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Among the nine subtypes of human voltage-gated sodium (Nav) channels, the brain and cardiac isoforms, Nav1.1 and Nav1.5, each carry more than 400 missense mutations respectively associated with epilepsy and cardiac disorders. High-resolution structures are required for structure-function relationship dissection of the disease variants. We report the cryo-EM structures of the full-length human Nav1.1-β4 complex at 3.3 Å resolution here and the Nav1.5-E1784K variant in the accompanying paper. Up to 341 and 261 disease-related missense mutations in Nav1.1 and Nav1.5, respectively, are resolved. Comparative structural analysis reveals several clusters of disease mutations that are common to both Nav1.1 and Nav1.5. Among these, the majority of mutations on the extracellular loops above the pore domain and the supporting segments for the selectivity filter may impair structural integrity, while those on the pore domain and the voltage-sensing domains mostly interfere with electromechanical coupling and fast inactivation. Our systematic structural delineation of these mutations provides important insight into their pathogenic mechanism, which will facilitate the development of precise therapeutic interventions against various sodium channelopathies. [ABSTRACT FROM AUTHOR]

Epilepsies have numerous specific mechanisms. The understanding of neural dynamics leading to seizures is important for disclosing pathological mechanisms and developing therapeutic approaches. We investigated electrographic activities and neural dynamics leading to convulsive seizures in patients and mouse models of Dravet syndrome (DS), a developmental and epileptic encephalopathy in which hypoexcitability of GABAergic neurons is considered to be the main dysfunction. We analyzed EEGs from DS patients carrying a SCN1A pathogenic variant, as well as epidural electrocorticograms, hippocampal local field potentials, and hippocampal single-unit neuronal activities in Scn1a+/- and Scn1aRH/+ DS mice. Strikingly, most seizures had low-voltage-fast onset in both patients and mice, which is thought to be generated by hyperactivity of GABAergic interneurons, the opposite of the main pathological mechanism of DS. Analyzing single-unit recordings, we observed that temporal disorganization of the firing of putative interneurons in the period immediately before the seizure (preictal) precedes the increase of their activity at seizure onset, together with the entire neuronal network. Moreover, we found early signatures of the preictal period in the spectral features of hippocampal and cortical field potential of Scn1a mice and of patients' EEG, which are consistent with the dysfunctions that we observed in single neurons and that allowed seizure prediction. Therefore, the perturbed preictal activity of interneurons leads to their hyperactivity at the onset of generalized seizures, which have low-voltage-fast features that are similar to those observed in other epilepsies and are triggered by hyperactivity of GABAergic neurons. Preictal spectral features may be used as predictive seizure biomarkers. [ABSTRACT FROM AUTHOR]

The sodium (Na+) leak channel (NALCN) is a member of the four-domain voltage-gated cation channel family that includes the prototypical voltage-gated sodium and calcium channels (NaVs and CaVs, respectively). Unlike NaVs and CaVs, which have four lateral fenestrations that serve as routes for lipophilic compounds to enter the central cavity to modulate channel function, NALCN has bulky residues (W311, L588, M1145, and Y1436) that block these openings. Structural data suggest that occluded fenestrations underlie the pharmacological resistance of NALCN, but functional evidence is lacking. To test this hypothesis, we unplugged the fenestrations of NALCN by substituting the four aforementioned residues with alanine (AAAA) and compared the effects of NaV, CaV, and NALCN blockers on both wild-type (WT) and AAAA channels. Most compounds behaved in a similar manner on both channels, but phenytoin and 2-aminoethoxydiphenyl borate (2-APB) elicited additional, distinct responses on AAAA channels. Further experiments using single alanine mutants revealed that phenytoin and 2-APB enter the inner cavity through distinct fenestrations, implying structural specificity to their modes of access. Using a combination of computational and functional approaches, we identified amino acid residues critical for 2-APB activity, supporting the existence of drug binding site(s) within the pore region. Intrigued by the activity of 2-APB and its analogues, we tested compounds containing the diphenylmethane/amine moiety on WT channels. We identified clinically used drugs that exhibited diverse activity, thus expanding the pharmacological toolbox for NALCN. While the low potencies of active compounds reiterate the pharmacological resistance of NALCN, our findings lay the foundation for rational drug design to develop NALCN modulators with refined properties. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Apneic events are frightening but largely benign events that often occur in infants. Here, we report apparent life-threatening apneic events in an infant with the homozygous SCN1AL263V missense mutation, which causes familial hemiplegic migraine type 3 in heterozygous family members, in the absence of epilepsy. Observations consistent with the events in the infant were made in an Scn1aL263V knock-in mouse model, in which apnea was preceded by a large brainstem DC-shift, indicative of profound brainstem depolarization. The L263V mutation caused gain of NaV1.1 function effects in transfected HEK293 cells. Sodium channel blockade mitigated the gain-of-function characteristics, rescued lethal apnea in Scn1aL263V mice, and decreased the frequency of severe apneic events in the patient. Hence, this study shows that SCN1AL263V can cause life-threatening apneic events, which in a mouse model were caused by profound brainstem depolarization. In addition to being potentially relevant to sudden infant death syndrome pathophysiology, these data indicate that sodium channel blockers may be considered therapeutic for apneic events in patients with these and other gain-of-function SCN1A mutations. [ABSTRACT FROM AUTHOR]

Voltage-gated sodium channels (Nav) undergo conformational shifts in response to membrane potential changes, a mechanism known as the electromechanical coupling. To delineate the structure-function relationship of human Nav channels, we have performed systematic structural analysis using human Nav1.7 as a prototype. Guided by the structural differences between wild-type (WT) Nav1.7 and an eleven mutation-containing variant, designated Nav1.7-M11, we generated three additional intermediate mutants and solved their structures at overall resolutions of 2.9-3.4 Å. The mutant with nine-point mutations in the pore domain (PD), named Nav1.7-M9, has a reduced cavity volume and a sealed gate, with all voltage-sensing domains (VSDs) remaining up. Structural comparison of WT and Nav1.7-M9 pinpoints two residues that may be critical to the tightening of the PD. However, the variant containing these two mutations, Nav1.7-M2, or even in combination with two additional mutations in the VSDs, named Nav1.7-M4, failed to tighten the PD. Our structural analysis reveals a tendency of PD contraction correlated with the right shift of the static inactivation I-V curves. We predict that the channel in the resting state should have a "tight" PD with down VSDs. [ABSTRACT FROM AUTHOR]

A sodium channel is composed of four similar domains, each containing a highly charged S4 helix that is driven outward (activates) in response to a depolarization. Functionally, the channel has two gates, called activation gate (a gate) and inactivation gate (I gate), both of which must be open for conduction to occur. The cytoplasmically located a gate opens after a depolarization has activated the S4s of (probably) all four domains. The I gate consists of a cytoplasmically located inactivation "particle" and a receptor for it in the channel. The receptor becomes available after some degree of S4 activation, and the particle diffuses in to inactivate the channel. The I gate usually closes when the a gate is open [open-state inactivation (Osi)] but also can close before the channel reaches the conducting state. This "closed-state inactivation" (Csi) is studied quantitatively in this paper to determine the degree of S4 activation required for (i) opening the a gate, and (ii) permitting the I gate to close. Csi is most prominent for small depolarizations, during which occupancy of the partially activated closed states is prolonged. Large depolarizations drive the S4s outward quickly, minimizing the duration of closed-state occupancy and making Csi small and Osi large. Based on these data and evidence in the literature, it is concluded that opening the a gate requires S4 activation in domains 1-3, with partial activation of the S4 of domain 4. Csi requires only S4 activation of domains 3 and 4, which does not open the a gate.

Epithelial Na+ channels are expressed widely in absorptive epithelia such as the renal collecting duct and the colon and play a critical role in fluid and electrolyte homeostasis. Recent studies have shown that these channels interact via PY motifs in the C terminals of their alpha, beta, and gamma subunits with the WW domains of the ubiquitin-protein ligase Nedd4. Mutation or deletion of these PY motifs (as occurs, for example, in the heritable form of hypertension known as Liddle's syndrome) leads to increased Na+ channel activity. Thus, binding of Nedd4 by the PY motifs would appear to be part of a physiological control system for down-regulation of Na+ channel activity. The nature of this control system is, however, unknown. In the present paper, we show that Nedd4 mediates the ubiquitin-dependent down-regulation of Na+ channel activity in response to increased intracellular Na+. We further show that Nedd4 operates downstream of Go in this feedback pathway. We find, however, that Nedd4 is not involved in the feedback control of Na+ channels by intracellular anions. Finally, we show that Nedd4 has no influence on Na+ channel activity when the Na+ and anion feedback systems are inactive. We conclude that Nedd4 normally mediates feedback control of epithelial Na+ channels by intracellular Na+, and we suggest that the increased Na+ channel activity observed in Liddle's syndrome is attributable to the loss of this regulatory feedback system.

There remains an urgent need for new therapies for treatment-resistant epilepsy. Sodium channel blockers are effective for seizure control in common forms of epilepsy, but loss of sodium channel function underlies some genetic forms of epilepsy. Approaches that provide bidirectional control of sodium channel expression are needed. MicroRNAs (miRNA) are small noncoding RNAs which negatively regulate gene expression. Here we show that genome-wide miRNA screening of hippocampal tissue from a rat epilepsy model, mice treated with the antiseizure medicine cannabidiol, and plasma from patients with treatment-resistant epilepsy, converge on a single target—miR-335- 5p. Pathway analysis on predicted and validated miR-335- 5p targets identified multiple voltage-gated sodium channels (VGSCs). Intracerebroventricular injection of antisense oligonucleotides against miR-335- 5p resulted in upregulation of Scn1a, Scn2a, and Scn3a in the mouse brain and an increased action potential rising phase and greater excitability of hippocampal pyramidal neurons in brain slice recordings, consistent with VGSCs as functional targets of miR-335- 5p. Blocking miR-335- 5p also increased voltage-gated sodium currents and SCN1A, SCN2A, and SCN3A expression in human induced pluripotent stem cell-derived neurons. Inhibition of miR-335- 5p increased susceptibility to tonic-clonic seizures in the pentylenetetrazol seizure model, whereas adeno-associated virus 9-mediated overexpression of miR-335- 5p reduced seizure severity and improved survival. These studies suggest modulation of miR-335- 5p may be a means to regulate VGSCs and affect neuronal excitability and seizures. Changes to miR-335- 5p may reflect compensatory mechanisms to control excitability and could provide biomarker or therapeutic strategies for different types of treatment-resistant epilepsy. [ABSTRACT FROM AUTHOR]

Voltage-gated sodium channel Nav1.6 plays a crucial role in neuronal firing in the central nervous system (CNS). Aberrant function of Nav1.6 may lead to epilepsy and other neurological disorders. Specific inhibitors of Nav1.6 thus have therapeutic potentials. Here we present the cryo-EM structure of human Nav1.6 in the presence of auxiliary subunits β1 and fibroblast growth factor homologous factor 2B (FHF2B) at an overall resolution of 3.1 Å. The overall structure represents an inactivated state with closed pore domain (PD) and all “up” voltage-sensing domains. A conserved carbohydrate–aromatic interaction involving Trp302 and Asn326, together with the β1 subunit, stabilizes the extracellular loop in repeat I. Apart from regular lipids that are resolved in the EM map, an unprecedented Y-shaped density that belongs to an unidentified molecule binds to the PD, revealing a potential site for developing Nav1.6-specific blockers. Structural mapping of disease-related Nav1.6 mutations provides insights into their pathogenic mechanism. [ABSTRACT FROM AUTHOR]

Teleost fishes and urodele amphibians can regenerate amputated appendages, whereas this ability is restricted to digit tips in adult mammals. One key component of appendage regeneration is reinnervation of the wound area. However, how innervation is regulated in injured appendages of adult vertebrates has seen limited research attention. From a forward genetics screen for temperature-sensitive defects in zebrafish fin regeneration, we identified a mutation that disrupted regeneration while also inducing paralysis at the restrictive temperature. Genetic mapping and complementation tests identify a mutation in the major neuronal voltage-gated sodium channel (VGSC) gene scn8ab. Conditional disruption of scn8ab impairs early regenerative events, including blastema formation, but does not affect morphogenesis of established regenerates. Whereas scn8ab mutations reduced neural activity as expected, they also disrupted axon regrowth and patterning in fin regenerates, resulting in hypoinnervation. Our findings indicate that the activity of VGSCs plays a proregenerative role by promoting innervation of appendage stumps. [ABSTRACT FROM AUTHOR]

Expansion of the GGGGCC hexanucleotide repeat in the chromosome 9 open reading frame 72 (C9orf72) gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS). As in other forms of ALS, selective hyperexcitability of the motor cortex has been implicated as a cause of the motor neuron death in C9orf72-associated ALS. Here, we show that proline-arginine (PR) poly-dipeptides generated from C9orf72 repeat expansions increase the intrinsic excitability in pyramidal neurons of the motor cortex but not in the principal neurons of the visual cortex, somatosensory cortex, or hippocampus. We further show that this effect is attributable to PR-induced enhancement of the persistent sodium current primarily through an Nav1.2-β1-β4 complex. Reconstitution assays reveal that an auxiliary subunit, β4, plays a crucial role in the PR-mediated modulation of human Nav1.2 channel activity. Moreover, compared with the visual cortex, binding of PR poly-dipeptide to Nav1.2 is stronger in the motor cortex, where β4 is highly expressed. Taken together, these studies suggest a cellular mechanism underlying cortical hyperexcitability in C9orf72 ALS by providing evidence that PR poly-dipeptides induce hyperexcitability in cortical motor neurons by modulating the Nav1.2 channel complex. [ABSTRACT FROM AUTHOR]

Nav1.5 initiates the cardiac action potential. Alterations of its activation and inactivation properties due to mutations can cause severe, life-threatening arrhythmias. Yet despite intensive research efforts, many functional aspects of this cardiac channel remain poorly understood. For instance, Nav1.5 undergoes extensive posttranslational modification in vivo, but the functional significance of these modifications is largely unexplored, especially under pathological conditions. This is because most conventional approaches are unable to insert metabolically stable posttranslational modification mimics, thus preventing a precise elucidation of the contribution by these modifications to channel function. Here, we overcome this limitation by using protein semisynthesis of Nav1.5 in live cells and carry out complementary molecular dynamics simulations. We introduce metabolically stable phosphorylation mimics on both wild-type (WT) and two pathogenic long-QT mutant channel backgrounds and decipher functional and pharmacological effects with unique precision. We elucidate the mechanism by which phosphorylation of Y1495 impairs steady-state inactivation in WT Nav1.5. Surprisingly, we find that while the Q1476R patient mutation does not affect inactivation on its own, it enhances the impairment of steady-state inactivation caused by phosphorylation of Y1495 through enhanced unbinding of the inactivation particle. We also show that both phosphorylation and patient mutations can impact Nav1.5 sensitivity toward the clinically used antiarrhythmic drugs quinidine and ranolazine, but not flecainide. The data highlight that functional effects of Nav1.5 phosphorylation can be dramatically amplified by patient mutations. Our work is thus likely to have implications for the interpretation of mutational phenotypes and the design of future drug regimens. [ABSTRACT FROM AUTHOR]

Voltage-gated sodium (NaV) channels control excitable cell functions. While structural investigations have revealed conformation details of different functional states, the mechanisms of both activation and slow inactivation remain unclear. Here, we identify residue T140 in the S4-S5 linker of the bacterial voltage-gated sodium channel NaChBac as critical for channel activation and drug effects on inactivation. Mutations at T140 either attenuate activation or render the channel nonfunctional. Propofol, a clinical anesthetic known to inhibit NaChBac by promoting slow inactivation, binds to a pocket between the S4-S5 linker and S6 helix in a conformation-dependent manner. Using 19F-NMR to quantify site-specific binding by saturation transfer differences (STDs), we found strong STDs in inactivated, but not activated, NaChBac. Molecular dynamics simulations show a highly dynamic pocket in the activated conformation, limiting STD buildup. In contrast, drug binding to this pocket promotes and stabilizes the inactivated states. Our results provide direct experimental evidence showing distinctly different associations between the S4-S5 linker and S6 helix in activated and inactivated states. Specifically, an exchange occurs between interaction partners T140 and N234 of the same subunit in activation, and T140 and N225 of the domainswapped subunit in slow inactivation. The drug action on slow inactivation of prokaryotic NaV channels seems to have a mechanism similar to the recently proposed "door-wedge" action of the isoleucine-phenylalanine-methionine (IFM) motif on the fast inactivation of eukaryotic NaV channels. Elucidating this gating mechanism points to a possible direction for conformation-dependent drug development. [ABSTRACT FROM AUTHOR]

Excitability--a threshold-governed transient in transmembrane voltage--is a fundamental physiological process that controls the function of the heart, endocrine, muscles, and neuronal tissues. The 1950s Hodgkin and Huxley explicit formulation provides a mathematical framework for understanding excitability, as the consequence of the properties of voltage-gated sodium and potassium channels. The Hodgkin-Huxley model is more sensitive to parametric variations of protein densities and kinetics than biological systems whose excitability is apparently more robust. It is generally assumed that the model's sensitivity reflects missing functional relations between its parameters or other components present in biological systems. Here we experimentally assembled excitable membranes using the dynamic clamp and voltage-gated potassium ionic channels (Kv1.3) expressed in Xenopus oocytes. We take advantage of a theoretically derived phase diagram, where the phenomenon of excitability is reduced to two dimensions defined as combinations of the Hodgkin-Huxley model parameters, to examine functional relations in the parameter space. Moreover, we demonstrate activity dependence and hysteretic dynamics over the phase diagram due to the impacts of complex slow inactivation kinetics. The results suggest that maintenance of excitability amid parametric variation is a low-dimensional, physiologically tenable control process. In the context of model construction, the results point to a potentially significant gap between high-dimensional models that capture the full measure of complexity displayed by ion channel function and the lower dimensionality that captures physiological function. [ABSTRACT FROM AUTHOR]

Voltage-gated ion channels endow membranes with excitability and the means to propagate action potentials that form the basis of all neuronal signaling. We determined the structure of a voltage-gated sodium channel, two-pore channel 3 (TPC3), which generates ultralong action potentials. TPC3 is distinguished by activation only at extreme membrane depolarization (V50 ~ +75 mV), in contrast to other TPCs and NaV channels that activate between -20 and 0 mV. We present electrophysiological evidence that TPC3 voltage activation depends only on voltage sensing domain 2 (VSD2) and that each of the three gating arginines in VSD2 reduces the activation threshold. The structure presents a chemical basis for sodium selectivity, and a constricted gate suggests a closed pore consistent with extreme voltage dependence. The structure, confirmed by our electrophysiology, illustrates the configuration of a bona fide resting state voltage sensor, observed without the need for any inhibitory ligand, and independent of any chemical or mutagenic alteration. [ABSTRACT FROM AUTHOR]

Valproic acid (VPA) is an anticonvulsant drug that is also used to treat migraines and bipolar disorder. Its proposed biological targets include human voltage-gated sodium channels, among other membrane proteins. We used the prokaryotic NavMs sodium channel, which has been shown to be a good exemplar for drug binding to human sodium channels, to examine the structural and functional interactions of VPA. Thermal melt synchrotron radiation circular dichroism spectroscopic binding studies of the full-length NavMs channel (which includes both pore and voltage sensor domains), and a pore-only construct, undertaken in the presence and absence of VPA, indicated that the drug binds to and destabilizes the channel, but not the poreonly construct. This is in contrast to other antiepileptic compounds that have previously been shown to bind in the central hydrophobic core of the pore region of the channel, and that tend to increase the thermal stability of both pore-only constructs and full-length channels. Molecular docking studies also indicated that the VPA binding site is associated with the voltage sensor, rather than the hydrophobic cavity of the pore domain. Electrophysiological studies show that VPA influences the block and inactivation rates of the NavMs channel, although with lower efficacy than classical channel-blocking compounds. It thus appears that, while VPA is capable of binding to these voltage-gated sodium channels, it has a very different mode and site of action than other anticonvulsant compounds. [ABSTRACT FROM AUTHOR]

Dravet Syndrome is a severe childhood epileptic disorder caused by haploinsufficiency of the SCN1A gene encoding brain voltage-gated sodium channel NaV1.1. Symptoms include treatment-refractory epilepsy, cognitive impairment, autistic-like behavior, and premature death. The specific loci of NaV1.1 function in the brain that underlie these global deficits remain unknown. Here we specifically deleted Scn1a in the hippocampus using the Cre-Lox method in weanling mice. Local gene deletion caused selective reduction of inhibitory neurotransmission measured in dentate granule cells. Mice with local NaV1.1 reduction had thermally evoked seizures and spatial learning deficits, but they did not have abnormalities of locomotor activity or social interaction. Our results show that local gene deletion in the hippocampus can induce two of the most severe dysfunctions of Dravet Syndrome: Epilepsy and cognitive deficit. Considering these results, the hippocampus may be a potential target for future gene therapy for Dravet Syndrome. [ABSTRACT FROM AUTHOR]

The rationale for genome-wide association study (GWAS) results is sequence variation in cis-regulatory elements (CREs) modulating a target gene's expression as the major cause of trait variation. To understand the complete molecular landscape of one of these GWAS loci, we performed in vitro reporter screens in cardiomyocyte cell lines for CREs overlapping nearly all common variants associated with any of five independent QT interval (QTi)- associated GWAS hits at the SCN5A-SCN10A locus. We identified 13 causal CRE variants using allelic reporter activity, cardiomyocyte nuclear extract-based binding assays, overlap with human cardiac tissue DNaseI hypersensitive regions, and predicted impact of sequence variants on DNaseI sensitivity. Our analyses identified at least one high-confidence causal CRE variant for each of the five sentinel hits that could collectively predict SCN5A cardiac gene expression and QTi association. Although all 13 variants could explain SCN5A gene expression, the highest statistical significance was obtained with seven variants (inclusive of the five above). Thus, multiple, causal, mutually associated CRE variants can underlie GWAS signals. [ABSTRACT FROM AUTHOR]

The human voltage-gated sodium channel, hNaV1.5, is responsible for the rapid upstroke of the cardiac action potential and is target for antiarrhythmic therapy. Despite the clinical relevance of hNaV1.5- targeting drugs, structure-based molecular mechanisms of promising or problematic drugs have not been investigated at atomic scale to inform drug design. Here, we used Rosetta structural modeling and docking as well as molecular dynamics simulations to study the interactions of antiarrhythmic and local anesthetic drugs with hNaV1.5. These calculations revealed several key drug binding sites formed within the pore lumen that can simultaneously accommodate up to two drug molecules. Molecular dynamics simulations identified a hydrophilic access pathway through the intracellular gate and a hydrophobic access pathway through a fenestration between DIII and DIV. Our results advance the understanding of molecular mechanisms of antiarrhythmic and local anesthetic drug interactions with hNaV1.5 and will be useful for rational design of novel therapeutics. [ABSTRACT FROM AUTHOR]

Potency of drug action is usually determined by binding to a specific receptor site on target proteins. In contrast to this conventional paradigm, we show here that potency of local anesthetics (LAs) and antiarrhythmic drugs (AADs) that block sodium channels is controlled by fenestrations that allow drug access to the receptor site directly from the membrane phase. Voltage-gated sodium channels initiate action potentials in nerve and cardiac muscle, where their hyperactivity causes pain and cardiac arrhythmia, respectively. LAs and AADs selectively block sodium channels in rapidly firing nerve and muscle cells to relieve these conditions. The structure of the ancestral bacterial sodium channel NaVAb, which is also blocked by LAs and AADs, revealed fenestrations connecting the lipid phase of the membrane to the central cavity of the pore. We cocrystallized lidocaine and flecainide with NavAb, which revealed strong drugdependent electron density in the central cavity of the pore. Mutation of the contact residue T206 greatly reduced drug potency, confirming this site as the receptor for LAs and AADs. Strikingly, mutations of the fenestration cap residue F203 changed fenestration size and had graded effects on resting-state block by flecainide, lidocaine, and benzocaine, the potencies of which were altered from 51- to 2.6-fold in order of their molecular size. These results show that conserved fenestrations in the pores of sodium channels are crucial pharmacologically and determine the level of resting-state block by widely used drugs. Fine-tuning drug access through fenestrations provides an unexpected avenue for structure-based design of ion-channel- blocking drugs. [ABSTRACT FROM AUTHOR]

Dravet syndrome is a catastrophic, pharmacoresistant epileptic encephalopathy. Disease onset occurs in the first year of life, followed by developmental delay with cognitive and behavioral dysfunction and substantially elevated risk of premature death. The majority of affected individuals harbor a loss-of-functionmutation in one allele of SCN1A, which encodes the voltage-gated sodium channel NaV1.1. Brain NaV1.1 is primarily localized to fast-spiking inhibitory interneurons; thus the mechanism of epileptogenesis in Dravet syndrome is hypothesized to be reduced inhibitory neurotransmission leading to brain hyperexcitability. We show that selective activation of NaV1.1 by venom peptide Hm1a restores the function of inhibitory interneurons from Dravet syndrome mice without affecting the firing of excitatory neurons. Intracerebroventricular infusion of Hm1a rescues Dravet syndrome mice from seizures and premature death. This precision medicine approach, which specifically targets the molecular deficit in Dravet syndrome, presents an opportunity for treatment of this intractable epilepsy. [ABSTRACT FROM AUTHOR]

Neocortical pyramidal neurons express several distinct subtypes of voltage-gated Na+ channels. In mature cells, Nav1.6 is the dominant channel subtype in the axon initial segment (AIS) as well as in the nodes of Ranvier. Action potentials (APs) are initiated in the AIS, and it has been proposed that the high excitability of this region is related to the unique characteristics of the Nav1.6 channel. Knockout or loss-of-function mutation of the Scn8a gene is generally lethal early in life because of the importance of this subtype in noncortical regions of the nervous system. Using the Cre/loxP system, we selectively deleted Nav1.6 in excitatory neurons of the forebrain and characterized the excitability of Nav1.6-deficient layer 5 pyramidal neurons by patch-clamp and Na+ and Ca2+ imaging recordings. We now report that, in the absence of Nav1.6 expression, the AIS is occupied by Nav1.2 channels. However, APs are generated in the AIS, and differences in AP propagation to soma and dendrites are minimal. Moreover, the channels that are expressed in the AIS still show a clear hyperpolarizing shift in voltage dependence of activation, compared with somatic channels. The only major difference between Nav1.6-null and wild-type neurons was a strong reduction in persistent sodium current. We propose that the molecular environment of the AIS confers properties on whatever Na channel subtype is present and that some other benefit must be conferred by the selective axonal presence of the Nav1.6 channel. [ABSTRACT FROM AUTHOR]

Since the availability of the first crystal structure of a bacterial Na+ channel in 2011, understanding selectivity across this family of membrane proteins has been the subject of intense research efforts. Initially, free energy calculations based on molecular dynamics simulations revealed that although sodium ions can easily permeate the channel with their first hydration shell almost intact, the selectivity filter is too narrow for efficient conduction of hydrated potassium ions. This steric view of selectivity was subsequently questioned bymicrosecond atomic trajectories,which proved that the selectivity filter appears to the permeating ions as a highly degenerate, liquid-like environment. Although this liquid-like environment looks optimal for rapid conduction of Na+, it seems incompatible with efficient discrimination between similar ion species, such as Na+ and K+, through steric effects. Here extensive molecular dynamics simulations, combined with Markov state model analyses, reveal that at positive membrane potentials, potassium ions trigger a conformational change of the selectivity toward a nonconductive metastable state. It is this transition of the selectivity filter, and not steric effects, that prevents the outward flux of K+ at positive membrane potentials. This description of selectivity, triggered by the nature of the permeating ions, might have implications on the current understanding of how ion channels, and in particular bacterial Na+ channels, operate at the atomic scale. [ABSTRACT FROM AUTHOR]

Voltage-gated sodium channels are essential for carrying electrical signals throughout the body, and mutations in these proteins are responsible for a variety of disorders, including epilepsy and pain syndromes. As such, they are the target of a number of drugs used for reducing pain or combatting arrhythmias and seizures. However, these drugs affect all sodium channel subtypes found in the body. Designing compounds to target select sodium channel subtypes will provide a new therapeutic pathway and would maximize treatment efficacy while minimizing side effects. Here, we examine the binding preferences of nine compounds known to be sodium channel pore blockers in molecular dynamics simulations. We use the approach of replica exchange solute tempering (REST) to gain a more complete understanding of the inhibitors' behavior inside the pore of NavMs, a bacterial sodium channel, and NavPas, a eukaryotic sodium channel. Using these simulations, we are able to show that both charged and neutral compounds partition into the bilayer, but neutral forms more readily cross it. We show that there are two possible binding sites for the compounds: (i) a site on helix 6, which has been previously determined by many experimental and computational studies, and (ii) an additional site, occupied by protonated compounds in which the positively charged part of the drug is attracted into the selectivity filter. Distinguishing distinct binding poses for neutral and charged compounds is essential for understanding the nature of pore block and will aid the design of subtype-selective sodium channel inhibitors. [ABSTRACT FROM AUTHOR]

The bacterial flagellar motor (BFM) rotates hundreds of times per second to propel bacteria driven by an electrochemical ion gradient. The motor consists of a rotor 50 nm in diameter surrounded by up to 11 ion-conducting stator units, which exchange between motors and a membrane-bound pool. Measurements of the torque-speed relationship guide the development of models of the motor mechanism. In contrast to previous reports that speed near zero torque is independent of the number of stator units, we observe multiple speeds that we attribute to different numbers of units near zero torque in both Na+- and H+- driven motors. We measure the full torque-speed relationship of one and two H+ units in Escherichia coli by selecting the number of H+ units and controlling the number of Na+ units in hybrid motors. These experiments confirm that speed near zero torque in H+-driven motors increases with the stator number. We also measured 75 torque-speed curves for Na+-driven chimeric motors at different ion-motive force and stator number. Torque and speed were proportional to ion-motive force and number of stator units at all loads, allowing all 77 measured torque-speed curves to be collapsed onto a single curve by simple rescaling. [ABSTRACT FROM AUTHOR]

Bacterial voltage-gated sodium channels (BacNavs) serve as models of their vertebrate counterparts. BacNavs contain conserved voltagesensing and pore-forming domains, but they are homotetramers of four identical subunits, rather than pseudotetramers of four homologous domains. Here, we present structures of two NavAb mutants that capture tightly closed and open states at a resolution of 2.8-3.2 Å. Introduction of two humanizingmutations in the S6 segment (NavAb/FY: T206F and V213Y) generates a persistently closed form of the activation gate in which the intracellular ends of the four S6 segments are drawn tightly together to block ion permeation completely. This construct also revealed the complete structure of the four-helix bundle that forms the C-terminal domain. In contrast, truncation of the C-terminal 40 residues in NavAb/1-226 captures the activation gate in an open conformation, revealing the open state of a BacNav with intact voltage sensors. Comparing these structures illustrates the full range of motion of the activation gate, from closed with its orifice fully occluded to open with an orifice of ~10 Å. Molecular dynamics and free-energy simulations confirm designation of NavAb/1-226 as an open state that allows permeation of hydrated Na+, and these results also support a hydrophobic gating mechanism for control of ion permeation. These two structures allow completion of a closed-open-inactivated conformational cycle in a single voltage-gated sodium channel and give insight into the structural basis for state-dependent binding of sodium channel-blocking drugs. [ABSTRACT FROM AUTHOR]

Voltage-gated sodium channels (Navs) play crucial roles in excitable cells. Although vertebrate Nav function has been extensively studied, the detailed structural basis for voltage-dependent gating mechanisms remain obscure. We have assessed the structural changes of the Nav voltage sensor domain using lanthanide-based resonance energy transfer (LRET) between the rat skeletal muscle voltage-gated sodium channel (Nav1.4) and fluorescently labeled Nav1.4-targeting toxins. We generated donor constructs with genetically encoded lanthanide-binding tags (LBTs) inserted at the extracellular end of the S4 segment of each domain (with a single LBT per construct). Three different Bodipy-labeled, Nav1.4- targeting toxins were synthesized as acceptors: β-scorpion toxin (Ts1)-Bodipy, KIIIA-Bodipy, and GIIIA-Bodipy analogs. Functional Nav-LBT channels expressed in Xenopus oocytes were voltage-clamped, and distinct LRET signals were obtained in the resting and slow inactivated states. Intramolecular distances computed from the LRET signals define a geometrical map of Nav1.4 with the bound toxins, and reveal voltage-dependent structural changes related to channel gating. [ABSTRACT FROM AUTHOR]

Monogenic epilepsies with wide-ranging clinical severity have been associated with mutations in voltage-gated sodium channel genes. In the Scn2aQ54 mouse model of epilepsy, a focal epilepsy phenotype is caused by transgenic expression of an engineered NaV1.2 mutation displaying enhanced persistent sodium current. Seizure frequency and other phenotypic features in Scn2aQ54 mice depend on genetic background. We investigated the neurophysiological and molecular correlates of strain-dependent epilepsy severity in this model. Scn2aQ54 mice on the C57BL/6J background (B6.Q54) exhibit a mild disorder, whereas animals intercrossed with SJL/J mice (F1.Q54) have a severe phenotype. Whole-cell recording revealed that hippocampal pyramidal neurons from B6.Q54 and F1.Q54 animals exhibit spontaneous action potentials, but F1.Q54 neurons exhibited higher firing frequency and greater evoked activity compared with B6.Q54 neurons. These findings correlated with larger persistent sodium current and depolarized inactivation in neurons from F1.Q54 animals. Because calcium/calmodulin protein kinase II (CaMKII) is known to modify persistent current and channel inactivation in the heart, we investigated CaMKII as a plausible modulator of neuronal sodium channels. CaMKII activity in hippocampal protein lysates exhibited a strain-dependence in Scn2aQ54 mice with higher activity in F1.Q54 animals. Heterologously expressed NaV1.2 channels exposed to activated CaMKII had enhanced persistent current and depolarized channel inactivation resembling the properties of F1.Q54 neuronal sodium channels. By contrast, inhibition of CaMKII attenuated persistent current, evoked a hyperpolarized channel inactivation, and suppressed neuronal excitability. We conclude that CaMKII-mediated modulation of neuronal sodium current impacts neuronal excitability in Scn2aQ54 mice and may represent a therapeutic target for the treatment of epilepsy. [ABSTRACT FROM AUTHOR]

KCNQ2/3 (Kv7.2/7.3) channels and voltage-gated sodium channels (VGSCs) are enriched in the axon initial segment (AIS) where they bind to ankyrin-G and coregulate membrane potential in central nervous system neurons. The molecular mechanisms supporting coordinated regulation of KCNQ and VGSCs and the cellular mechanisms governing KCNQ trafficking to the AIS are incompletely understood. Here, we show that fibroblast growth factor 14 (FGF14), previously described as a VGSC regulator, also affects KCNQ function and localization. FGF14 knockdown leads to a reduction of KCNQ2 in the AIS and a reduction in whole-cell KCNQ currents. FGF14 positively regulates KCNQ2/3 channels in a simplified expression system. FGF14 interacts with KCNQ2 at a site distinct from the FGF14-VGSC interaction surface, thus enabling the bridging of NaV1.6 and KCNQ2. These data implicate FGF14 as an organizer of channel localization in the AIS and provide insight into the coordination of KCNQ and VGSC conductances in the regulation of membrane potential. [ABSTRACT FROM AUTHOR]

Voltage-gated sodium channels (NaV) play an important role in general anesthesia. Electrophysiology measurements suggest that volatile anesthetics such as isoflurane inhibit NaV by stabilizing the inactivated state or altering the inactivation kinetics. Recent computational studies suggested the existence of multiple isoflurane binding sites in NaV, but experimental binding data are lacking. Here we use site-directed placement of 19F probes in NMR experiments to quantify isoflurane binding to the bacterial voltage-gated sodium channel NaChBac. 19F probes were introduced individually to S129 and L150 near the S4-S5 linker, L179 and S208 at the extracellular surface, T189 in the ion selectivity filter, and all phenylalanine residues. Quantitative analyses of 19F NMR saturation transfer difference (STD) spectroscopy showed a strong interaction of isoflurane with S129, T189, and S208; relatively weakly with L150; and almost undetectable with L179 and phenylalanine residues. An orientation preference was observed for isoflurane bound to T189 and S208, but not to S129 and L150.We conclude that isoflurane inhibits NaChBac by two distinct mechanisms: (i) as a channel blocker at the base of the selectivity filter, and (ii) as a modulator to restrict the pivot motion at the S4-S5 linker and at a critical hinge that controls the gating and inactivation motion of S6. [ABSTRACT FROM AUTHOR]

Clustering of voltage-gated sodium channels (VGSCs) within the neuronal axon initial segment (AIS) is critical for efficient action potential initiation. Although initially inserted into both somatodendritic and axonal membranes, VGSCs are concentrated within the axon through mechanisms that include preferential axonal targeting and selective somatodendritic endocytosis. How the endocytic machinery specifically targets somatic VGSCs is unknown. Here, using knockdown strategies, we show that noncanonical FGF13 binds directly to VGSCs in hippocampal neurons to limit their somatodendritic surface expression, although exerting little effect on VGSCs within the AIS. In contrast, homologous FGF14, which is highly concentrated in the proximal axon, binds directly to VGSCs to promote their axonal localization. Single-point mutations in FGF13 or FGF14 abrogating VGSC interaction in vitro cannot support these specific functions in neurons. Thus, our data showhow the concerted actions of FGF13 and FGF14 regulate the polarized localization of VGSCs that supports efficient action potential initiation. [ABSTRACT FROM AUTHOR]

The regulation of the axonal proteome is key to generate and maintain neural function. Fast and slow axoplasmic waves have been known for decades, but alternative mechanisms to control the abundance of axonal proteins based on local synthesis have also been identified. The presence of the endoplasmic reticulum has been documented in peripheral axons, but it is still unknown whether this localized organelle participates in the delivery of axonal membrane proteins. Voltage-gated sodium channels are responsible for action potentials and aremostly concentrated in the axon initial segment and nodes of Ranvier. Despite their fundamental role, little is known about the intracellular trafficking mechanisms that govern their availability in mature axons. Here we describe the secretory machinery in axons and its contribution to plasma membrane delivery of sodiumchannels. The distribution of axonal secretory components was evaluated in axons of the sciatic nerve and in spinal nerve axons after in vivo electroporation. Intracellular protein trafficking was pharmacologically blocked in vivo and in vitro. Axonal voltage-gated sodium channel mRNA and local trafficking were examined by RT-PCR and a retentionrelease methodology. We demonstrate that mature axons contain components of the endoplasmic reticulum and other biosynthetic organelles. Axonal organelles and sodium channel localization are sensitive to local blockade of the endoplasmic reticulum to Golgi transport. More importantly, secretory organelles are capable of delivering sodium channels to the plasma membrane in isolated axons, demonstrating an intrinsic capacity of the axonal biosynthetic route in regulating the axonal proteome in mammalian axons. [ABSTRACT FROM AUTHOR]

The article discusses the gating currents of voltage-gated proton channel (HV1), which is a molecule that uses proton gradients to store or transduce energy. It highlights role of HV1 in health and disease of diverse tissues and species and studying physical components of ion-channel gating. Topics measurement of gating currents from Hv1, sodium and potassium channel gating and Cole-Moore effect.

The article presents the author's comments on the study "Protonation state of inhibitors determines interaction sites within voltage-gated sodium channels," by A. Buyan and colleagues in a 2018 issue. The author says that the study provides a systematic exploration of the binding of six molecules known to act as sodium channel pore blockers. The author notes that the study does not consider the issue of state dependent affinities while observing voltage gated sodium-selective channels (VGSCs).

The axon initial segment (AIS) is a structure at the start of the axon with a high density of sodium and potassium channels that defines the site of action potential generation. It has recently been shown that this structure is plastic and can change its position along the axon, as well as its length, in a homeostatic manner. Chronic activity-deprivation paradigms in a chick auditory nucleus lead to a lengthening of the AIS and an increase in neuronal excitability. On the other hand, a long-term increase in activity in dissociated rat hippocampal neurons results in an outwardmovement of the AIS and a decrease in the cell's excitability. Here, we investigated whether the AIS is capable of undergoing structural plasticity in rat hippocampal organotypic slices, which retain the diversity of neuronal cell types present at postnatal ages, including chandelier cells. These interneurons exclusively target the AIS of pyramidal neurons and form rows of presynaptic boutons along them. Stimulating individual CA1 pyramidal neurons that express channelrhodopsin-2 for 48 h leads to an outward shift of the AIS. Intriguingly, both the pre- and postsynaptic components of the axo-axonic synapses did not change position after AIS relocation. We used computational modeling to explore the functional consequences of this partial mismatch and found that it allows the GABAergic synapses to strongly oppose action potential generation, and thus downregulate pyramidal cell excitability. We propose that this spatial arrangement is the optimal configuration for a homeostatic response to long-term stimulation. [ABSTRACT FROM AUTHOR]

Voltage-gated sodium (Nav) channels are important targets in the treatment of a range of pathologies. Bacterial channels, for which crystal structures have been solved, exhibit modulation by local anesthetic and anti-epileptic agents, allowing molecular-level investigations into sodium channel-drug interactions. These structures reveal no basis for the "hinged lid"-based fast inactivation, seen in eukaryotic Nav channels. Thus, they enable examination of potential mechanisms of use- or state-dependent drug action based on activation gating, or slower pore-based inactivation processes. Multimicrosecond simulations of NavAb reveal high-affinity binding of benzocaine to F203 that is a surrogate for FS6, conserved in helix S6 of Domain IV of mammalian sodium channels, as well as low-affinity sites suggested to stabilize different states of the channel. Phenytoin exhibits a different binding distribution owing to preferential interactions at the membrane and water–protein interfaces. Two drug-access pathways into the pore are observed: via lateral fenestrations connecting to the membrane lipid phase, as well as via an aqueous pathway through the intracellular activation gate, despite being closed. These observations provide insight into drug modulation that will guide further developments of Nav inhibitors. [ABSTRACT FROM AUTHOR]

Voltage-gated sodium channels are important targets for the development of pharmaceutical drugs, because mutations in different human sodium channel isoforms have causal relationships with a range of neurological and cardiovascular diseases. In this study, functional electrophysiological studies show that the prokaryotic sodium channel from Magnetococcus marinus (NavMs) binds and is inhibited by eukaryotic sodium channel blockers in a manner similar to the human Nav1.1 channel, despite millions of years of divergent evolution between the two types of channels. Crystal complexes of the NavMs pore with several brominated blocker compounds depict a common antagonist binding site in the cavity, adjacent to lipid-facing fenestrations proposed to be the portals for drug entry. In silico docking studies indicate the full extent of the blocker binding site, and electrophysiology studies of NavMs channels with mutations at adjacent residues validate the location. These results suggest that the NavMs channel can be a valuable tool for screening and rational design of human drugs. [ABSTRACT FROM AUTHOR]

Voltage-gated Na+ channels play an essential role in electrical signaling in the nervous system and are key pharmacological targets for a range of disorders. The recent solution of X-ray structures for the bacterial channel NavAb has provided an opportunity to study functional mechanisms at the atomic level. This channel's selectivity filter exhibits an EEEE ring sequence, characteristic of mammalian Ca2+, not Na+, channels. This raises the fundamentally important question: just what makes a Na+ channel conduct Na+ ions? Here we explore ion permeation on multimicrosecond timescales using the purpose-built Anton supercomputer. We isolate the likely protonation states of the EEEE ring and observe a striking flexibility of the filter that demonstrates the necessity for extended simulations to study conduction in this channel. We construct free energy maps to reveal complex multi-ion conduction via knock-on and "pass-by" mechanisms, involving concerted ion and glutamate side chain movements. Simulations in mixed ionic solutions reveal relative energetics for Na+, K+, and Ca2+ within the pore that are consistent with the modest selectivity seen experimentally. We have observed conformational changes in the pore domain leading to asymmetrical collapses of the activation gate, similar to proposed inactivated structures of NavAb, with helix bending involving conserved residues that are critical for slow inactivation. These structural changes are shown to regulate access to fenestrations suggested to be pathways for lipophilic drugs and provide deeper insight into the molecular mechanisms connecting drug activity and slow inactivation. [ABSTRACT FROM AUTHOR]

Loss-of-function mutations in the human voltage-gated sodium channel NaV1.7 result in a congenital indifference to pain. Selective inhibitors of NaV1.7 are therefore likely to be powerful analgesics for treating a broad range of pain conditions. Herein we describe the identification of μ-SLPTX-Ssm6a, a unique 46-residue peptide from centipede venom that potently inhibits NaV1.7 with an IC50 of ∼25 nM. μ-SLPTX-Ssm6a has more than 150-fold selectivity for NaV1.7 over all other human NaV subtypes, with the exception of NaV1.2, for which the selectivity is 32-fold. μ-SLPTX-Ssm6a contains three disulfide bonds with a unique connectivity pattern, and it has no significant sequence homology with any previously characterized peptide or protein. μ-SLPTX-Ssm6a proved to be a more potent analgesic than morphine in a rodent model of chemicalinduced pain, and it was equipotent with morphine in rodent models of thermal and acid-induced pain. This study establishes μ-SPTX-Ssm6a as a promising lead molecule for the development of novel analgesics targeting NaV1.7, which might be suitable for treating a wide range of human pain pathologies. [ABSTRACT FROM AUTHOR]

Pyrethroid insecticides are widely used as one of the most effective control measures in the global fight against agricultural arthropod pests and mosquito-borne diseases, including malaria and dengue. They exert toxic effects by altering the function of voltage-gated sodium channels, which are essential for proper electrical signaling in the nervous system. A major threat to the sustained use of pyrethroids for vector control is the emergence of mosquito resistance to pyrethroids worldwide. Here, we report the successful expression of a sodium channel, AaNav1-1, from Aedes aegypti in Xenopus oocytes, and the functional examination of nine sodium channel mutations that are associated with pyrethroid resistance in various Ae. aegypti and Anopheles gambiae populations around the world. Our analysis shows that five of the nine mutations reduce AaNav1-1 sensitivity to pyrethroids. Computer modeling and further mutational analysis revealed a surprising finding: Although two of the five confirmed mutations map to a previously proposed pyrethroid-receptor site in the house fly sodium channel, the other three mutations are mapped to a second receptor site. Discovery of this second putative receptor site provides a dual-receptor paradigm that could explain much of the molecular mechanisms of pyrethroid action and resistance as well as the high selectivity of pyrethroids on insect vs. mammalian sodium channels. Results from this study could impact future prediction and monitoring of pyrethroid resistance in mosquitoes and other arthropod pests and disease vectors. [ABSTRACT FROM AUTHOR]

Determination of a high-resolution 3D structure of voltage-gated sodium channel NaVAb opens the way to elucidating the mechanism of ion conductance and selectivity. To examine permeation of Na+ through the selectivity filter of the channel, we performed large-scale molecular dynamics simulations of NaVAb in an explicit, hydrated lipid bilayer at 0 mV in 150 mM NaCl, for a total simulation time of 21.6 μs. Although the cytoplasmic end of the pore is closed, reversible influx and efflux of Na+ through the selectivity filter occurred spontaneously during simulations, leading to equilibrium movement of Na+ between the extracellular medium and the central cavity of the channel. Analysis of Na+ dynamics reveals a knock-on mechanism of ion permeation characterized by alternating occupancy of the channel by 2 and 3 Na+ ions, with a computed rate of translocation of (6 ± 1) × 106 ions∙s-1 that is consistent with expectations from electrophysiological studies. The binding of Na+ is intimately coupled to conformational isomerization of the four E177 side chains lining the extracellular end of the selectivity filter. The reciprocal coordination of variable numbers of Na+ ions and carboxylate groups leads to their condensation into ionic clusters of variable charge and spatial arrangement. Structural fluctuations of these ionic clusters result in a myriad of ion binding modes and foster a highly degenerate, liquid-like energy landscape propitious to Na+ diffusion. By stabilizing multiple ionic occupancy states while helping Na+ ions diffuse within the selectivity filter, the conformational flexibility of E177 side chains underpins the knock-on mechanism of Na+ permeation. [ABSTRACT FROM AUTHOR]

The crystal structure of the open conformation of a bacterial voltage-gated sodium channel pore from Magnetococcus sp. (NaVMs) has provided the basis for a molecular dynamics study defining the channel's full ion translocation pathway and conductance process, selectivity, electrophysiological characteristics, and ion-binding sites. Microsecond molecular dynamics simulations permitted a complete time-course characterization of the protein in a membrane system, capturing the plethora of conductance events and revealing a complex mixture of single and multi-ion phenomena with decoupled rapid bidirectional water transport. The simulations suggest specific localization sites for the sodium ions, which correspond with experimentally determined electron density found in the selectivity filter of the crystal structure. These studies have also allowed us to identify the ion conductance mechanism and its relation to water movement for the NavMs channel pore and to make realistic predictions of its conductance properties. The calculated single-channel conductance and selectivity ratio correspond closely with the electrophysiology measurements of the NavMs channel expressed in HEK 293 cells. The ion translocation process seen in this voltage-gated sodium channel is clearly different from that exhibited by members of the closely related family of voltage-gated potassium channels and also differs considerably from existing proposals for the conductance process in sodium channels. These studies simulate sodium channel conductance based on an experimentally determined structure of a sodium channel pore that has a completely open transmembrane pathway and activation gate. [ABSTRACT FROM AUTHOR]

The X-ray structure of the bacterial voltage-gated sodium channel NavAb has been reported in a conformation with a closed conduction pore. Comparison between this structure and the activated-open and resting-closed structures of the voltage-gated Kv1.2 potassium channel suggests that the voltage-sensor domains (VSDs) of the reported structure are not fully activated. Using the aforementioned structures of Kv1.2 as templates, molecular dynamics simulations are used to identify analogous functional conformations of NavAb. Specifically, starting from the NavAb crystal structure, conformations of the membrane-bound channel are sampled along likely pathways for activation of the VSD and opening of the pore domain. Gating charge computations suggest that a structural rearrangement comparable to that occurring between activated-open and resting-closed states is required to explain experimental values of the gating charge, thereby confirming that the reported VSD structure is likely an intermediate along the channel activation pathway. Our observation that the X-ray structure exhibits a low pore domain-opening propensity further supports this notion. The present molecular dynamics study also identifies conformations of NavAb that are seemingly related to the resting-closed and activated-open states. Our findings are consistent with recent structural and functional studies of the orthologous channels NavRh, NaChBac, and NavMs and offer possible structures for the functionally relevant conformations of NavAb. [ABSTRACT FROM AUTHOR]

Neuronal activity regulates brain development and synaptic plasticity through N-methyl-D-aspartate receptors (NMDARs) and calcium-dependent signaling pathways. Intracellular sodium ([Na+]i) also exerts a regulatory influence on NMDAR channel activity, and [Na+]i may, therefore, function as a signaling molecule. In an attempt to mimic the influence of neuronal activity on synaptic plasticity, we used brevetoxin-2 (PbTx-2), a voltage-gated sodium channel (VGSC) gating modifier, to manipulate [Na+]i in cerebrocortical neurons. The acute application of PbTx-2 produced concentration-dependent increments in both intracellular [Na+] and [Ca2+]. Pharmacological evaluation showed that PbTx-2–induced Ca2+ influx primarily involved VGSC activation and NMDAR-mediated entry. Additionally, PbTx-2 robustly potentiated NMDA-induced Ca2+ influx. PbTx-2–exposed neurons showed enhanced neurite outgrowth, increased dendritic arbor complexity, and increased dendritic filopodia density. The appearance of spontaneous calcium oscillations, reflecting synchronous neuronal activity, was accelerated by PbTx-2 treatment. Parallel to this response, PbTx-2 increased cerebrocortical neuron synaptic density. PbTx-2 stimulation of neurite outgrowth, dendritic arborization, and synaptogenesis all exhibited bidirectional concentration–response profiles. This profile paralleled that of NMDA, which also produced bidirectional concentration–response profiles for neurite outgrowth and synaptogenesis. These data are consistent with the hypothesis that PbTx-2–enhanced neuronal plasticity involves NMDAR-dependent signaling. Our results demonstrate that PbTx-2 mimics activity-dependent neuronal structural plasticity in cerebrocortical neurons through an increase in [Na+]i, up-regulation of NMDAR function, and engagement of downstream Ca2+-dependent signaling pathways. These data suggest that VGSC gating modifiers may represent a pharmacologic strategy to regulate neuronal plasticity through NMDAR-dependent mechanisms. [ABSTRACT FROM AUTHOR]

Painful peripheral neuropathy often occurs without apparent underlying cause. Gain-of-function variants of sodium channel Nav1.7 have recently been found in ~30% of cases of idiopathic painful small-fiber neuropathy. Here, we describe mutations in Nav1.8, another sodium channel that is specifically expressed in dorsal root ganglion (DRG) neurons and peripheral nerve axons, in patients with painful neuropathy. Seven Na„1.8 mutations were identified in 9 subjects within a series of 104 patients with painful predominantly small-fiber neuropathy. Three mutations met criteria for potential pathogenicity based on predictive algorithms and were assessed by voltage and current clamp. Functional profiling showed that two of these three Nav1.8 mutations enhance the channel's response to depolarization and produce hyperexcitability in DRG neurons. These observations suggest that mutations of Nav1.8 contribute to painful peripheral neuropathy. [ABSTRACT FROM AUTHOR]

Voltage-gated sodium (NaV) and potassium (KV) channels are critical components of neuronal action potential generation and propagation. Here, we report that NaVβ1 encoded by SCN1b, an integral subunit of NaV channels, coassembles with and modulates the biophysical properties of KV1 and KV7 channels, but not KV3 channels, in an isoform-specific manner. Distinct domains of NaVβ1 are involved in modulation of the different KV channels. Studies with channel chimeras demonstrate that NaVβ1-mediated changes in activation kinetics and voltage dependence of activation require interaction of NaVβ1 with the channel's voltage-sensing domain, whereas changes in inactivation and deactivation require interaction with the channel's pore domain. A molecular model based on docking studies shows NaVβ1 lying in the crevice between the voltage-sensing and pore domains of KV channels, making significant contacts with the S1 and S5 segments. Cross-modulation of NaV and KV channels by NaVβ1 may promote diversity and flexibility in the overall control of cellular excitability and signaling. [ABSTRACT FROM AUTHOR]