of the difficulty of capturing the functionally relevant form of the toxin-channel complex. Here, we engineered the model sodium channel NaVAb with voltage-shifting mutations and the toxin-binding site of human NaV1.7, an attractive pain target. This mutant chimera enabled us to determine the cryoelectron microscopy (cryo-EM) structure of the channel functionally arrested by tarantula toxin. Our structure reveals a high-affinity resting-state-specific toxin-channel interaction between a key lysine residue that serves as a "stinger" and penetrates a triad of carboxyl groups in the S3-S4 linker of the voltage sensor. By unveiling this high-affinity binding mode, our studies establish a high-resolution channel-docking and resting-state locking mechanism for huwentoxin-IV and provide guidance for developing future resting-state-targeted analgesic drugs.

Computational design of transmembrane pores.

Transmembrane channels and pores have key roles in fundamental biological processes1 and in biotechnological applications such as DNA nanopore sequencing2-4, resulting in considerable interest in the design of pore-containing proteins. Synthetic amphiphilic peptides have been found to form ion channels5,6, and there have been recent advances in de novo membrane protein design7,8 and in redesigning naturally occurring channel-containing proteins9,10. However, the de novo design of stable, well-defined transmembrane protein pores that are capable of conducting ions selectively or are large enough to enable the passage of small-molecule fluorophores remains an outstanding challenge11,12. Here we report the computational design of protein pores formed by two concentric rings of α-helices that are stable and monodisperse in both their water-soluble and their transmembrane forms. Crystal structures of the water-soluble forms of a 12-helical pore and a 16-helical pore closely match the computational design models. Patch-clamp electrophysiology experiments show that, when expressed in insect cells, the transmembrane form of the 12-helix pore enables the passage of ions across the membrane with high selectivity for potassium over sodium; ion passage is blocked by specific chemical modification at the pore entrance. When incorporated into liposomes using in vitro protein synthesis, the transmembrane form of the 16-helix pore-but not the 12-helix pore-enables the passage of biotinylated Alexa Fluor 488. A cryo-electron microscopy structure of the 16-helix transmembrane pore closely matches the design model. The ability to produce structurally and functionally well-defined transmembrane pores opens the door to the creation of designer channels and pores for a wide variety of applications.

Structure and Pharmacology of Voltage-Gated Sodium and Calcium Channels.

Voltage-gated sodium and calcium channels are evolutionarily related transmembrane signaling proteins that initiate action potentials, neurotransmission, excitation-contraction coupling, and other physiological processes. Genetic or acquired dysfunction of these proteins causes numerous diseases, termed channelopathies, and sodium and calcium channels are the molecular targets for several major classes of drugs. Recent advances in the structural biology of these proteins using X-ray crystallography and cryo-electron microscopy have given new insights into the molecular basis for their function and pharmacology. Here we review this recent literature and integrate findings on sodium and calcium channels to reveal the structural basis for their voltage-dependent activation, fast and slow inactivation, ion conductance and selectivity, and complex pharmacology at the atomic level. We conclude with the theme that new understanding of the diseases and therapeutics of these channels will be derived from application of the emerging structural principles from these recent structural analyses.

Structure of the Cardiac Sodium Channel.

Voltage-gated sodium channel Nav1.5 generates cardiac action potentials and initiates the heartbeat. Here, we report structures of NaV1.5 at 3.2-3.5 Å resolution. NaV1.5 is distinguished from other sodium channels by a unique glycosyl moiety and loss of disulfide-bonding capability at the NaVß subunit-interaction sites. The antiarrhythmic drug flecainide specifically targets the central cavity of the pore. The voltage sensors are partially activated, and the fast-inactivation gate is partially closed. Activation of the voltage sensor of Domain III allows binding of the isoleucine-phenylalanine-methionine (IFM) motif to the inactivation-gate receptor. Asp and Ala, in the selectivity motif DEKA, line the walls of the ion-selectivity filter, whereas Glu and Lys are in positions to accept and release Na+ ions via a charge-delocalization network. Arrhythmia mutation sites undergo large translocations during gating, providing a potential mechanism for pathogenic effects. Our results provide detailed insights into Nav1.5 structure, pharmacology, activation, inactivation, ion selectivity, and arrhythmias.

Structural Basis for Diltiazem Block of a Voltage-Gated Ca2+ Channel.

Diltiazem is a widely prescribed Ca2+ antagonist drug for cardiac arrhythmia, hypertension, and angina pectoris. Using the ancestral CaV channel construct CaVAb as a molecular model for X-ray crystallographic analysis, we show here that diltiazem targets the central cavity of the voltage-gated Ca2+ channel underneath its selectivity filter and physically blocks ion conduction. The diltiazem-binding site overlaps with the receptor site for phenylalkylamine Ca2+ antagonist drugs such as verapamil. The dihydropyridine Ca2+ channel blocker amlodipine binds at a distinct site and allosterically modulates the binding sites for diltiazem and Ca2+ Our studies resolve two distinct binding poses for diltiazem in the absence and presence of amlodipine. The binding pose in the presence of amlodipine may mimic a high-affinity binding configuration induced by voltage-dependent inactivation, which is favored by dihydropyridine binding. In this binding pose, the tertiary amino group of diltiazem projects upward into the inner end of the ion selectivity filter, interacts with ion coordination Site 3 formed by the backbone carbonyls of T175, and alters binding of Ca2+ to ion coordination Sites 1 and 2. Altogether, our results define the receptor site for diltiazem and elucidate the mechanisms for pore block and allosteric modulation by other Ca2+ channel-blocking drugs at the atomic level. SIGNIFICANCE STATEMENT: Calcium antagonist drugs that block voltage-gated calcium channels in heart and vascular smooth muscle are widely used in the treatment of cardiovascular diseases. Our results reveal the chemical details of diltiazem binding in a blocking position in the pore of a model calcium channel and show that binding of another calcium antagonist drug alters binding of diltiazem and calcium. This structural information defines the mechanism of drug action at the atomic level and provides a molecular template for future drug discovery.

Resting-State Structure and Gating Mechanism of a Voltage-Gated Sodium Channel.

Voltage-gated sodium (NaV) channels initiate action potentials in nerve, muscle, and other electrically excitable cells. The structural basis of voltage gating is uncertain because the resting state exists only at deeply negative membrane potentials. To stabilize the resting conformation, we inserted voltage-shifting mutations and introduced a disulfide crosslink in the VS of the ancestral bacterial sodium channel NaVAb. Here, we present a cryo-EM structure of the resting state and a complete voltage-dependent gating mechanism. The S4 segment of the VS is drawn intracellularly, with three gating charges passing through the transmembrane electric field. This movement forms an elbow connecting S4 to the S4-S5 linker, tightens the collar around the S6 activation gate, and prevents its opening. Our structure supports the classical "sliding helix" mechanism of voltage sensing and provides a complete gating mechanism for voltage sensor function, pore opening, and activation-gate closure based on high-resolution structures of a single sodium channel protein.