The crystal structure of a voltage-gated sodium channel.

Voltage-gated sodium (Na(V)) channels initiate electrical signalling in excitable cells and are the molecular targets for drugs and disease mutations, but the structural basis for their voltage-dependent activation, ion selectivity and drug block is unknown. Here we report the crystal structure of a voltage-gated Na(+) channel from Arcobacter butzleri (NavAb) captured in a closed-pore conformation with four activated voltage sensors at 2.7 Å resolution. The arginine gating charges make multiple hydrophilic interactions within the voltage sensor, including unanticipated hydrogen bonds to the protein backbone. Comparisons to previous open-pore potassium channel structures indicate that the voltage-sensor domains and the S4-S5 linkers dilate the central pore by pivoting together around a hinge at the base of the pore module. The NavAb selectivity filter is short, ∼4.6 Å wide, and water filled, with four acidic side chains surrounding the narrowest part of the ion conduction pathway. This unique structure presents a high-field-strength anionic coordination site, which confers Na(+) selectivity through partial dehydration via direct interaction with glutamate side chains. Fenestrations in the sides of the pore module are unexpectedly penetrated by fatty acyl chains that extend into the central cavity, and these portals are large enough for the entry of small, hydrophobic pore-blocking drugs. This structure provides the template for understanding electrical signalling in excitable cells and the actions of drugs used for pain, epilepsy and cardiac arrhythmia at the atomic level.

Electrical signals (termed action potentials) encode and process information within the nervous system and regulate a wide range of physiological processes[1,2]. The voltage-gated ion channels (VGICs) that mediate electrical signaling have distinct functional roles[1,2]. Voltage-gated sodium (NaV) channels initiate action potentials. Voltage-gated calcium (CaV) channels initiate processes such as synaptic transmission, muscle contraction, and hormone secretion in response to membrane depolarization. Voltage-gated potassium (KV) channels terminate action potentials and return the membrane potential to its resting value. The NaV channels are mutated in inherited epilepsy, migraine, periodic paralysis, cardiac arrhythmia, and chronic pain syndromes[3]. These channels are molecular targets of drugs used in local anesthesia and in treatment of genetic and sporadic NaV channelopathies in brain, skeletal muscle, and heart[4]. The rapid activation, Na+-selectivity, and drug sensitivity of NaV channels are unique among VGICs[2].

VGICs share a conserved architecture in which four subunits or homologous domains create a central ion-conducting pore surrounded by four voltage-sensors[5]. The voltage-sensing domain (VSD) is composed of the S1-S4 segments, while the pore module is formed by the S5 and S6 segments with a P-loop between them[5]. The S4 segments place charged amino acids within the membrane electric field that undergo outward displacement in response to depolarization and initiate opening of the central pore[6,7]. Although the architecture of KV channels has been established at high-resolution[8,9], the structural basis for rapid, voltage-dependent activation of VGICs remains uncertain[7,9], and the structures responsible for Na+-selective conductance and drug block in NaV channels are unknown. The primary pore-forming subunits of NaV and CaV proteins in vertebrates are composed of approximately 2,000 amino acid residues in four linked homologous domains[5]. The bacterial NaChBac channel family is an important model for structure-function studies of more complex vertebrate NaV and CaV channels[10,11]. NaChBac is a homotetramer, and its pharmacological profile is similar to NaV and CaV channels.[10,12] Bacterial NaV channels are highly Na+-selective, but they can be converted into Ca2+-selective forms through simple mutagenesis[13]. The NaChBac family represents the likely ancestor of vertebrate NaV and CaV channels. Through analysis of the three-dimensional structure of NavAb from Arcobacter butzleri, we provide the first insights into the structural basis of voltage-dependent gating, ion selectivity, and drug block in NaV and CaV channels.

Structure of NavAb in a membrane environment

NavAb is a member of the NaChBac family and functions as a voltage-gated sodium-selective ion channel (Supplementary Fig. 1, 2). Vertebrate CaV channels require solubilization in digitonin and NaV channels require specific lipids to retain function when purified[14,15]. Accordingly, we solubilized NavAb in digitonin, crystallized it in a lipid-based bicelle system, and determined its structure at 2.70 Å resolution (Supplementary Fig. 3, 4, 5, 6; Table 1). NavAb crystallized as a dimer-of-dimers with 28 lipid molecules bound per tetramer (Supplementary Figure 3, 6b). Crystal-packing suggests a membrane-like environment (Supplementary Fig. 6a). NavAb VSDs interact noncovalently with the pore module of a neighboring subunit (Fig. 1a), and crystallographic temperature factors highlight their dynamic nature (Supplementary Fig. 6c).

Figure 1

Structure of NavAb and the activated VSD

a, Structural elements in NavAb. One subunit is highlighted (1-6, transmembrane segments S1-S6). The nearest VSD has been removed for clarity. b, Side and top-views of the VSD illustrating the extracellular negative charge-cluster (red, ENC), the intracellular negative charge-cluster (red, INC), hydrophobic constriction site (green, HCS), residues of the S1N helix (cyan) and phenylalanines of the S2-S3 loop (purple). S4 segment and gating charges (R1-R4) are in yellow. c-e, Hydrogen bonding of gating-charges, dotted lines (<3.5 Å). Fo-Fc omit maps are contoured over E96 and R1-R4 at 1, 1, 1.5, 2.5 and 1.75 σ, respectively. f, S3-S4 loop. Colored according to crystallographic temperature factors of the main-chain (blue < 50 Å2 to red > 150 Å2). An Fo-Fc omit map is contoured at 1.5 σ (grey) and 2.5 σ (pink).

Structure of the activated voltage-sensor

S4 segments in VSDs consist of repeated motifs of a positively charged residue, usually arginine, followed by two hydrophobic residues[5-7]. The R2 and R3 “gating charges” in NavAb are positioned to interact with a conserved extracellular negative-charge cluster (ENC), while the R4 gating charge interacts with a conserved intracellular negative-charge cluster (INC; Fig. 1b). These structural features, in conjunction with disulphide-locking experiments[16,17], indicate that the VSDs are activated. These ion-pair interactions are expected to stabilize and catalyze S4 movement in the membrane electric field[7,18,19]. Highly conserved Arg63 in the S2 segment also interacts with R4 and the INC (Fig. 1e), which may stabilize the INC and modulate its electrostatics[20]. NavAb has a spectrum of additional gating charge interactions. R1 interacts with Glu96, R2 forms a hydrogen bond with the backbone carbonyl of Val89 in S3, and R3 forms hydrogen bonds with Asn25 and Met29 in S1, and Ser87 in S3 (Fig. 1c-e). This network of hydrogen bonds (Supplementary Fig. 7a) should complement exchange of ion-pair partners and provide a low energy pathway for S4 movement. The R2-backbone interaction would escape detection in mutagenesis experiments (Fig. 1c) and could have unrecognized significance in passage of gating charges through the gating pore (Fig. 1b).

The S4 segment in NavAb forms a 310-helix from R1 to R4. This conformation places all four gating charges in a straight line on one side of S4 (Fig. 1b), such that they could move linearly through the central portion of the gating pore, rather than in a spiral pattern[7,17-19]. The S3 segment is a straight α-helix, and the S3-S4 loop displays a dynamic connection to S4 (Fig. 1f). The lack of structural rigidity within the S3-S4 loop (Fig. 1f) suggests that it moves relatively freely in response to large S4 movements during activation.

Our structural analysis further reveals that the S1N helix and S2-S3 loop shield the intracellular surface of the VSD (Fig. 1b and Supplementary Fig. 8). The S2-S3 loop is conserved among VGICs, and two prominent Phe side-chains likely stabilize the VSD in the membrane during gating transitions (Fig. 1b, Supplementary Fig. 7, 8)[9]. The S1N-to-S3 region may behave as a modular unit during activation. In contrast to the sheltered intracellular surface of the VSD, a large aqueous cleft extends ~10 Å into the membrane above the hydrophobic constriction site (HCS, Fig. 1b). The HCS contains highly conserved residues (Ile22, Phe56 and Val84; Supplementary Fig. 7) that seal the VSD against ion leakage during S4 movement (Fig. 1b). The NavAb VSD therefore illustrates two important concepts from structure-function studies of NaV channels: a large external vestibule accessible to hydrophilic reagents and a focused membrane electric field over the intracellular half of the VSD[6,7].

Despite their separation over one billion years of evolution, the VSDs of NavAb and KV1.2 display highly similar conformations (Supplementary Fig. 8a). R4 of NavAb is in an equivalent position to K5 in KV1.2 (Supplementary Fig. 8a), the most outward location of K5 during voltage-sensor activation[20]. This observation implies that the NavAb and KV1.2 VSDs are both activated.

The NavAb activation gate is closed

The pore of NavAb is closed, providing the first view of a closed pore in a VGIC (Fig. 2a and Supplementary Fig. 3). Met221 completely occludes the ion conduction pathway, as confirmed by unbiased experimentally-phased electron-density maps (Supplementary Fig. 4c). The S6 helices of NavAb superimpose well with other closed-pore structures and are distinct from the open-pore KV1.2 structure (Fig. 2a, b). A subtle iris-like dilation of the activation gate may be sufficient to open the pore, and the surrounding cuff of S4-S5 linkers may prevent larger pore opening (Fig. 2a-c).

Figure 2

NavAb pore module

a, Pore-lining S6 helices of NavAb (yellow) and the closed MlotiK (3BEH), KcsA (1K4C) and NaK (2AHY) channels. Cα locations of Met221 defines a common radius for the closed activation gate (red circle). b, Comparison of S6 helices of NavAb and KV1.2/2.1 (2R9R). c, Interaction of S6 with S4-S5 linkers (NavAb, top; KV1.2/2.1, bottom). d, Architecture of the NavAb pore. Glu177 side-chains (purple sticks); pore volume, grey. e, Electrostatic potential colored from −10 to 10 kT (red to blue).

It is surprising to have a closed pore in a VGIC with activated voltage-sensors at 0 mV. Our NavAb structures were obtained by introducing a Cys at two locations near the intracellular end of S6 (Ile217Cys and Met221Cys). Evidently, these substitutions allowed us to trap the NavAb channel in the pre-open state previously invoked in kinetic models of VGIC gating (Supplementary Discussion)[21-23].

Architecture of the pore and selectivity filter

VGICs are selective for specific cations yet conduct these ions at nearly the rate of free diffusion[2]. Our NavAb structure uncovers a basis for selectivity and high conductance of NaV channels. The NavAb pore module consists of an outer funnel-like vestibule, a selectivity filter, a central cavity, and an intracellular activation gate (Fig. 2d, Supplementary Fig. 4b). The large central cavity in NavAb accommodates a Na+ ion with one hydration shell and presents a hydrophobic surface over which ions should rapidly diffuse (Fig. 2e, Supplementary Figs. 1, 9). The pore (P)-helices are positioned to stabilize cations in the central cavity through helical-dipole interactions (Fig. 2d, Supplementary Fig. 4b), as for K+-channels[24,25]. Strikingly, a second pore-helix (P2-helix) forms an extracellular funnel in NavAb (Fig. 2d). This unique P2-helix is not seen in K+ channels and may represent a conserved structural element in the outer vestibule of NaV and CaV channels.

The ion conduction pathway in NavAb is strongly electronegative and the selectivity filter forms the narrowest constriction near the extracellular side of the membrane (Fig. 2d, e, 3; Supplementary Fig. 9). Classic permeation studies suggested a high field-strength anionic site with dimensions of ~3.1 × 5.1 Å for the selectivity filter in NaV channels[26,27] and 5.5 × 5.5 Å in CaV channels[28]. Mutagenesis studies implicated Glu side-chains as key determinants of ion selectivity in these channels[29-33]. In NavAb, the four Glu177 side-chains form a ~6.5 × 6.5 Å rectangle with an orifice of ~4.6 × 4.6 Å defined by van der Waals surfaces (Fig. 3a, Supplementary Fig. 9d). Remarkably, Glu177 aligns with Glu residues that determine ion selectivity in NaV and CaV channels (Fig. 3e).

Figure 3

Structure of the NavAb selectivity filter

a, Top view of the selectivity filter. Symmetry-related molecules are colored white and yellow; P-helix residues are colored green. Hydrogen bonds between Thr175 and Trp179 are indicated by grey dashes. Electron-densities from Fo-Fc omit maps are contoured at 4.0 σ (blue and grey) and subtle differences can be appreciated (small arrows). b, Side view of the selectivity filter. Glu177 (purple) interactions with Gln172, Ser178 and the backbone of Ser180 are shown in the far subunit. Fo-Fc omit map, 4.75 σ (blue); putative cations or water molecules (red spheres, IonEX). Electron-density around Leu176 (grey; Fo-Fc omit map at 1.75 σ) and a putative water molecule is shown (grey sphere). Na+-coordination sites: SiteHFS, SiteCEN and SiteIN. c, Superposition of NavAb and a K+-channel selectivity filter: NavAb Glu177 side-chains (purple) and backbone carbonyls (*); K+-channel (blue, 1K4C), site 3 and site 4 backbone carbonyls (ref. 35) (*). This structural alignment is based on P-helices. d, Enlarged view of SiteCEN and SiteIN: water molecules, grey spheres; dotted lines, ~2.5 Å. e, Selectivity filter sequence alignment. E177 homologs, purple; outer-ring of negatively charged residues (red).

The Glu177 side-chains of NavAb are supported by an elaborate architecture. The P-helix ends with the conserved Thr175, which accepts a hydrogen bond (3.0 Å) from the conserved Trp179 of a neighboring subunit (Fig. 3a). This landmark interaction staples together adjacent subunits at the selectivity filter. The residues between Thr175 and Trp179 form a tight turn and expose backbone carbonyls of Thr175 and Leu176 to conducted ions (Fig. 3b). The Glu177 side-chains form hydrogen bonds with the backbone amides of Ser180 (2.6 Å) and Met181 (3.1 Å) from the P2-helix (Fig. 3b, Supplementary Fig. 10). An extensive network of additional interactions (Supplementary Fig. 10), including hydrogen bonds between Gln172 from the P-helix and the carbonyl of Glu177 (Fig. 3a, b), further stabilizes the selectivity filter. Due to the dimer-of-dimers arrangement, the Glu177 and Ser178 side-chains of NavAb are in two slightly different environments (small arrows, Fig. 3a, Supplementary Fig. 11), consistent with functional nonequivalence of the corresponding glutamates in CaV channels[31-33].

In agreement with the low affinity of NaV channels for permeant ions (Kd for Na+>350 mM[34]), no extra density was observed beside the Glu177 side-chains. Instead, strong electron densities were found above Glu177 at a distance of >4 Å. These densities likely represent cations or solvent molecules (IonEX, Fig. 3b) positioned above the selectivity filter by its intense electronegativity (Fig. 2e).

Ion permeation and selectivity

NavAb represents a prototype for understanding Na+ selectivity and permeation. Analysis of the pore radius indicates that a partially hydrated Na+ ion can be accommodated at the high field-strength site formed by the Glu177 side-chains (SiteHFS; Fig. 3a,b, Supplementary Fig. 9d). The much smaller K+-channel filter can fit inside the NavAb selectivity filter (Fig. 3c). Careful inspection of electron density suggests four well-bound water molecules 2.5 Å from the Leu176 carbonyls (SiteCEN, Fig. 3b). Remarkably, these four water molecules occupy the same positions as site 3 carbonyls from K+-channels (Fig. 3c, d)[35]. A distance of 2.5 Å is also found between the backbone carbonyls of Thr175 from NavAb and the site 4 carbonyls of K+-channels (Fig. 3c, d)[35]. Similar to other Na+ complexes (Supplementary Fig. 12)[36-40], a Na+ ion surrounded by a square array of four water molecules could interact with backbone carbonyls of Leu176 (SiteCEN) or Thr175 (SiteIN) (Fig. 3d, Supplementary Fig. 12). Therefore, unlike K+-channels, the NavAb selectivity filter appears to select and conduct Na+ ions in a mostly hydrated form.

The NavAb structure fits closely with Hille’s single-ion pore model for NaV channels, in which a high-field-strength anion partially dehydrates the permeating ion[2,34]. According to Eisenman’s theory[41], a Na+ ion would approach the SiteHFS more closely than the larger K+ ion, allowing more efficient removal of water and faster permeation (Fig. 3a, b)[34]. A Na+ ion could fit in-plane between the Glu177 side-chains, with one side-chain coordinating the Na+ ion directly and neighboring Glu177 side-chains acting as hydrogen bond acceptors for in-plane water molecule(s)[26,27,34]. With two waters remaining axial to the ion, this arrangement would approximate trigonal bipyramidal coordination[38]. Since only one Glu177 side-chain engages the permeating ion directly, this transient complex would be inherently asymmetric. When the permeating ion escapes SiteHFS, full rehydration occurs along the water-lined sites formed by the backbone carbonyls of Leu176 (SiteCEN) and Thr175 (SiteIN; Fig. 3b,d, Supplementary Fig. 12).

Free diffusion then allows the hydrated Na+ ion to enter the central cavity and move through the open activation gate into the cytoplasm[34]. The selectivity-filter structure of NavAb concentrates barriers to ion flow into ~5 Å (Fig. 3b, Supplementary Fig. 9d), which should promote high flux rates[34]. This permeation mechanism likely reflects the high free energy of Na+ hydration, where further removal of solvating waters would present too high an energy barrier. In sharp contrast, K+-selective channels conduct nearly fully-dehydrated K+ ions through direct interactions with backbone carbonyls in a long, narrow, multi-ion pore[35,36]. The selectivity filters of vertebrate NaV and CaV channels likely resemble NavAb, and amino acid substitutions within this framework must impart Na+ versus Ca2+selectivity (Supplementary Discussion)[13,29-33].

Interaction sites of pore blockers

NavAb provides a foundation to interpret pharmacological mechanisms. From the extracellular side, the Glu177 side-chains of NavAb represent the blocking site of Nav channels by protons and guanidinium moieties of tetrodotoxin and saxitoxin[2,42], as well as the site where divalent cations and protons bind and block CaV channels (Fig. 3)[31-33]. From the intracellular side, local anesthetics, antiarrhythmics, and antiepileptic drugs block NaV and CaV channels[2,4] by entering through the open intracellular mouth of the pore and binding to an overlapping receptor site on the S6 segments[43-45]. Alignment of NavAb S6 segments with vertebrate NaV and CaV channels reveals a high degree of sequence similarity (Supplementary Fig. 7b), and drug molecules could easily fit into the large central cavity (Fig. 3, Supplementary Fig. 9). Use-dependent block is enhanced by repetitive opening of the pore to provide drug access[2,46], and the local anesthetic etidocaine is an open-channel blocker of NaChBac[12]. The tight seal observed at the intracellular activation gate in NavAb illustrates why pore opening is required for access of large or hydrophilic drugs to the S6 receptor site (Fig. 2, Supplementary Fig. 4c).

Fenestrations provide hydrophobic access to the pore

Membrane lipids modulate the structure and function of VGICs[8,9,47,48]. However, NavAb presents a completely unexpected type of lipid interaction that has profound implications. The NavAb central cavity reveals four lateral openings leading from the membrane to the lumen of the closed pore (Fig. 4). These fenestrations measure ~8 × 10 Å, and could become larger depending upon nearby side-chain conformations (Phe203, Fig. 4). Lipids penetrate through these side portals and lie deep within the central cavity, occluding the ion conduction pathway in NavAb (Fig. 4, red). Because acyl-chain containing detergents were never used in preparation of NavAb crystals, these electron densities are assigned as acyl chains of membrane phospholipids. Similar fenestrations were not observed in the open-pore structure of KV1.2 (refs 8,9), raising the possibility that these lipid chains withdraw and the fenestrations close in the open state.

Figure 4

Membrane access to the central cavity in NavAb

a, Side-view through the pore module illustrating fenestrations (portals) and hydrophobic access to central cavity. Phe203 side-chains, yellow sticks. Surface representations of NavAb residues aligning with those implicated in drug binding and block, Thr206, blue; Met209, green; Val213, orange. Membrane boundaries, grey lines. Electron-density from an Fo-Fc omit map is contoured at 2.0 σ. b, Top-view sectioned below the selectivity filter, colored as in a.

The lateral pore fenestrations in NavAb lead directly to the drug binding sites within the central cavity and abut residues that are important for drug binding in NaV and CaV channels (Fig. 4, Supplementary Fig. 7b)[43,44]. These NavAb portals appear compatible with passage of small neutral or hydrophobic drugs such as phenytoin[49] and benzocaine[46], which can gain access to their receptor site in closed channels[2,46]. We propose that pore fenestrations may be directly involved in voltage-dependent drug block according to the “modulated receptor model”[46]. Our findings highlight the potential for lipids and other hydrophobic molecules to influence the function of ion channels from the lipid phase of the membrane.

Structural basis for central pore gating

The domain-swapped arrangement of the VSD around the pore allows the S4-S5 linker to couple S4 movements to activation of VGICs (Fig. 1a)[9]. Kinetic models indicate that all four voltage-sensors activate and then the central pore opens in a concerted transition[21-23]. An essential element of this gating model is a state in which all four VSDs have activated but the pore remains closed[21-23]. It is likely that we have captured this pre-open state in our crystals (Supplementary Discussion). NavAb therefore provides a unique opportunity to consider the structural basis for coupling of VSD activation to pore-opening.

When activated VSDs of NavAb and KV1.2 are overlaid (Supplementary Fig. 8a), the S4-S5 linkers superimpose precisely, but the pore domains diverge at the foot of S5 (Fig. 5a). Superposition of the pore domains demonstrates an equivalent displacement of the VSDs (Supplementary Fig. 13). These comparisons lead to a working model for pore opening. First, during activation, the S4-S5 linker and VSD move together as a modular unit (Fig. 5a). Second, a single molecular hinge at the base of S5 mediates the closed-to-open pore transition (Fig. 5a, b). Third, tight structural-coupling is maintained between the S5 and S6 segments (Supplementary Fig. 13a). This model suggests that rotation of the VSD and S4-S5 linker as a structural unit pulls the S5-S6 helices outward to open the pore (Fig. 5b, Supplementary Fig. 13b). Because of their tight structural-coupling, displacement of the S5-S6 segments from one subunit forces the neighboring subunits to move similarly, leading to concerted pore opening. During this transition, the amphipathic S4-S5 linker pivots along the plane of the membrane interface (Fig. 5b, Supplementary Fig. 7, 13b). In contrast to KV1.2, the S6 helices in NavAb have not fully engaged their interaction site on the S4-S5 linker (Fig. 2c), in agreement with NavAb’s pre-open state. The rolling motion of the VSDs around the pore produces displacements up to ~10 Å at the intracellular side (Fig. 5b, Supplementary Fig. 13b), which may influence movements of the S1N helix and the conserved S2-S3 loop.

Figure 5

Model for activation gate opening

a, Superposition of the NavAb and KV1.2/2.1 based on their VSDs (cylinders). b, Superposition of NavAb and KV1.2/2.1 tetrameric pore modules (PM) viewed from the membrane: S5 gating hinge, *. c-d, S1 interaction with P-helix. .S1 Thr is P-helix is 2.9 Å neighboring subunit in KV1.2/2.1 (), but >4.5 Å in NavAb.

In NavAb, a 310-helix extends from R1 to R4 (Fig. 1b). In KV1.2, a 310-helix encompasses R3 to K5 (equivalent to NavAb R2 to R4), but the remaining S4 segment is α-helical[9]. Conceivably, energy derived from voltage-driven translocation of S4 may be stored in the higher-energy 310-helix, and then released to help drive pore opening. The VSDs in KV1.2 are displaced outward (~2 Å) compared to the pre-open NavAb structure (Fig. 5b), which could account for the small gating current associated with concerted pore-opening[6]. At the extracellular side of the VSD, an S1 threonine residue hydrogen bonds (2.9 Å) with the P-helix of a neighboring subunit in KV1.2 (Fig. 5d), providing a conserved contact point that allows the VSD to perform mechanical work on the pore[50]. The equivalent S1 threonine in NavAb has not yet engaged the P-helix (Fig. 5c). This interaction may therefore represent an essential step in activation gating that has not yet occurred in the pre-open state of NavAb.

Conclusion

The structure of NavAb provides key insights into the molecular basis of voltage-sensing, ion conductance, and voltage-dependent gating in a historic class of ion channels[1,2]. A new network of interactions within the VSD appears well-positioned to catalyze gating charge movements during activation. Our model for electromechanical coupling reveals a rolling motion of the VSD and its connecting S4-S5 linker around the pore. The NavAb selectivity filter illustrates the basis for selective Na+ conductance through a water-lined pore featuring a high-field-strength anionic site. Finally, hydrophobic access from the membrane phase has been uncovered as a potentially important pathway for drug binding and modulation of VGICs.