Eukaryotic Voltage-gated Sodium Channels Research Articles

N-type fast inactivation of a eukaryotic voltage-gated sodium channel

Voltage-gated sodium (NaV) channels initiate action potentials. Fast inactivation of NaV channels, mediated by an Ile-Phe-Met motif, is crucial for preventing hyperexcitability and regulating firing frequency. Here we present cryo-electron microscopy structure of NaVEh from the coccolithophore Emiliania huxleyi, which reveals an unexpected molecular gating mechanism for NaV channel fast inactivation independent of the Ile-Phe-Met motif. An N-terminal helix of NaVEh plugs into the open activation gate and blocks it. The binding pose of the helix is stabilized by multiple electrostatic interactions. Deletion of the helix or mutations blocking the electrostatic interactions completely abolished the fast inactivation. These strong interactions enable rapid inactivation, but also delay recovery from fast inactivation, which is ~160-fold slower than human NaV channels. Together, our results provide mechanistic insights into fast inactivation of NaVEh that fundamentally differs from the conventional local allosteric inhibition, revealing both surprising structural diversity and functional conservation of ion channel inactivation.

Possible Mechanism of Ion Selectivity in Eukaryotic Voltage-Gated Sodium Channels

The outer pore of Nav1.x channels is lined by the selectivity-filter ring Asp-Glu-Lys-Ala (DEKA), an outer ring of carboxylates, and two inner rings of backbone carbonyls. A key role of Lys in the Na+/K+ selectivity is known, but the mechanism is unclear. Here, contacts involving DEKA residues in 15 cryo-EM structures of Nav1.x channels were analyzed and Monte Carlo (MC) energy minimizations of models with the DEKA residues in different protonation states, with or without Na+ or K+, were performed. In MC-minimized structures, protonated Lys+ was salt-bridged with Glu, whereas deprotonated Lys•• "dunked" to the inner rings. When Na+ was pulled through the outer pore, it was inevitably chelated by Glu and Lys•• at the narrow pore levels. Lys•• further escorted Na+ to the inner rings and in several steps mutual dispositions of the DEKA residues are similar to those seen in cryo-EM structures. Analogous results were obtained in models with DEKA mutants, which have high, but not low Na+/K+ selectivity. When K+ was pulled through the pore, it was also chelated between Glu and Lys••, but respective distances were larger and K+ energy was higher than in models with Na+. The computations suggest that salt-bridged Lys+ and Glu block the pore. Approaching Na+ would knock out H+, squeeze between Glu and Lys••, and move down escorted by Lys••, whereas the displaced H+ would stay nearby in a H-bond involving Glu or/and Asp. When Na+ leaves the outer pore, reprotonated Lys•• would rejoin Glu to complete the permeation cycle.

Computational Study of Ion Selectivity in Eukaryotic Voltage-Gated Sodium Channels

The outer pore of Nav1.x channels is lined by the selectivity-filter ring Asp-Glu-Lys-Ala (DEKA), an outer ring of carboxylates, and two inner rings of backbone carbonyls. A key role of Lys in the Na/K selectivity is known, but the mechanism is unclear. The DEKA residues may exist in different protonation forms. Here contacts involving DEKA residues in 15 cryo-EM structures of Nav1.x channels were analyzed. The analysis suggests that either Asp or Glu are protonated in the majority of the structures. Monte Carlo (MC) energy minimizations of models with the DEKA residues in different protonation states, with or without Na+ or K+ were further performed. In MC-minimized structures protonated Lys+ was salt bridged with Glu-, whereas deprotonated Lys·· “dunked” to the inner rings. When Na+ was pulled through the outer pore, it was inevitably chelated by Glu- and Lys·· at the narrow pore levels. Lys·· further escorted Na+ to the inner rings and in several steps mutual dispositions of the DEKA residues are similar to those seen in cryo-EM structures. Analogous results were obtained in models with DEKA mutants, which have high, but not low Na/K selectivity. When K+ was pulled through the pore, it was also chelated between Glu- and Lys··, but respective distances were expectedly larger and K+ energy was higher than in models with Na+. The computations suggest that salt-bridged Lys+ and Glu- block the pore. Approaching Na+ would knock-out H+, squeeze between Glu- and Lys·· and move down escorted by Lys··, whereas the displaced H+ would stay nearby in an H-bond involving Glu or/and Asp. When Na+ leaves the outer pore, Lys·· would be re-protonated and rejoin Glu- to complete the permeation cycle. Supported by NSERC (RGPIN-2020-07100) and RSF (17-15-01292-П).

Exploring Noncanonical Coupling in Voltage-Gated Sodium Channels

Eukaryotic voltage-gated sodium channels (Nav) contain four homologous domains I-IV (DI-IV), each is comprised of six transmembrane segments (S1-S6) analogous to a voltage-gated potassium channel (Kv) subunit. A common structural architecture shared between the Nav and many Kv channels is the domain-swapped assembly, which results in interfaces between voltage sensor domains (VSD) and pore domains (PD) of different domains. The VSD enables channel sensitivity to voltage. The canonical mechanism that couples the VSD to the PD includes the S4-S5 linker that ultimately links movements of the segment S4 VSD to the segment S6 helix of the PD. A noncanonical pathway comprised of a noncovalent network connects the VSD to the PD in the Shaker K+ channel. Here, we aimed to elucidate the possibility of a noncanonical pathway in Nav1.4. Single point mutations were introduced into positions of Nav1.4 that have structural analogs to the VSD-PD pathway that are known to disrupt the noncanonical pathway in Shaker. We studied L1445H (DIV-S4), V267W (DI-S5), and L266A (DI-S5), which are analogous to positions L366, S411 and S412, respectively, in Shaker. Initially, Nav1.4 was expressed without the β-subunit in Xenopuslaevis oocytes. Compared to the wild-type, the mutants had the same voltage dependence in conductance (G-V) and showed a slight voltage shift in steady-state fast inactivation. The time constants of fast inactivation settling were two to three times slower. To study steady-state slow inactivation, a 60 s depolarizing pulse was followed by a recovery interval for fast inactivation (20 ms hyperpolarizing pulse) and tested with a 0 mV pulse. The mutants showed a clear enhancement of the steady-state slow inactivation. Together, the data indicates that perturbations introduced into the Nav1.4 DIV-S4 and DI-S5 interface have an impact on the channel's function. (Support: NIHGM030376)

Bases of Bacterial Sodium Channel Selectivity Among Organic Cations

Hille’s (1971) seminal study of organic cation selectivity of eukaryotic voltage-gated sodium channels showed a sharp size cut-off for ion permeation, such that no ion possessing a methyl group was permeant. Using the prokaryotic channel, NaChBac, we found some similarity and two peculiar differences in the selectivity profiles for small polyatomic cations. First, we identified a diverse group of minimally permeant cations for wildtype NaChBac, ranging in sizes from ammonium to guanidinium and tetramethylammonium; and second, for both ammonium and hydrazinium, the charge-conserving selectivity filter mutation (E191D) yielded substantial increases in relative permeability (PX/PNa). The relative permeabilities varied inversely with relative Kd calculated from 1D Potential of Mean Force profiles (PMFs) for the single cations traversing the channel. Several of the cations bound more strongly than Na+, and hence appear to act as blockers, as well as charge carriers. Consistent with experimental observations, the E191D mutation had little impact on Na+ binding to the selectivity filter, but disrupted the binding of ammonium and hydrazinium, consequently facilitating ion permeation across the NaChBac-like filter. We concluded that for prokaryotic sodium channels, a fine balance among filter size, binding affinity, occupancy, and flexibility seems to contribute to observed functional differences.

Covalent linkage of bacterial voltage-gated sodium channels

BackgroundBacterial sodium channels are important models for understanding ion permeation and selectivity. However, their homotetrameric structure limits their use as models for understanding the more complex eukaryotic voltage-gated sodium channels (which have a pseudo-heterotetrameric structure formed from an oligomer composed of four domains). To bridge this gap we attempted to synthesise oligomers made from four covalently linked bacterial sodium channel monomers and thus resembling their eukaryotic counterparts.ResultsWestern blot analyses revealed NaChBac oligomers to be inherently unstable whereas intact expression of NavMs oligomers was possible. Immunodectection using confocal microscopy and electrophysiological characterisation of NavMs tetramers confirmed plasma membrane localisation and equivalent functionality with wild type NavMs channels when expressed in human embryonic kidney cells.ConclusionThis study has generated new tools for the investigation of eukaryotic channels. The successful covalent linkage of four bacterial Nav channel monomers should permit the introduction of radial asymmetry into the structure of bacterial Nav channels and enable the known structures of these channels to be used to gain unique insights into structure-function relationships of their eukaryotic counterparts.

Eukaryotic Voltage-Gated Sodium Channels: On Their Origins, Asymmetries, Losses, Diversification and Adaptations.

The appearance of voltage-gated, sodium-selective channels with rapid gating kinetics was a limiting factor in the evolution of nervous systems. Two rounds of domain duplications generated a common 24 transmembrane segment (4 × 6 TM) template that is shared amongst voltage-gated sodium (Nav1 and Nav2) and calcium channels (Cav1, Cav2, and Cav3) and leak channel (NALCN) plus homologs from yeast, different single-cell protists (heterokont and unikont) and algae (green and brown). A shared architecture in 4 × 6 TM channels include an asymmetrical arrangement of extended extracellular L5/L6 turrets containing a 4-0-2-2 pattern of cysteines, glycosylated residues, a universally short III-IV cytoplasmic linker and often a recognizable, C-terminal PDZ binding motif. Six intron splice junctions are conserved in the first domain, including a rare U12-type of the minor spliceosome provides support for a shared heritage for sodium and calcium channels, and a separate lineage for NALCN. The asymmetrically arranged pores of 4x6 TM channels allows for a changeable ion selectivity by means of a single lysine residue change in the high field strength site of the ion selectivity filter in Domains II or III. Multicellularity and the appearance of systems was an impetus for Nav1 channels to adapt to sodium ion selectivity and fast ion gating. A non-selective, and slowly gating Nav2 channel homolog in single cell eukaryotes, predate the diversification of Nav1 channels from a basal homolog in a common ancestor to extant cnidarians to the nine vertebrate Nav1.x channel genes plus Nax. A close kinship between Nav2 and Nav1 homologs is evident in the sharing of most (twenty) intron splice junctions. Different metazoan groups have lost their Nav1 channel genes altogether, while vertebrates rapidly expanded their gene numbers. The expansion in vertebrate Nav1 channel genes fills unique functional niches and generates overlapping properties contributing to redundancies. Specific nervous system adaptations include cytoplasmic linkers with phosphorylation sites and tethered elements to protein assemblies in First Initial Segments and nodes of Ranvier. Analogous accessory beta subunit appeared alongside Nav1 channels within different animal sub-phyla. Nav1 channels contribute to pace-making as persistent or resurgent currents, the former which is widespread across animals, while the latter is a likely vertebrate adaptation.

Veratridine binding to a transmembrane helix of sodium channel Nav1.4 determined by solid-state NMR.

The multi-step ligand action to a target protein is an important aspect when understanding mechanisms of ligand binding and discovering new drugs. However, structurally capturing such complex mechanisms is challenging. This is particularly true for interactions between large membrane proteins and small molecules. One such large membrane of interest is Nav1.4, a eukaryotic voltage-gated sodium channel. Domain 4 segment 6 (D4S6) of Nav1.4 is a transmembrane α-helical segment playing a key role in channel gating regulation, and is targeted by a neurotoxin, veratridine (VTD). VTD has been suggested to exhibit a two-step action to activate Nav1.4. Here, we determine the NMR structure of a selectively 13C-labeled peptide corresponding to D4S6 and its VTD binding site in lipid bilayers determined by using magic-angle spinning solid-state NMR. By 13C NMR, we obtain NMR structural constraints as 13C chemical shifts and the 1H-2H dipolar couplings between the peptide and deuterated lipids. The peptide backbone structure and its location with respect to the membrane are determined under the obtained NMR structural constraints aided by replica exchange molecular dynamics simulations with an implicit membrane/solvent system. Further, by measuring the 1H-2H dipolar couplings to monitor the peptide-lipid interaction, we identify a VTD binding site on D4S6. When superimposed to a crystal structure of a bacterial sodium channel NavRh, the determined binding site is the only surface exposed to the protein exterior and localizes beside the second-step binding site reported in the past. Based on these results, we propose that VTD initially binds to these newly-determined residues on D4S6 from the membrane hydrophobic domain, which induces the first-step channel opening followed by the second-step blocking of channel inactivation of Nav1.4. Our findings provide new detailed insights of the VTD action mechanism, which could be useful in designing new drugs targeting D4S6.

Predicting Structural Details of the Sodium Channel Pore Basing on Animal Toxin Studies.

Eukaryotic voltage-gated sodium channels play key roles in physiology and are targets for many toxins and medically important drugs. Physiology, pharmacology, and general architecture of the channels has long been the subject of intensive research in academia and industry. In particular, animal toxins such as tetrodotoxin, saxitoxin, and conotoxins have been used as molecular probes of the channel structure. More recently, X-ray structures of potassium and prokaryotic sodium channels allowed elaborating models of the toxin-channel complexes that integrated data from biophysical, electrophysiological, and mutational studies. Atomic level cryo-EM structures of eukaryotic sodium channels, which became available in 2017, show that the selectivity filter structure and other important features of the pore domain have been correctly predicted. This validates further employments of toxins and other small molecules as sensitive probes of fine structural details of ion channels.

Three-dimensional Modelling of the Voltage-gated Sodium Ion Channel from Anopheles gambiae Reveals Spatial Clustering of Evolutionarily Conserved Acidic Residues at the Extracellular Sites.

Background: The eukaryotic voltage-gated sodium channel(e-Nav) is a large asymmetric transmembrane protein with important functions concerning neurological function. No structure has been resolved at high resolution for this protein.Methods: A homology model of the transmembrane and extracellular regions of an Anopheles gambiae para-like channel with emphasis on the pore entrance has been constructed, based upon the templates provided by a prokaryotic sodium channel and a potassium two-pore channel. The latter provides a template for the extracellular regions, which are located above the entrance to the pore, which is likely to open at a side of a dome formed by these loops.Results: A model created with this arrangement shows a structure similar to low-resolution cryo-electron microscope images of a related structure. The pore entrance also shows favorable electrostatic interface.Conclusion: Residues responsible for the negative charge around the pore have been traced in phylogeny to highlight their importance. This model is intended for the study of pore-blocking toxins..

Structure of a eukaryotic voltage-gated sodium channel at near-atomic resolution.

Voltage-gated sodium (Nav) channels are responsible for the initiation and propagation of action potentials. They are associated with a variety of channelopathies and are targeted by multiple pharmaceutical drugs and natural toxins. Here, we report the cryogenic electron microscopy structure of a putative Nav channel from American cockroach (designated NavPaS) at 3.8 angstrom resolution. The voltage-sensing domains (VSDs) of the four repeats exhibit distinct conformations. The entrance to the asymmetric selectivity filter vestibule is guarded by heavily glycosylated and disulfide bond-stabilized extracellular loops. On the cytoplasmic side, a conserved amino-terminal domain is placed below VSDI, and a carboxy-terminal domain binds to the III-IV linker. The structure of NavPaS establishes an important foundation for understanding function and disease mechanism of Nav and related voltage-gated calcium channels.

Open and Closed States of the NaVAb Activation Gate

The bacterial voltage-gated sodium channel NavAb is a structural model for eukaryotic voltage-gated sodium channels, and it has been crystallized in both the pre-open and inactivated states (Payanadeh et al 2011, 2012). These structures revealed the general architecture of bacterial voltage-gated sodium channels and provided insight into mechanisms of voltage-sensing, electromechanical coupling, and the selective conductance of sodium ions. Comparison with structures of other bacterial sodium channels (Zhang et al 2012, Bagneris et al 2013) has also informed hypotheses of activation gating, an opening transition now thought to involve twisting movements of the S6 helix that ultimately result in an iris-like dilation of the intracellular ends of the four S6 segments. We sought to capture closed and open states in the same channel protein through x-ray crystallography and electrophysiological studies of mutations in the S6 of NavAb. Our studies show that paired mutations of hydrophobic residues in the S6 helix can uncouple voltage-sensor movement from pore opening and thereby lock the channel in a permanently closed state in which the intracellular ends of the four S6 helices interact tightly with each other. On the other hand, truncation of the intracellular extensions of the S6 segments results in an open conformation of the activation gate. These analyses allow for comparison of the closed, open, and inactivated states of NavAB's activation gate, a first for a voltage-gated sodium channel. Our results help confirm and sharpen the previously hypotheses for the pore-opening transition of sodium channels and reveal key conformational changes that connect the closed, open, and inactivated states of NavAb.

The Sodium Ion Binding Region at the Focus of P1 Helices Attracts both Charged and Electroneutral Ligands of Sodium Channels

The inner pore of eukaryotic voltage-gated sodium channels is targeted by ligands of dramatically different chemical structures. These include organic cations such as a local anesthetic lidocaine and electroneutral drugs such as an anticonvulsant carbamazepine. Mutations of the critical phenylalanine residue in helix IVS6 and some other inner pore-facing residues are known to affect action of both charged and neutral ligands. The structural cause of this ligand-binding promiscuity of sodium channels is unclear. Here we used the X-ray structure of a prokaryotic sodium channel NavMs to model the pore domain of the Nav1.x channels. We further employed the Monte Carlo energy-minimization method to perform intensive docking of lidocaine and carbamazepine from thousands starting points in the inner-pore region. The sodium ion NaIII, which is located between the four backbone carbonyls at the C-ends of P1 helices and does not make direct contacts with the channel protein, attracted carbamazepine, but repelled lidocaine. Therefore we further docked electroneutral ligands (lamotrigine, carbamazepine, phenytoin, lacosamide and bisphenol A) and cationic ligands (lidocaine, QX-314, cocaine, quinidine, and sipatrigine) in the channel models, respectively, with and without NaIII. In our models all the ligands interacted with the phenylalanine residue in IVS6 and most of the ligands also interacted with the tyrosine residue in IVS6. Some ligands extended their moieties in the III/IV sidewalk (fenestration). The electroneutral ligands bound the sodium ion with their electronegative groups and lacosamide chelated this ion. The ligand-bound ion remained close to the NaIII position due to attraction to the pore-facing backbone carbonyls. The same region attracted the charged group of the cationic ligands. In the predicted binding modes even small-size ligands would block the ion permeation by the electrostatic and steric mechanisms. Our study proposes a common pharmacophore for the diverse ligands. It includes a cation (the ligand's ammonium group or the ligand-bound sodium ion) and an aromatic moiety, which are usually linked by four bonds. Supported by NSERC and RFBR.

Congruent pattern of accessibility identifies minimal pore gate in a non-symmetric voltage-gated sodium channel

Opening and closing of the central ion-conducting pore in voltage-dependent ion channels is gated by changes in membrane potential. Although a gate residue in the eukaryotic voltage-gated sodium channel has been identified, the minimal molecular determinants of this gate region remain unknown. Here, by measuring the closed- and open-state reactivity of MTSET to substituted cysteines in all the pore-lining helices, we show that the state-dependent accessibility is delineated by four hydrophobic residues at homologous positions in each domain. Introduced cysteines above these sites do not react with intracellular MTSET while the channels are closed and yet are rapidly modified while the channels are open. These findings, in conjunction with state-dependent metal cross-bridging, support the notion that the gate residues in each of the four S6 segments of the eukaryotic sodium channel form an occlusion for ions in the closed state and are splayed open on activation.

Investigating the size and dynamics of voltage-gated sodium channel fenestrations

Eukaryotic voltage-gated sodium channels (VGSCs) are essential for the initiation and propagation of action potentials in electrically excitable cells, and are important pharmaceutical targets for the treatment of neurological disorders such as epilepsy, cardiac arrhythmias, and chronic pain. Evidence suggests that small, hydrophobic, VGSC-blocking drugs can gain access to binding residues within the central cavity of these channels by passing through lateral, lipid-filled “fenestrations” which run between the exterior of the protein and its central pore. Here, we use molecular dynamics simulations to investigate how the size and shape of fenestrations change over time in several bacterial VGSC models and a homology model of Nav1.4. We show that over the course of the simulations, the size of the fenestrations is primarily influenced by rapid protein motions, such as amino acid side-chain rotation, and highlight that differences between fenestration bottleneck-contributing residues are the primary cause of variations in fenestration size between the 6 bacterial models. In the eukaryotic channel model, 2 fenestrations are wide, but 2 are narrow due to differences in the amino acid sequence in the 4 domains. Lipid molecules are found to influence the size of the fenestrations by protruding acyl chains into the fenestrations and displacing amino acid side-chains. Together, the results suggest that fenestrations provide viable pathways for small, flexible, hydrophobic drugs.

Modulation of Inactivation Kinetics of the Bacterial Sodium Channel Nachbac Suggests a Complex Mode of Inhibition by Isoflurane

The bacterial sodium channel NaChBac is a prokaryotic ancestor of eukaryotic voltage-gated sodium channels (Nav), which are critical for action potential generation and propagation in nervous and cardiac tissue. Like all mammalian Nav isoforms examined thus far, NaChBac is inhibited by clinically relevant concentrations of the inhaled volatile anesthetic isoflurane, and inhibition is accompanied by enhancement of slow or C-type inactivation (Ouyang et al., 2007). However, a detailed mechanistic explanation of the interplay between C-type inactivation and isoflurane inhibition is lacking. To shed light on the relationship between inactivation and inhibition, we introduced point mutations known to alter inactivation in NaChBac (G219A, G219P, G229A, and S195E), expressed the channels in HEK293FT cells, and applied isoflurane during whole-cell patch clamp recording. Preliminary data support the idea that isoflurane acts by multiple mechanisms invovling multiple sites on NaChBac. Channel mutations that enhance inactivation show greater current reduction by isoflurane, suggesting that isoflurane binds more favorably to inactivated channels. However, isoflurane exhibits both tonic and use-dependent block of the essentially non-inactivating NaChBac G219P mutant, indicating that binding also occurs in the closed/resting and open/conducting states. A detailed biophysical and pharmacological profile of these NaChBac inactivation variants together with a recent molecular dynamics simulation study showing isoflurane interacting with NaChBac at three distinct binding sites (Carnavale et al, 2013) supports multiple mechanisms of state-dependent inhibtion. Such functional data help clarify the complex pharmacological effects of volatile anesthetics on Nav channels, and contribute to better understanding of C-type inactivation in these proteins. Supported by NIH grant GM 58055 (RS and HH) and German Research Foundation fellowship 5767/1-1 (TM).

Crystal Structure of a Pore Only Sodium Channel

Voltage-gated sodium channels (VGSC) are central to neuromuscular physiology generating the leading phase of action potentials in excitable cells. A variety of marine bacteria produce voltage gated sodium channels that share features with eukaryotic VGSCs and voltage-gated calcium channels (VGCCs). These bacterial channels provide tractable subjects for detailed structural analysis of common features that impact VGSC and VGCC selectivity and gating. We previously reported a structural based design approach in which dissection of the ion conducting module of bacterial VGSCs from the voltage sensor domain created a set of stand-alone ‘pore only’ channels (Shaya et al. PNAS 2011). These ‘pore-only’ channels self-assemble as tetramers and form functional, ion selective channels when incorporated into lipid vesicles. Here, we present the crystal structure of a bacterial ‘pore-only’ sodium channel. The structure encompasses both the transmembrane and the cytoplasmic parts of the protein and reveals new features that impact channel assembly, gating, and ion permeation.

Architecture and Pore Block of Eukaryotic Voltage-Gated Sodium Channels in View of NavAb Bacterial Sodium Channel Structure

The X-ray structure of the bacterial sodium channel NavAb provides a new template for the study of sodium and calcium channels. Unlike potassium channels, NavAb contains P2 helices in the outer-pore region. Because the sequence similarity between eukaryotic and prokaryotic sodium channels in this region is poor, the structural similarity is unclear. We analyzed it by using experimental data on tetrodotoxin block of sodium channels. Key tetrodotoxin-binding residues are outer carboxylates in repeats I, II, and IV, three positions downstream from the selectivity-filter residues. In a NavAb-based model of Nav1 channels derived from the sequence alignment without insertions/deletions, the outer carboxylates did not face the pore and therefore did not interact with tetrodotoxin. The hypothesis that the evolutionary appearance of Nav1 channels involved point deletions in an ancestral channel between the selectivity filter and the outer carboxylates allowed building of a NavAb-based model with tetrodotoxin-channel contacts similar to those proposed previously. This hypothesis also allowed us to reproduce in Nav1 the folding-stabilizing contacts between long-side chain residues in P1 and P2, which are seen in NavAb. The NavAb-based inner-pore model of Nav1 preserved major features of our previous KcsA-based models, including the access pathway for ligands through the repeat III/IV interface and their interactions with specific residues. Thus, structural properties of eukaryotic voltage-gated sodium channels that are suggested by functional data were reproduced in the NavAb-based models built by using the unaltered template structure but with adjusted sequence alignment. Sequences of eukaryotic calcium channels aligned with NavAb without insertions/deletions, which suggests that NavAb is a promising basis for the modeling of calcium channels.

Expression, Purification and Biophysical Characterization of a Superfamily of Prokaryotic Voltage-gated Sodium Channels

Eukaryotic voltage-gated sodium channels are monomeric membrane proteins comprised of four pseudo-repeats of domains containing six transmembrane segments, and have a molecular weight of >200 KDa. Their size and complexity makes them an arduous target for production in heterologous expression systems, a necessary step in acquiring the amounts of protein needed for biophysical and structural characterization. The simplified, single domain bacterial sodium channel containing six transmembrane segments isolated from Bacillus halodurans, NaChBac, can be expressed in E. coli in yields suitable for biophysical characterization and may enable successful crystallization and 3-D structure determination. Upon purification in 0.1% DDM, NaChBac is functional and associates to form a stable homotetramer (Nurani et al. (2008) Biochemistry 31:8114-8121). Seven different bacterial sodium channels with significant homology to NaChBac have been expressed in E. coli, purified in high-yield and characterized for secondary structure, thermal stability and drug binding. The experimental ease of obtaining a pure and homogeneous sample varies amongst the superfamily of sodium channels studied. The bacterial sodium channels are extremely thermal stable but individual members differ in their long-term stability when stored at room temperature, 4°C and −80°C, and their ability to bind the drug mibefradil. These channels also differ in their ability to form stable tetramers upon purification in different detergents. The differences and similarities found in this superfamily of sodium channels may prove valuable for determining general structural features important for specific voltage-gated sodium channel functions. The ability to express, purify and reconstitute multiple active bacterial sodium channels in membrane-mimic environments provides an arsenal of resources for elucidating structural features and identifying residues important for sustaining function in voltage-gated sodium channels. (Supported by a grants from the BBSRC to the MPSI consortium and BAW)

Design, Production and Characterisation of a Thermally-stable Mutant of the Bacterial Voltage Gated Sodium Channel Nachbac

NaChBac from B. halodurans is a bacterial homologue of the eukaryotic voltage-gated sodium channels which has been expressed and purified from E. coli. We have previously shown (Nurani et al (2008) Biochemistry 31:8114-8121) that this membrane protein,, purified from E. coli, forms a mostly helical, tetrameric detergent-solubilisable protein that is capable of binding the drug mibefradil and inducing sodium flux when reconstituted into vesicles. The tetrameric quaternary structure of NaChBac differentiates it from the single-chain eukaryotic sodium channels.