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Background and purpose Corpus cavernosum smooth muscle (CCSM) exhibits phasic contractions that are coordinated by ion channels. Mouse models are commonly used to study erectile dysfunction, but there are few published electrophysiological studies of mouse CCSM. We describe, for the first time, voltage-dependent sodium (NaV ) currents in mouse CCSM and investigate their function. Experimental approach Electrophysiological, pharmacological, and immunocytochemical studies on isolated CCSM cells. Tension measurements in whole tissue. Key results A fast, voltage-dependent sodium current was induced by depolarising steps. Steady-state activation and inactivation curves revealed a window current between -60 and -30 mV. Two populations of NaV currents, ('TTX-sensitive') and ('TTX-insensitive'), were distinguished. TTX-sensitive current showed 48% block with the NaV -subtype-specific blockers ICA-121431 (NaV 1.1-1.3), PF-05089771 (NaV 1.7), and 4,9-anhydro-TTX (NaV 1.6). TTX-insensitive current was insensitive to A803467, a NaV 1.8 blocker. Immunocytochemistry confirmed the expression of NaV 1.5 and NaV 1.4 in freshly dispersed CCSM cells. Veratridine, a NaV activator, reduced time-dependent inactivation of the current and increased the duration of evoked action potentials. Veratridine induced phasic contractions in CCSM strips. This effect was reversible with TTX and nifedipine but not by KB-R7943. Conclusion and implications We report, for the first time, a fast voltage-dependent sodium current in mouse CCSM. Stimulation of this current increases the contractility of corpus cavernosum in vitro, suggesting that it may contribute to the mechanisms of detumescence, and potentially serve as a clinically relevant target for pharmaceutical intervention in erectile dysfunction. Further work will be necessary to define its role.

Background and Purpose Voltage-gated sodium (Na-V) channels are expressed de novo in carcinomas where their activity promotes invasiveness. Breast and colon cancer cells express the neonatal splice variant of Na(V)1.5 (nNa(V)1.5), which has several amino acid substitutions in the domain I voltage-sensor compared with its adult counterpart (aNa(V)1.5). This study aimed to determine whether nNa(V)1.5 channels could be distinguished pharmacologically from aNa(V)1.5 channels. Experimental Approach Cells expressing either nNa(V)1.5 or aNa(V)1.5 channels were exposed to low MW inhibitors, an antibody or natural toxins, and changes in electrophysiological parameters were measured. Stable expression in EBNA cells and transient expression in Xenopus laevis oocytes were used. Currents were recorded by whole-cell patch clamp and two-electrode voltage-clamp, respectively. Key Results Several clinically used blockers of Na-V channels (lidocaine, procaine, phenytoin, mexiletine, ranolazine, and riluzole) could not distinguish between nNa(V)1.5 or aNa(V)1.5 channels. However, two tarantula toxins (HaTx and ProTx-II) and a polyclonal antibody (NESOpAb) preferentially inhibited currents elicited by either nNa(V)1.5 or aNa(V)1.5 channels by binding to the spliced region of the channel. Furthermore, the amino acid residue at position 211 (aspartate in aNa(V)1.5/lysine in nNa(V)1.5), that is, the charge reversal in the spliced region of the channel, played a key role in the selectivity, especially in antibody binding. Conclusion and Implications We conclude that the cancer-related nNa(V)1.5 channel can be distinguished pharmacologically from its nearest neighbour, aNa(V)1.5 channels. Thus, it may be possible to design low MW compounds as antimetastatic drugs for non-toxic therapy of nNa(V)1.5-expressing carcinomas.

BACKGROUND AND PURPOSE The purpose of this study was to determine the role of NaV 1.7 in action potential conduction in C-fibres in the bronchial branches of the human vagus nerve. EXPERIMENTAL APPROACH Bronchial branches of the vagus nerve were dissected from human donor tissue. The C-wave of the electrically evoked compound action potential was quantified in the absence and presence of increasing concentrations of the selective NaV 1.7 blocking drugs, PF-05089771 and ST-2262, as well as the NaV 1.1, 1.2, and 1.3 blocking drug ICA121-431. The efficacy and potency of these inhibitors were compared to the standard NaV 1 blocker, tetrodotoxin. We then compared the relative potencies of the NaV 1 blockers in inhibiting the C-wave of the compound action potential, with their ability to inhibit parasympathetic cholinergic contraction of human isolated bronchi, a response previously shown to be strictly dependent on NaV 1.7 channels. KEY RESULTS The selective NaV 1.7 blockers inhibited the C-wave of the compound action potential with potencies similar to that observed in the NaV 1.7 bronchial contractions assay. Using rt-PCR, we noted that NaV 1.7 mRNA was strongly expressed and transported down the vagus nerve bundles. CONCLUSIONS AND IMPLICATIONS NaV 1.7 blockers can prevent action potential conduction in the majority of vagal C-fibres arising from human bronchi. Blockers of NaV 1.7 channels may therefore have value in inhibiting the responses to excessive airway C-fibre activation in inflammatory airway disease, responses that include coughing as well as reflex bronchoconstriction and secretions.

Neurological and neuronal disorders are associated with structural, biochemical, or electrical abnormalities in the nervous system. Many neurological diseases have not yet been discovered. Interventions used for the treatment of these disorders include avoidance measures, lifestyle changes, physiotherapy, neurorehabilitation, pain management, medication, and surgery. In the sodium channelopathies, alterations in the structure, expression, and function of voltage-gated sodium channels (VGSCs) are considered as the causes of neurological and neuronal diseases. Online databases, including Scopus, Science Direct, Google Scholar, and PubMed were assessed for studies published between 1977 and 2020 using the keywords of review, sodium channels blocker, neurological diseases, and neuronal diseases. VGSCs consist of one α subunit and two β subunits. These subunits are known to regulate the gating kinetics, functional characteristics, and localization of the ion channel. These channels are involved in cell migration, cellular connections, neuronal pathfinding, and neurite outgrowth. Through the VGSC, the action potential is triggered and propagated in the neurons. Action potentials are physiological functions and passage of impermeable ions. The electrophysiological properties of these channels and their relationship with neurological and neuronal disorders have been identified. Subunit mutations are involved in the development of diseases, such as epilepsy, multiple sclerosis, autism, and Alzheimer's disease. Accordingly, we conducted a review of the link between VGSCs and neurological and neuronal diseases. Also, novel therapeutic targets were introduced for future drug discoveries.

Three analogues of To042, a tocainide-related lead compound recently reported for the treatment of myotonia, were synthesized and evaluated in vitro as skeletal muscle sodium channel blockers possibly endowed with enhanced use-dependent behavior. Patch-clamp experiments on hNav1.4 expressed in HEK293 cells showed that N -[(naphthalen-1-yl)methyl]-4-[(2,6-dimethyl)phenoxy]butan-2-amine, the aryloxyalkyl bioisoster of To042, exerted a higher use-dependent block than To042 thus being able to preferentially block the channels in over-excited membranes while preserving healthy tissue function. It also showed the lowest active transport across BBB according to the results of P-glycoprotein (P-gp) interacting activity evaluation and the highest cytoprotective effect on HeLa cells. Quantum mechanical calculations and dockings gave insights on the most probable conformation of the aryloxyalkyl bioisoster of To042 in solution and the target residues involved in the binding, respectively. Both approaches indicated the conformations that might be both adopted in both the unbound and bound state of the ligand. Overall, N -[(naphthalen-1-yl)methyl]-4-[(2,6-dimethyl)phenoxy]butan-2-amine exhibits an interesting toxico-pharmacological profile and deserves further investigation.

BACKGROUND AND PURPOSE Microglia-related inflammation is associated with the pathology of Parkinson's disease. Functional voltage-gated sodium channels (VGSCs) are involved in regulating microglial function. Here, we aim to investigate the effects of scorpion venom heat-resistant synthesized peptide (SVHRSP) on 6-hydroxydopamine (6-OHDA)-induced Parkinson's disease-like mouse model and reveal its underlying mechanism. EXPERIMENTAL APPROACH Unilateral brain injection of 6-OHDA was performed to establish Parkinson's disease mouse model. After behaviour test, brain tissues were collected for morphological analysis and protein/gene expression examination. Primary microglia culture was used to investigate the role of sodium channel Nav 1.6 in the regulation of microglia inflammation by SVHRSP. KEY RESULTS SVHRSP treatment attenuated motor deficits, dopamine neuron degeneration, activation of glial cells and expression of pro-inflammatory cytokines induced by 6-OHDA lesion. Primary microglia activation and the production of pro-inflammatory cytokines induced by lipopolysaccharide (LPS) were also suppressed by SVHRSP treatment. In addition, SVHRSP could inhibit mitogen-activated protein kinases (MAPKs) pathway, which plays pivotal roles in the pro-inflammatory response. Notably, SVHRSP treatment suppressed the overexpression of microglial Nav 1.6 induced by 6-OHDA and LPS. Finally, it was shown that the anti-inflammatory effect of SVHRSP in microglia was Nav 1.6 dependent and was related to suppression of sodium current and probably the consequent Na+ /Ca2+ exchange. CONCLUSIONS AND IMPLICATIONS SVHRSP might inhibit neuroinflammation and protect dopamine neurons via down-regulating microglial Nav 1.6 and subsequently suppressing intracellular Ca2+ accumulation to attenuate the activation of MAPKs signalling pathway in microglia.

Funder: University of Surrey; Id: http://dx.doi.org/10.13039/501100003513, Understanding cardiomyocyte ion channel expression is crucial to understanding normal cardiac electrophysiology and underlying mechanisms of cardiac pathologies particularly arrhythmias. Hitherto, equine cardiac ion channel expression has rarely been investigated. Therefore, we aim to predict equine cardiac ion channel gene expression. Raw RNAseq data from normal horses from 9 datasets was retrieved from ArrayExpress and European Nucleotide Archive and reanalysed. The normalised (FPKM) read counts for a gene in a mix of tissue were hypothesised to be the average of the expected expression in each tissue weighted by the proportion of the tissue in the mix. The cardiac-specific expression was predicted by estimating the mean expression in each other tissues. To evaluate the performance of the model, predicted gene expression values were compared to the human cardiac gene expression. Cardiac-specific expression could be predicted for 91 ion channels including most expressed Na+ channels, K+ channels and Ca2+ -handling proteins. These revealed interesting differences from what would be expected based on human studies. These differences included predominance of NaV 1.4 rather than NaV 1.5 channel, and RYR1, SERCA1 and CASQ1 rather than RYR2, SERCA2, CASQ2 Ca2+ -handling proteins. Differences in channel expression not only implicate potentially different regulatory mechanisms but also pathological mechanisms of arrhythmogenesis.

Low-frequency pulsed magnetic field (LF-PMF) application is a non-invasive, easy, and inexpensive treatment method in pain management. However, the molecular mechanism underlying the effect of LF-PMF on pain is not fully understood. Considering the obvious dysregulations of gene expression observed in certain types of voltage-gated sodium channels (VGSCs) in pain conditions, the present study tested the hypothesis that LF-PMF shows its pain-relieving effect by regulating genes that code VGSCs proteins. Five experimental rat groups (Control, Streptozotocin-induced experimental painful diabetic neuropathy (PDN), PDN Sham, PDN 10 Hz PMF, and PDN 30 Hz PMF) were established. After the pain formation in PDN groups, the magnetic field groups were exposed to 10/30 Hz, 1.5 mT PMF for 4 weeks, an hour daily. Progression of pain was evaluated using behavioral pain tests during the entire experimental processes. After the end of PMF treatment, SCN9A (NaV1.7 ), SCN10A (NaV1.8 ), SCN11A (NaV1.9 ), and SCN3A (NaV1.3 ) gene expression level changes were determined by analyzing real-time polymerase chain reaction results. We found that 10 Hz PMF application was more effective than 30 Hz on pain management. In addition, NaV1.7 and NaV1.3 transcriptions were upregulated while NaV1.8 and NaV1.9 were downregulated in painful conditions. Notably, the downregulated expression of the genes encoding NaV1.8 and NaV1.9 were re-regulated and increased to control level by 10 Hz PMF application. Consequently, it may be deduced that 10 Hz PMF application reduces pain by modulating certain VGSCs at the transcriptional level. © 2021 Bioelectromagnetics Society.

Background and purpose Protoxin II (ProTx II) is a high affinity gating modifier that is thought to selectively block the Nav 1.7 voltage-dependent Na+ channel, a major therapeutic target for the control of pain. We aimed at producing ProTx II analogues entitled with novel functionalities for cell distribution studies and biochemical characterization of its Nav channel targets. Experimental approach We took advantage of the high affinity properties of the peptide, combined to its slow off rate, to design a number of new tagged analogues useful for imaging and biochemistry purposes. We used high-throughput automated patch-clamp to identify the analogues best matching the native properties of ProTx II and validated them on various Nav -expressing cells in pull-down and cell distribution studies. Key results Two of the produced ProTx II analogues, Biot-ProTx II and ATTO488-ProTx II, best recapitulate the pharmacological properties of unlabelled ProTx II, while other analogues remain high affinity blockers of Nav 1.7. The biotinylated version of ProTx II efficiently works for the pull-down of several Nav isoforms tested in a concentration-dependent manner, while the fluorescent ATTO488-ProTx II specifically labels the Nav 1.7 channel over other Nav isoforms tested in various experimental conditions. Conclusions and implications The properties of these ProTx II analogues as tools for Nav channel purification and cell distribution studies pave the way for a better understanding of ProTx II channel receptors in pain and their pathophysiological implications in sensory neuronal processing. The new fluorescent ProTx II should also reveal itself useful for the design of new drug screening strategies.

Objective Fibroblast growth factor homologous factors (FHFs) are brain and cardiac sodium channel-binding proteins that modulate channel density and inactivation gating. A recurrent de novo gain-of-function missense mutation in the FHF1(FGF12) gene (p.Arg52His) is associated with early infantile epileptic encephalopathy 47 (EIEE47; Online Mendelian Inheritance in Man database 617166). To determine whether the FHF1 missense mutation is sufficient to cause EIEE and to establish an animal model for EIEE47, we sought to engineer this mutation into mice. Methods The Arg52His mutation was introduced into fertilized eggs by CRISPR (clustered regularly interspaced short palindromic repeats) editing to generate Fhf1R52H /F+ mice. Spontaneous epileptiform events in Fhf1R52H /+ mice were assessed by cortical electroencephalography (EEG) and video monitoring. Basal heart rhythm and seizure-induced arrhythmia were recorded by electrocardiography. Modulation of cardiac sodium channel inactivation by FHF1BR52H protein was assayed by voltage-clamp recordings of FHF-deficient mouse cardiomyocytes infected with adenoviruses expressing wild-type FHF1B or FHF1BR52H protein. Results All Fhf1R52H /+ mice experienced seizure or seizurelike episodes with lethal ending between 12 and 26 days of age. EEG recordings in 19-20-day-old mice confirmed sudden unexpected death in epilepsy (SUDEP) as severe tonic seizures immediately preceding loss of brain activity and death. Within 2-53 s after lethal seizure onset, heart rate abruptly declined from 572 ± 16 bpm to 108 ± 15 bpm, suggesting a parasympathetic surge accompanying seizures that may have contributed to SUDEP. Although ectopic overexpression of FHF1BR52H in cardiomyocytes induced a 15-mV depolarizing shift in voltage of steady-state sodium channel inactivation and slowed the rate of channel inactivation, heart rhythm was normal in Fhf1R52H /+ mice prior to seizure. Significance The Fhf1 missense mutation p.Arg52His induces epileptic encephalopathy with full penetrance in mice. Both Fhf1 (p.Arg52His) and Scn8a (p.Asn1768Asp) missense mutations enhance sodium channel Nav 1.6 currents and induce SUDEP with bradycardia in mice, suggesting an FHF1/Nav 1.6 functional axis underlying altered brain sodium channel gating in epileptic encephalopathy.

Na v 1.5, the primary voltage-gated Na + (Na v ) channel in heart, is a major target for class I antiarrhythmic agents. Here we present the cryo-EM structure of full-length human Na v 1.5 bound to quinidine, a class Ia antiarrhythmic drug, at 3.3 A resolution. Quinidine is positioned right beneath the selectivity filter in the pore domain and coordinated by residues from repeats I, III, and IV. Pore blockade by quinidine is achieved through both direct obstruction of the ion permeation path and induced rotation of an invariant Tyr residue that tightens the intracellular gate. Structural comparison with a truncated rat Na v 1.5 in the presence of flecainide, a class Ic agent, reveals distinct binding poses for the two antiarrhythmics within the pore domain. Our work reported here, along with previous studies, reveals the molecular basis for the mechanism of action of class I antiarrhythmic drugs.

BACKGROUND Aedes aegypti is a remarkably effective mosquito vector of epidemiologically important arboviral diseases including dengue fever, yellow fever and Zika. The present spread of resistance against pyrethroids, the primary insecticides used for mosquito control, in global populations of this species is of great concern. The voltage‐gated sodium channel (VGSC) in the nervous system is the known target site of pyrethroids in insects. Past studies have revealed several amino‐acid substitutions in this channel that confer pyrethroid resistance, which are known as knockdown resistance (kdr) mutations. RESULTS This study investigated a laboratory colony of Ae. aegypti, MCNaeg, established from larvae collected in Rio de Janeiro, Brazil in 2016. The MCNaeg colony showed strong resistance against pyrethroids without laboratory selection. Of the two VGSC gene haplotypes present within this colony, one harbored three known kdr mutations, V410L, V1016I, and F1534C, and the other harbored only the known F1534C mutation. In latter haplotype, we also found novel amino‐acid substations including V253F. Previous molecular modeling and electrophysiological studies suggest that this residue serves a pyrethroid‐sensing site in the second receptor, PyR2. Our genetical analysis showed that the haplotype harboring V253F and F1534C is associated with equal or slightly stronger resistance than the other triple kdr haplotype to both Type I and Type II pyrethroids. CONCLUSION The novel substitution V253F is potentially involved in pyrethroid resistance in Ae. aegypti. Further studies are needed to elucidate the role of this substitution in the pyrethroid susceptibility of VGSC. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry., We found a novel amino‐acid substitution V253F in the voltage‐gated sodium channel in Aedes aegypti which is potentially involved in pyrethroid resistance of this vector mosquito

Juvenile (postnatal day 16) mice lacking Nav 1.6 channels (null-mutant Scn8admu ) have reduced photoreceptor function, which is unexpected given that Nav channels have not been detected in mouse photoreceptors and do not contribute appreciably to photoreceptor function in adults. We demonstrate that acute block of Nav channels with intravitreal TTX in juvenile (P16) wild-type mice has no effect on photoreceptor function. However, reduced light activity by prolonged dark adaptation from P8 caused significant reduction in photoreceptor function at P16. Injecting TTX into the retrobulbar space at P16 to specifically block Nav channels in the optic nerve also caused a reduction in photoreceptor function comparable to that seen at P16 in null-mutant Scn8a mice. In both P16 null-mutant Scn8admu and retrobulbar TTX-injected wild type mice, photoreceptor function was restored following intravitreal injection of the TrkB receptor agonist 7,8 dihydroxyflavone, linking Nav -dependent retrograde transport to TrkB-dependent neurotrophic factor production pathways as a modulatory influence of photoreceptor function at P16. We also found that in Scn8admu mice, photoreceptor function recovers by P22-25 despite more precarious general health of the animal. Retrobulbar injection of TTX in the wild type still reduced the photoreceptor response at this age but to a lesser extent, suggesting that Nav -dependent modulation of photoreceptor function is largely transient, peaking soon after eye opening. Together, these results suggest that the general photosensitivity of the retina is modulated following eye opening by retrograde transport through activity-dependent retinal ganglion cell axonal signaling targeting TrkB receptors.

Background and purpose Sodium channel inhibitors can be used to treat hyperexcitability-related diseases, including epilepsies, pain syndromes, neuromuscular disorders and cardiac arrhythmias. The applicability of these drugs is limited by their nonspecific effect on physiological function. They act mainly by sodium channel block and in addition by modulation of channel kinetics. While channel block inhibits healthy and pathological tissue equally, modulation can preferentially inhibit pathological activity. An ideal drug designed to target the sodium channels of pathological tissue would act predominantly by modulation. Thus far, no such drug has been described. Experimental approach Patch-clamp experiments with ultra-fast solution exchange and photolabeling-coupled electrophysiology were applied to describe the unique mechanism of riluzole on Nav1.4 sodium channels. In silico docking experiments were used to study the molecular details of binding. Key results We present evidence that riluzole acts predominantly by non-blocking modulation. We propose that, being a relatively small molecule, riluzole is able to stay bound to the binding site, but nonetheless stay off the conduction pathway, by residing in one of the fenestrations. We demonstrate how this mechanism can be recognized. Conclusions and implications Our results identify riluzole as the prototype of this new class of sodium channel inhibitors. Drugs of this class are expected to selectively prevent hyperexcitability, while having minimal effect on cells firing at a normal rate from a normal resting potential.

Key points A human NaV 1.6 construct was established to study the biophysical consequences of the R1617Q mutation on NaV 1.6 identified in patients with unclassified epileptic encephalopathy and severe intellectual disability. The R1617Q mutation disrupts the inactivation process of the channel, and more specifically, slows the current decay, increases the persistent sodium current that was blocked by tetrodotoxin and riluzole, and disrupts the inactivation voltage-dependence and increases the kinetics of recovery. In native hippocampal neurons, the R1617Q mutation exhibited a significant increase in action potentials triggered in response to stimulation and a significant increase in the number of neurons that exhibited spontaneous activity compared to neurons expressing WT channels that were inhibited by riluzole. The abnormally persistent current activity caused by the disruption of the channel inactivation process in NaV 1.6/R1617Q may result in epileptic encephalopathy in patients. Abstract The voltage-gated sodium channel NaV 1.6 is the most abundantly expressed sodium channel isoform in the central nervous system. It plays a critical role in saltatory and continuous conduction. Although over 40 NaV 1.6 mutations have been linked to epileptic encephalopathy, only a few have been functionally analysed. In the present study, we characterized a NaV 1.6 mutation (R1617Q) identified in patients with epileptic encephalopathy and intellectual disability. R1617Q substitutes an arginine for a glutamine in the S4 segment of domain IV, which plays a major role in coupling the activation and inactivation of sodium channels. We used patch-clamp to show that R1617Q is a gain-of-function mutation. It is typified by slower inactivation kinetics and a loss of inactivation of voltage-dependence, which result in a 2.5-fold increase in the window current. In addition, sodium currents exhibited an enhanced rate of recovery from inactivation, most likely due to the destabilization of the inactivation state. The alterations in the fast inactivation caused a significant increase in the persistent sodium current. Overexpression of R1617Q in rat hippocampal neurons resulted in an increase in action potential firing activity that was inhibited by riluzole, consistent with the gain-of-function observed. We conclude that the R1617Q mutation causes neuronal hyperexcitability and may result in epileptic encephalopathy.

Objective Many antiseizure drugs (ASDs) act on voltage-dependent sodium channels, and the molecular basis of these effects is well established. In contrast, how ASDs act on the level of neuronal networks is much less understood. Methods In the present study, we determined the effects of eslicarbazepine (S-Lic) on different types of inhibitory neurons, as well as inhibitory motifs. Experiments were performed in hippocampal slices from both sham-control and chronically epileptic pilocarpine-treated rats. Results We found that S-Lic causes an unexpected reduction of feed-forward inhibition in the CA1 region at high concentrations (300 µM), but not at lower concentrations (100 µM). Concurrently, 300 but not 100 μM S-Lic significantly reduced maximal firing rates in putative feed-forward interneurons located in the CA1 stratum radiatum of sham-control and epileptic animals. In contrast, feedback inhibition was not inhibited by S-Lic. Instead, application of S-Lic, in contrast to previous data for other drugs like carbamazepine (CBZ), resulted in a lasting potentiation of feedback inhibitory post-synaptic currents (IPSCs) only in epileptic and not in sham-control animals, which persisted after washout of S-Lic. We hypothesized that this plasticity of inhibition might rely on anti-Hebbian potentiation of excitatory feedback inputs onto oriens-lacunosum moleculare (OLM) interneurons, which is dependent on Ca2+ -permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Indeed, we show that blocking Ca2+ -permeable AMPA receptors completely prevents upmodulation of feedback inhibition. Significance These results suggest that S-Lic affects inhibitory circuits in the CA1 hippocampal region in unexpected ways. In addition, ASD actions may not be sufficiently explained by acute effects on their target channels, rather, it may be necessary to take plasticity of inhibitory circuits into account.

Objective Human variants in voltage‐gated sodium channel (VGSC) α and β subunit genes are linked to developmental and epileptic encephalopathies (DEEs). Inherited, biallelic, loss‐of‐function variants in SCN1B, encoding the β1/β1B subunits, are linked to early infantile DEE (EIEE52). De novo, monoallelic variants in SCN1A (Nav1.1), SCN2A (Nav1.2), SCN3A (Nav1.3), and SCN8A (Nav1.6) are also linked to DEEs. While these VGSC‐linked DEEs have similar presentations, they have diverse mechanisms of altered neuronal excitability. Mouse models have suggested that Scn2a‐, Scn3a‐, and Scn8a‐linked DEE variants are, in general, gain of function, resulting in increased persistent or resurgent sodium current (INa) and pyramidal neuron hyperexcitability. In contrast, Scn1a‐linked DEE variants, in general, are loss‐of‐function, resulting in decreased INa and hypoexcitability of fast‐spiking interneurons. VGSC β1 subunits associate with Nav1.1, Nav1.2, Nav1.3, and Nav1.6 and are expressed throughout the brain, raising the possibility that insults to both pyramidal and interneuron excitability may drive EIEE52 pathophysiology. Methods We investigated excitability defects in pyramidal and parvalbumin‐positive (PV +) interneurons in the Scn1b −/− model of EIEE52. We also used Scn1bFL/FL mice to delete Scn1b in specific neuronal populations. Results Scn1b −/− cortical PV + interneurons were hypoexcitable, with reduced INa density. Scn1b −/− cortical pyramidal neurons had population‐specific changes in excitability and impaired INa density. Scn1b deletion in PV + neurons resulted in 100% lethality, whereas deletion in Emx1 + or Camk2a + neurons did not affect survival. Interpretation This work suggests that SCN1B‐linked DEE variants impact both excitatory and inhibitory neurons, leading to the increased severity of EIEE52 relative to other DEEs.

This study aimed to investigate and compare cell growth manners and functional differences of primary cortical neurons cultured on either poly-d-lysine (PDL) and or Matrigel, to delineate the role of extracellular matrix on providing resemblance to in vivo cellular interactions in nervous tissue. Primary cortical neurons, obtained from embryonic day 15 mice pups, seeded either on PDL- or Matrigel-coated culture ware were investigated by DIC/bright field and fluorescence/confocal microscopy for their morphology, 2D and 3D structure, and distribution patterns. Patch clamp, western blot, and RT-PCR studies were performed to investigate neuronal firing thresholds and sodium channel subtypes Nav1.2 and Nav1.6 expression. Cortical neurons cultured on PDL coating possessed a 2D structure composed of a few numbers of branched and tortuous neurites that contacted with each other in one to one manner, however, neurons on Matrigel coating showed a more complicated dimensional network that depicted tight, linear axonal bundles forming a 3D interacted neuron-astrocyte construction. This difference in growth patterns also showed a significant alteration in neuronal firing threshold which was recorded between 80 Iinj 120 pA on PDL and 2 Iinj 160 pA on Matrigel. Neurons grown up on Matrigel showed increased levels of sodium channel protein expression of Nav1.2 and Nav1.6 compared to neurons on PDL. These results have demonstrated that a 3D interacted neuron-astrocyte construction on Matrigel enhances the development of Nav1.2 and Nav1.6 in vitro and decreases neuronal firing threshold by 40 times compared to conventional PDL, resembling in vivo neuronal networks and hence would be a better in vitro model of adult neurons.

All cells maintain a voltage across their plasma membranes. Only excitable cells, however, can generate action potentials, the rapid, transient changes in membrane potential that spread along the surface of these unique cells. Action potential generation and propagation occurs through, and is regulated by, the function of voltage-gated ion channels – proteins with ion-selective pores that span the cell membrane. Ion channels undergo changes in their structural conformation in response to changes in the electrical field across the membrane. These structural changes cause the opening of pores – channels – through which ions can flow down their electrochemical gradient. The charge carried by ions creates an electrical current and rapidly alters the membrane potential with time- and voltage-dependent properties. This rapid, transient membrane potential change is called the action potential. Action potentials transmit information within neurons, trigger contractions within muscle cells, and lead to exocytosis in secretory cells. Key Concepts: All cells maintain a voltage difference across their plasma membranes. Action potentials are all-or-nothing, transient changes in membrane potentials of electrically excitable cells that carry important cellular information. Influx of sodium ions through voltage-gated sodium channels is responsible for the upstroke of the action potential, whereas efflux of potassium ions through voltage-gated potassium channels is responsible for the falling phase. Propagation of action potentials depends on gating kinetics of ion channels and intracellular and membrane resistances. Keywords: membrane potential; ionic current; threshold; refractoriness; length constant

Background:Telethonin (TCAP) is a Z-disk protein that maintains cytoskeletal integrity and various signaling pathways in cardiomyocytes. TCAP is shown to modulate α-subunit of the human cardiac sodium channel (hNav 1.5) by direct interactions. Several TCAP variants are found in cardiomyopathies. We sought to investigate whether TCAP variants are associated with arrhythmia syndromes., Methods:Mutational analyses for TCAP were performed in 303 Japanese patients with Brugada syndrome, arrhythmogenic right ventricular cardiomyopathy, and J-wave pattern ECG. Using patch-clamp techniques, electrophysiological characteristics of hNav 1.5 were studied in HEK-293 cells stably expressing hNav 1.5 and transiently transfected with wild-type (WT) or variant TCAP., Results:We identified two TCAP variants, c.145G>A:p.E49K and c.458G>A:p.R153H, in four individuals. p.E49K was found in two patients with ARVC or BrS. p.R153H was found in two patients with BrS or J-wave pattern ECG. No patient had variant hNav 1.5. Patch-clamp experiments demonstrated that peak sodium currents were significantly reduced in cells expressing p.R153H and p.E49K compared with WT-TCAP (66%, p.R153H; 72%, p.E49K). Voltage dependency of peak IV curve was rightward-shifted by 5 mV in cells expressing p.E49K compared with WT-TCAP. Voltage dependency of activation was not leftward-shifted by p.R153H, while voltage dependency of steady-state inactivation was leftward-shifted by p.E49K., Conclusions:We found two TCAP variants in the patients with BrS, J-wave pattern ECG, and ARVC that can cause loss-of-function of the hNav 1.5 in heterologous expression systems. Our observation suggests that these variants might impair INa and be associated with the patients' electrophysiological phenotypes. Further studies linking our experimental data to clinical phenotypes are warranted.

Genetic variants in brain-expressed voltage-gated sodium channels (SCNs) have emerged as one of the most frequent causes of Mendelian forms of epilepsy and neurodevelopmental disorders (NDDs). This review explores the biological concepts that underlie sodium channel NDDs, explains their phenotypic heterogeneity, and appraises how this knowledge may inform clinical practice. We observe that excitatory/inhibitory neuronal expression ratios of sodium channels are important regulatory mechanisms underlying brain development, homeostasis, and neurological diseases. We hypothesize that a detailed understanding of gene expression, variant tolerance, location, and function, as well as timing of seizure onset can aid the understanding of how variants in SCN1A, SCN2A, SCN3A, and SCN8A contribute to seizure aetiology and inform treatment choice. We propose a model in which variant type, development-specific gene expression, and functions of SCNs explain the heterogeneity of sodium channel associated NDDs. Understanding of basic disease mechanisms and detailed knowledge of variant characteristics have increasing influence on clinical decision making, enabling us to stratify treatment and move closer towards precision medicine in sodium channel epilepsy and NDDs. WHAT THIS PAPER ADDS: Sodium-channel disorder heterogeneity is explained by variant-specific gene expression timing and function. Gene tolerance and location analyses aid sodium channel variant interpretation. Sodium-channel variant characteristics can contribute to clinical decision making.

Brugada syndrome (BrS) is an inherited channelopathy responsible for almost 20% of sudden cardiac deaths in patients with nonstructural cardiac diseases. Approximately 70% of BrS patients, the causative gene mutation(s) remains unknown. In this study, we used whole exome sequencing to investigate candidate mutations in a family clinically diagnosed with BrS. A heterozygous 1616G>A substitution (R539Q mutation) was identified in the transmembrane protein 168 (TMEM168) gene of symptomatic individuals. Similar to endogenous TMEM168, both TMEM168 wild-type (WT) and mutant proteins that were ectopically induced in HL-1 cells showed nuclear membrane localization. A significant decrease in Na+ current and Nav 1.5 protein expression was observed in HL-1 cardiomyocytes expressing mutant TMEM168. Ventricular tachyarrhythmias and conduction disorders were induced in the heterozygous Tmem168 1616G>A knock-in mice by pharmacological stimulation, but not in WT mice. Na+ current was reduced in ventricular cardiomyocytes isolated from the Tmem168 knock-in heart, and Nav 1.5 expression was also impaired. This impairment was dependent on increased Nedd4-2 binding to Nav 1.5 and subsequent ubiquitination. Collectively, our results show an association between the TMEM168 1616G>A mutation and arrhythmogenesis in a family with BrS.

Objective: To pinpoint the earliest cellular defects underlying seizure onset (epileptogenic period) during perinatal brain development in a new zebrafish model of Dravet syndrome (DS) and to investigate potential disease-modifying activity of the 5HT2 receptor agonist fenfluramine. Methods: We used CRISPR/Cas9 mutagenesis to introduce a missense mutation, designed to perturb ion transport function in all channel isoforms, into scn1lab, the zebrafish orthologue of SCN1A (encoding voltage-gated sodium channel alpha subunit 1). We performed behavioral analysis and electroencephalographic recordings to measure convulsions and epileptiform discharges, followed by single-cell RNA-Seq, morphometric analysis of transgenic reporter-labeled γ-aminobutyric acidergic (GABAergic) neurons, and pharmacological profiling of mutant larvae. Results: Homozygous mutant (scn1labmut/mut ) larvae displayed spontaneous seizures with interictal, preictal, and ictal discharges (mean = 7.5 per 20-minute recording; P < .0001; one-way analysis of variance). Drop-Seq analysis revealed a 2:1 shift in the ratio of glutamatergic to GABAergic neurons in scn1labmut/mut larval brains versus wild type (WT), with dynamic changes in neuronal, glial, and progenitor cell populations. To explore disease pathophysiology further, we quantified dendritic arborization in GABAergic neurons and observed a 40% reduction in arbor number compared to WT (P < .001; n = 15 mutant, n = 16 WT). We postulate that the significant reduction in inhibitory arbors causes an inhibitory to excitatory neurotransmitter imbalance that contributes to seizures and enhanced electrical brain activity in scn1labmut/mut larvae (high-frequency range), with subsequent GABAergic neuronal loss and astrogliosis. Chronic fenfluramine administration completely restored dendritic arbor numbers to normal in scn1labmut/mut larvae, whereas similar treatment with the benzodiazepine diazepam attenuated seizures, but was ineffective in restoring neuronal cytoarchitecture. BrdU labeling revealed cell overproliferation in scn1labmut/mut larval brains that were rescued by fenfluramine but not diazepam. Significance: Our findings provide novel insights into early mechanisms of DS pathogenesis, describe dynamic cell population changes in the scn1labmut/mut brain, and present first-time evidence for potential disease modification by fenfluramine. Keywords: Dravet syndrome; epileptogenesis; fenfluramine; sodium channel; zebrafish. publishedVersion

BACKGROUND Apolygus lucorum (Meyer-Dur) is a serious worldwide agricultural pest, especially for Bt cotton in China. Pyrethroids, neonicotinoids and organophosphates are the most effective insecticides to control piercing and sucking insects, including A. lucorum. The voltage-gated sodium channel (Nav ) is major target site of pyrethroids. Extensive alternative splicing and RNA editing, two major post-transcriptional mechanisms, contribute to generate different functional sodium channel variants. In our research, we characterized the sodium channel variants of A. lucorum. RESULTS In this study, we isolated numerous sodium channel variants that cover the entire coding region of the VGSC gene from A. lucorum. All clones could be grouped into 47 splice types based on the presence of nine alternative exons (exons j, n, o, a, p, b, s, q and t). Exons j, b and t were located independently, while exons n, o, a and p were located adjacently, as were exons s and q. We also found 35 nucleotide changes in different positions in individual variants, of which 18 nucleotide changes were A-to-I RNA editing, 11 nucleotide changes were likely due to U-to-C or C-to-U editing, and the others were likely natural sequence polymorphisms in the population. Furthermore, we expressed all of the variants in Xenopus oocytes. Eighteen of them were expressed in oocytes and sensitive to tetrodotoxin. CONCLUSION Our results provide a functional basis for understanding how A. lucorum sodium channel variants work in regulating channel expression, pharmacology and gating properties for agricultural insects. Apolygus lucorum is widely distributed in cotton production. Our results suggest how AlNav (the sodium channel of A.lucorum) variants work in regulating channel expression, pharmacology and sodium channel gating for agricultural insects in the future. © 2020 Society of Chemical Industry.

The sodium channel NaX (encoded by the SCN7A gene) was originally identified in the heart and skeletal muscle and is structurally similar to the other voltage-gated sodium channels but does not appear to be voltage gated. Although NaX is expressed at high levels in cardiac and skeletal muscle, little information exists on the function of NaX in these tissues. Transcriptional profiling of ion channels in the heart in a subset of patients with Brugada syndrome revealed an inverse relationship between the expression of NaX and NaV 1.5 suggesting that, in cardiac myocytes, the expression of these channels may be linked. We propose that NaX plays a role in excitation-contraction coupling based on our experimental observations. Here we show that in cardiac myocytes, NaX is expressed in a striated pattern on the sarcolemma in regions corresponding to the sarcomeric M-line. Knocking down NaX expression decreased NaV 1.5 mRNA and protein and reduced the inward sodium current (INa+ ) following cell depolarization. When the expression of NaV 1.5 was knocked down, ~85% of the INa+ was reduced consistent with the observations that NaV 1.5 is the main voltage-gated sodium channel in cardiac muscle and that NaX likely does not directly participate in mediating the INa+ following depolarization. Silencing NaV 1.5 expression led to significant upregulation of NaX mRNA. Similar to NaV 1.5, NaX protein levels were rapidly downregulated when the intracellular [Ca2+ ] was increased either by CaCl2 or caffeine. These data suggest that a relationship exists between NaX and NaV 1.5 and that NaX may play a role in excitation-contraction coupling.

Objective SCN8A encephalopathy is a developmental and epileptic encephalopathy (DEE) caused by de novo gain-of-function mutations of sodium channel Nav 1.6 that result in neuronal hyperactivity. Affected individuals exhibit early onset drug-resistant seizures, developmental delay, and cognitive impairment. This study was carried out to determine whether reducing the abundance of the Scn8a transcript with an antisense oligonucleotide (ASO) would delay seizure onset and prolong survival in a mouse model of SCN8A encephalopathy. Methods ASO treatment was tested in a conditional mouse model with Cre-dependent expression of the pathogenic patient SCN8A mutation p.Arg1872Trp (R1872W). This model exhibits early onset of seizures, rapid progression, and 100% penetrance. An Scn1a +/- haploinsufficient mouse model of Dravet syndrome was also treated. ASO was administered by intracerebroventricular injection at postnatal day 2, followed in some cases by stereotactic injection at postnatal day 30. Results We observed a dose-dependent increase in length of survival from 15 to 65 days in the Scn8a-R1872W/+ mice treated with ASO. Electroencephalographic recordings were normal prior to seizure onset. Weight gain and activity in an open field were unaffected, but treated mice were less active in a wheel running assay. A single treatment with Scn8a ASO extended survival of Dravet syndrome mice from 3 weeks to >5 months. Interpretation Reduction of Scn8a transcript by 25 to 50% delayed seizure onset and lethality in mouse models of SCN8A encephalopathy and Dravet syndrome. Reduction of SCN8A transcript is a promising approach to treatment of intractable childhood epilepsies. Ann Neurol 2020;87:339-346.

In excitable membranes, the clustering of voltage-gated sodium channels (VGSC) serves to enhance excitability at critical sites. The two most profoundly studied sites of channel clustering are the axon initial segment, where action potentials are generated and the node of Ranvier, where action potentials propagate along myelinated axons. The clustering of VGSC is found, however, in other highly excitable sites such as axonal terminals, postsynaptic membranes of dendrites and muscle fibers, and pre-myelinated axons. In this review, different examples of axonal as well as non-axonal clustering of VGSC are discussed and the underlying mechanisms are compared. Whether the clustering of channels is intrinsically or extrinsically induced, it depends on the submembranous actin-based cytoskeleton that organizes these highly specialized membrane microdomains through specific adaptor proteins.

Funder: University of Surrey; Id: http://dx.doi.org/10.13039/501100003513, The Scn5a gene encodes the major pore‐forming Nav1.5 (α) subunit, of the voltage‐gated Na+ channel in cardiomyocytes. The key role of Nav1.5 in action potential initiation and propagation in both atria and ventricles predisposes organisms lacking Scn5a or carrying Scn5a mutations to cardiac arrhythmogenesis. Loss‐of‐function Nav1.5 genetic abnormalities account for many cases of the human arrhythmic disorder Brugada syndrome (BrS) and related conduction disorders. A murine model with a heterozygous Scn5a deletion recapitulates many electrophysiological phenotypes of BrS. This study examines the relationships between its Scn5a+/− genotype, resulting transcriptional changes, and the consequent phenotypic presentations of BrS. Of 62 selected protein‐coding genes related to cardiomyocyte electrophysiological or homeostatic function, concentrations of mRNA transcribed from 15 differed significantly from wild type (WT). Despite halving apparent ventricular Scn5a transcription heterozygous deletion did not significantly downregulate its atrial expression, raising possibilities of atria‐specific feedback mechanisms. Most of the remaining 14 genes whose expression differed significantly between WT and Scn5a+/− animals involved Ca2+ homeostasis specifically in atrial tissue, with no overlap with any ventricular changes. All statistically significant changes in expression were upregulations in the atria and downregulations in the ventricles. This investigation demonstrates the value of future experiments exploring for and clarifying links between transcriptional control of Scn5a and of genes whose protein products coordinate Ca2+ regulation and examining their possible roles in BrS.

Prolongation of the cardiac action potential (AP) and early after depolarizations (EADs) are electrical anomalies of cardiomyocytes that can lead to lethal arrhythmias and are potential liabilities for existing drugs and drug candidates in development. For example, long QT syndrome‐3 (LQTS3) is caused by mutations in the Nav1.5 sodium channel that debilitate channel inactivation and cause arrhythmias. We tested the hypothesis that a useful drug (i.e., mexiletine) with potential liabilities (i.e., potassium channel inhibition and adverse reactions) could be re‐engineered by dynamic medicinal chemistry to afford a new drug candidate with greater efficacy and less toxicity. Human cardiomyocytes were generated from LQTS3 patient‐derived induced pluripotent stem cells (hIPSCs) and normal hIPSCs to determine beneficial (on‐target) and detrimental effects (off‐target) of mexiletine and synthetic analogs, respectively. The approach combined “drug discovery” and "hit to lead” refinement and showed that iterations of medicinal chemistry and physiological testing afforded optimized compound 22. Compared to mexiletine, compound 22 showed a 1.85‐fold greater AUC and no detectable CNS toxicity at 100 mg/kg. In vitro hepatic metabolism studies showed that 22 was metabolized via cytochrome P‐450, as previously shown, and by the flavin‐containing monooxygenase (FMO). Deuterated‐22 showed decreased metabolism and showed acceptable cardiovascular and physicochemical properties., Scheme for identification of Lead compounds.

Ion channels can regulate the plasma membrane potential (Vm ) and cell migration as a result of altered ion flux. However, the mechanism by which Vm regulates motility remains unclear. Here, we show that the Nav 1.5 sodium channel carries persistent inward Na+ current which depolarizes the resting Vm at the timescale of minutes. This Nav 1.5-dependent Vm depolarization increases Rac1 colocalization with phosphatidylserine, to which it is anchored at the leading edge of migrating cells, promoting Rac1 activation. A genetically encoded FRET biosensor of Rac1 activation shows that depolarization-induced Rac1 activation results in acquisition of a motile phenotype. By identifying Nav 1.5-mediated Vm depolarization as a regulator of Rac1 activation, we link ionic and electrical signaling at the plasma membrane to small GTPase-dependent cytoskeletal reorganization and cellular migration. We uncover a novel and unexpected mechanism for Rac1 activation, which fine tunes cell migration in response to ionic and/or electric field changes in the local microenvironment.

Objective Drug-resistant epilepsy causes great clinical danger and still lacks effective treatments. Methods Here, we used multifaceted approaches combining electrophysiology, optogenetics, and chemogenetics in a classic phenytoin-resistant epilepsy model to reveal the key target of subicular pyramidal neurons in phenytoin resistance. Results In vivo neural recording showed that the firing rate of pyramidal neurons in the subiculum, but not other hippocampal subregions, could not be inhibited by phenytoin in phenytoin-resistant rats. Selective inhibition of subicular pyramidal neurons by optogenetics or chemogenetics reversed phenytoin resistance, whereas selective activation of subicular pyramidal neurons induced phenytoin resistance. Moreover, long-term low-frequency stimulation at the subiculum, which is clinically feasible, significantly inhibited the subicular pyramidal neurons and reversed phenytoin resistance. Furthermore, in vitro electrophysiology revealed that off-target use of phenytoin on sodium channels of subicular pyramidal neurons was involved in the phenytoin resistance, and clinical neuroimaging data suggested the volume of the subiculum in drug-resistant patients was related to the usage of sodium channel inhibitors. Interpretation These results highlight that the subicular pyramidal neurons may be a key switch control of drug-resistant epilepsy and represent a new potential target for precise treatments. ANN NEUROL 2019;86:626-640.

There is growing evidence that cardiac dysfunction in patients with chronic epilepsy could play a pathogenic role in sudden unexpected death in epilepsy (SUDEP). Recent animal studies have revealed that epilepsy secondarily alters the expression of cardiac ion channels alongside abnormal cardiac electrophysiology and remodeling. These molecular findings represent novel evidence for an acquired cardiac channelopathy in epilepsy, distinct from inherited ion channels mutations associated with cardiocerebral phenotypes. Specifically, seizure activity has been shown to alter the messenger RNA (mRNA) and protein expression of voltage-gated sodium channels (Nav 1.1, Nav 1.5), voltage-gated potassium channels (Kv 4.2, Kv 4.3), sodium-calcium exchangers (NCX1), and nonspecific cation-conducting channels (HCN2, HCN4). The pathophysiology may involve autonomic dysfunction and structural cardiac disease, as both are independently associated with epilepsy and ion channel dysregulation. Indeed, in vivo and in vitro studies of cardiac pathology reveal a complex network of signaling pathways and transcription factors regulating ion channel expression in the setting of sympathetic overactivity, cardiac failure, and hypertrophy. Other mechanisms such as circulating inflammatory mediators or exogenous effects of antiepileptic medications lack evidence. Moreover, an acquired cardiac channelopathy may underlie the electrophysiologic cardiac abnormalities seen in chronic epilepsy, potentially contributing to the increased risk of malignant arrhythmias and sudden death. Therefore, further investigation is necessary to establish whether cardiac ion channel dysregulation similarly occurs in patients with epilepsy, and to characterize any pathogenic relationship with SUDEP.

The voltage-sensitive sodium channel (VSSC) is essential for the generation and propagation of action potentials. VSSC kinetics can be modified by producing different splice variants. The functionality of VSSC depends on features such as the voltage sensors, the selectivity filter and the inactivation loop. Mutations in Vssc conferring resistance to pyrethroid insecticides are known as knockdown resistance (kdr). We analysed the conservation of VSSC in both a broad scope and a narrow scope by three approaches: (1) we compared conservation of sequences and of differential exon use across orders of the Insecta; (2) we determined which kdr mutations were possible with a single nucleotide mutation in nine populations of Aedes aegypti; and (3) we examined the individual VSSC variation that exists within a population of Drosophila melanogaster. There is an increasing amount of transcript diversity possible from Diplura towards Diptera. The residues of the voltage sensors, selectivity filter and inactivation loop are highly conserved. The majority of exon sequences were >88.6% similar. Strain-specific differences in codon constraints exist for kdr mutations in nine strains of A. aegypti. Three Vssc mutations were found in one population of D. melanogaster. This study shows that, overall, Vssc is highly conserved across Insecta and within a population of an insect, but that important differences do exist.

Multiple Sclerosis is an autoimmune disorder causing neurodegeneration mostly in young adults. Thereby, myelin is lost in the inflammatory lesions leaving unmyelinated axons at a high risk to degenerate. Oligodendrocyte precursor cells maintain their regenerative capacity into adulthood and are able to remyelinate axons if they are properly activated and differentiate. Neuronal activity influences the success of myelination indicating a close interplay between neurons and oligodendroglia. The myelination profile determines the distribution of voltage‐gated ion channels along the axon. Here, we analyze the distribution of the sodium channel subunit Nav1.6 and the ultrastructure of axons after cuprizone‐induced demyelination in transgenic mice expressing GFP in oligodendroglial cells. Using this mouse model, we found an increased number of GFP‐expressing oligodendroglial cells compared to untreated mice. Analyzing the axons, we found an increase in the number of nodes of Ranvier in mice that had received cuprizone. Furthermore, we found an enhanced portion of unmyelinated axons showing vesicles in the cytoplasm. These vesicles were labeled with VGlut1, indicating that they are involved in axonal signaling. Our results highlight the flexibility of axons towards changes in the glial compartment and depict the structural changes they undergo upon myelin removal. These findings might be considered if searching for new neuroprotective therapies that aim at blocking neuronal activity in order to avoid interfering with the process of remyelination.

SCN4A gene mutations cause a number of neuromuscular phenotypes including myotonia. A subset of infants with myotonia‐causing mutations experience severe life‐threatening episodic laryngospasm with apnea. We have recently identified similar SCN4A mutations in association with sudden infant death syndrome. Laryngospasm has also been proposed as a contributory mechanism to some cases of sudden unexpected death in epilepsy (SUDEP). We report an infant with EEG‐confirmed seizures and recurrent apneas. Whole‐exome sequencing identified a known pathogenic mutation in the SCN4A gene that has been reported in several unrelated families with myotonic disorder. We propose that the SCN4A mutation contributed to the apneas in our case, irrespective of the underlying cause of the epilepsy. We suggest this supports the notion that laryngospasm may contribute to some cases of SUDEP, and implicates a possible shared mechanism between a proportion of sudden infant deaths and sudden unexpected deaths in epilepsy.

The MDA-MB-231 cell line was used as a model of triple negative breast cancer to investigate the interaction of β-adrenergic receptor (β-AR) and voltage-gated sodium channel (VGSC). There was significant (86%) overlap in their expression. Short-term (acute) application of the β-AR antagonist propranolol (25 μM) led to reduction of peak current and quickening of current inactivation (the latter occurred only in 5% fetal bovine serum). Long-term (48 hr) incubation with propranolol (25 μM) resulted in several changes in VGSC characteristics: shifts in (a) current-voltage relationship and (b) steady-state inactivation, both to more negative potentials and (c) the slowing of recovery from inactivation. We then investigated the effects of propranolol and ranolazine, a blocker of VGSC activity, alone and in combination, on lateral motility and Matrigel invasion. These assays were carried out under hypoxic conditions more representative of tumor progression. Propranolol (2.5 and 25 μM) and ranolazine (5 μM), and their combination inhibited lateral motility. Also, propranolol (25 μM) and ranolazine (5 μM), and their combination inhibited invasion. However, no synergy was observed in the pharmacological combinations for both assays. Propranolol also significantly decreased total neonatal Nav1.5 protein expression, the predominant VGSC subtype expressed in these cells. We conclude (a) that β-AR and VGSC are functionally coupled in MDA-MB-231 cells; (b) that propranolol has direct blocking action on the VGSC; (c) that the action of propranolol is modulated by serum; and (d) that the antimetastatic cellular effects of propranolol and ranolazine are not additive.

Key points: Voltage-gated sodium channels are critical for peripheral sensory neuron transduction and have been implicated in a number of painful and painless disorders. The β-scorpion toxin, Cn2, is selective for Na1.6 in dorsal root ganglion neurons. Na1.6 plays an essential role in peripheral sensory neurons, specifically at the distal terminals of mechanosensing fibres innervating the skin and colon. Na1.6 activation also leads to enhanced response to mechanical stimulus in vivo. This works highlights the use of toxins in elucidating pain pathways moreover the importance of non-peripherally restricted Na isoforms in pain generation. Abstract: Peripheral sensory neurons express multiple voltage-gated sodium channels (Na) critical for the initiation and propagation of action potentials and transmission of sensory input. Three pore-forming sodium channel isoforms are primarily expressed in the peripheral nervous system (PNS): Na1.7, Na1.8 and Na1.9. These sodium channels have been implicated in painful and painless channelopathies and there has been intense interest in them as potential therapeutic targets in human pain. Emerging evidence suggests Na1.6 channels are an important isoform in pain sensing. This study aimed to assess, using pharmacological approaches, the function of Na1.6 channels in peripheral sensory neurons. The potent and Na1.6 selective β-scorpion toxin Cn2 was used to assess the effect of Na1.6 channel activation in the PNS. The multidisciplinary approach included Ca imaging, whole-cell patch-clamp recordings, skin–nerve and gut–nerve preparations and in vivo behavioural assessment of pain. Cn2 facilitates Na1.6 early channel opening, and increased persistent and resurgent currents in large-diameter dorsal root ganglion (DRG) neurons. This promotes enhanced excitatory drive and tonic action potential firing in these neurons. In addition, Na1.6 channel activation in the skin and gut leads to increased response to mechanical stimuli. Finally, intra-plantar injection of Cn2 causes mechanical but not thermal allodynia. This study confirms selectivity of Cn2 on Na1.6 channels in sensory neurons. Activation of Na1.6 channels, in terminals of the skin and viscera, leads to profound changes in neuronal responses to mechanical stimuli. In conclusion, sensory neurons expressing Na1.6 are important for the transduction of mechanical information in sensory afferents innervating the skin and viscera.

Natural product biosynthetic pathways are composed of enzymes that use powerful chemistry to assemble complex molecules. Small molecule neurotoxins are examples of natural products with intricate scaffolds which often have high affinities for their biological targets. The focus of this Minireview is small molecule neurotoxins targeting voltage-gated sodium channels (VGSCs) and the state of knowledge on their associated biosynthetic pathways. There are three small molecule neurotoxin receptor sites on VGSCs associated with three different classes of molecules: guanidinium toxins, alkaloid toxins, and ladder polyethers. Each of these types of toxins have unique structural features which are assembled by biosynthetic enzymes and the extent of information known about these enzymes varies among each class. The biosynthetic enzymes involved in the formation of these toxins have the potential to become useful tools in the efficient synthesis of VGSC probes.

Voltage-dependent sodium (Na(V)) 1.8 channels regulate action potential generation in nociceptive neurons, identifying them as putative analgesic targets. Here, we show that Na(V)1.8 channel plasma membrane localization, retention, and stability occur through a direct interaction with the postsynaptic density-95/discs large/zonula occludens-1–and WW domain–containing scaffold protein called membrane-associated guanylate kinase with inverted orientation (Magi)-1. The neurophysiological roles of Magi-1 are largely unknown, but we found that dorsal root ganglion (DRG)–specific knockdown of Magi-1 attenuated thermal nociception and acute inflammatory pain and produced deficits in Na(V)1.8 protein expression. A competing cell-penetrating peptide mimetic derived from the Na(V)1.8 WW binding motif decreased sodium currents, reduced Na(V)1.8 protein expression, and produced hypoexcitability. Remarkably, a phosphorylated variant of the very same peptide caused an opposing increase in Na(V)1.8 surface expression and repetitive firing. Likewise, in vivo, the peptides produced diverging effects on nocifensive behavior. Additionally, we found that Magi-1 bound to sequence like a calcium-activated potassium channel sodium-activated (Slack) potassium channels, demonstrating macrocomplexing with Na(V)1.8 channels. Taken together, these findings emphasize Magi-1 as an essential scaffold for ion transport in DRG neurons and a central player in pain.—Pryce, K. D., Powell, R., Agwa, D., Evely, K. M., Sheehan, G. D., Nip, A., Tomasello, D. L., Gururaj, S., Bhattacharjee, A. Magi-1 scaffolds Na(V)1.8 and Slack K(Na) channels in dorsal root ganglion neurons regulating excitability and pain.