A voltage-gated sodium channel is essential for the positive selection of CD4(+) T cells.

The sustained entry of Ca(2+) into CD4(+)CD8(+) double-positive thymocytes is required for positive selection. Here we identified a voltage-gated Na(+) channel (VGSC) that was essential for positive selection of CD4(+) T cells. Pharmacological inhibition of VGSC activity inhibited the sustained Ca(2+) influx induced by positively selecting ligands and the in vitro positive selection of CD4(+) but not CD8(+) T cells. In vivo short hairpin RNA (shRNA)-mediated knockdown of the gene encoding a regulatory ß-subunit of a VGSC specifically inhibited the positive selection of CD4(+) T cells. Ectopic expression of VGSC in peripheral AND CD4(+) T cells bestowed the ability to respond to a positively selecting ligand, which directly demonstrated that VGSC expression was responsible for the enhanced sensitivity. Thus, active VGSCs in thymocytes provide a mechanism by which a weak positive selection signal can induce the sustained Ca(2+) signals required for CD4(+) T cell development.

The Sodium Channel B4-Subunits are Dysregulated in Temporal Lobe Epilepsy Drug-Resistant Patients.

Temporal lobe epilepsy (TLE) is the most common type of partial epilepsy referred for surgery due to antiepileptic drug (AED) resistance. A common molecular target for many of these drugs is the voltage-gated sodium channel (VGSC). The VGSC consists of four domains of pore-forming α-subunits and two auxiliary ß-subunits, several of which have been well studied in epileptic conditions. However, despite the ß4-subunits' role having been reported in some neurological conditions, there is little research investigating its potential significance in epilepsy. Therefore, the purpose of this work was to assess the role of SCN4ß in epilepsy by using a combination of molecular and bioinformatics approaches. We first demonstrated that there was a reduction in the relative expression of SCN4B in the drug-resistant TLE patients compared to non-epileptic control specimens, both at the mRNA and protein levels. By analyzing a co-expression network in the neighborhood of SCN4B we then discovered a linkage between the expression of this gene and K+ channels activated by Ca2+, or K+ two-pore domain channels. Our approach also inferred several potential effector functions linked to variation in the expression of SCN4B. These observations support the hypothesis that SCN4B is a key factor in AED-resistant TLE, which could help direct both the drug selection of TLE treatments and the development of future AEDs.

Significant association of rare variant p.Gly8Ser in cardiac sodium channel ß4-subunit SCN4B with atrial fibrillation.

Atrial fibrillation (AF) affects 33.5 million individuals worldwide. It accounts for 15% of strokes and increases risk of heart failure and sudden death. The voltage-gated cardiac sodium channel complex is responsible for the generation and conduction of the cardiac action potential, and composed of the main pore-forming α-subunit Nav 1.5 (encoded by the SCN5A gene) and one or more auxiliary ß-subunits, including Nav ß1 to Nav ß4 encoded by SCN1B to SCN4B, respectively. We and others identified loss-of-function mutations in SCN1B and SCN2B and dominant-negative mutations in SCN3B in patients with AF. Three missense variants in SCN4B were identified in sporadic AF patients and small nuclear families; however, the association between SCN4B variants and AF remains to be further defined. In this study, we performed mutational analysis in SCN4B using a panel of 477 AF patients, and identified one nonsynonymous genomic variant p.Gly8Ser in four patients. To assess the association between the p.Gly8Ser variant and AF, we carried out case-control association studies with two independent populations (944 AF patients vs. 9,81 non-AF controls in the first discovery population and 732 cases and 1,291 controls in the second replication population). Significant association was identified in the two independent populations and in the combined population (p = 4.16 × 10-4 , odds ratio [OR] = 3.14) between p.Gly8Ser and common AF as well as lone AF (p = 0.018, OR = 2.85). These data suggest that rare variant p.Gly8Ser of SCN4B confers a significant risk of AF, and SCN4B is a candidate susceptibility gene for AF.

Identification of rare variants in cardiac sodium channel ß4-subunit gene SCN4B associated with ventricular tachycardia.

Ventricular tachycardia (VT) causes sudden cardiac death, however, the majority of risk genes for VT remain unknown. SCN4B encodes a ß-subunit, Navß4, for the voltage-gated cardiac sodium channel complex involved in generation and conduction of the cardiac action potential. We hypothesized that genomic variants in SCN4B increase the risk of VT. We used high-resolution melt analysis followed by Sanger sequencing to screen 199 VT patients to identify nonsynonymous variants in SCN4B. Two nonsynonymous heterozygous variants in SCN4B were identified in VT patients, including p.Gly8Ser in four VT patients and p.Ala145Ser in one VT patient. Case-control association studies were used to assess the association between variant p.Gly8Ser and VT in two independent populations for VT (299 VT cases vs. 981 controls in population 1 and 270 VT patients vs. 639 controls in population 2). Significant association was identified between p.Gly8Ser and VT in population 1 (P = 1.21 × 10-4, odds ratio or OR = 11.04), and the finding was confirmed in population 2 (P = 0.03, OR = 3.62). The association remained highly significant in the combined population (P = 3.09 × 10-5, OR = 6.17). Significant association was also identified between p.Gly8Ser and idiopathic VT (P = 1.89 × 10-5, OR = 7.27). Functional analysis with Western blotting showed that both p.Gly8Ser and p.Ala145Ser variants significantly reduced the expression level of Navß4. Based on 2015 ACMG Standards and Guidelines, p.Gly8Ser and p.Ala145Ser can be classified as the pathogenic and likely pathogenic variant, respectively. Our data suggest that SCN4B is a susceptibility gene for common VT and idiopathic VT and link rare SCN4B variants with large effects (OR = 6.17-7.27) to common VT.

Parallel homodimer structures of the extracellular domains of the voltage-gated sodium channel ß4 subunit explain its role in cell-cell adhesion.

Voltage-gated sodium channels (VGSCs) are transmembrane proteins required for the generation of action potentials in excitable cells and essential for propagating electrical impulses along nerve cells. VGSCs are complexes of a pore-forming α subunit and auxiliary ß subunits, designated as ß1/ß1B-ß4 (encoded by SCN1B-4B, respectively), which also function in cell-cell adhesion. We previously reported the structural basis for the trans homophilic interaction of the ß4 subunit, which contributes to its adhesive function. Here, using crystallographic and biochemical analyses, we show that the ß4 extracellular domains directly interact with each other in a parallel manner that involves an intermolecular disulfide bond between the unpaired Cys residues (Cys58) in the loop connecting strands B and C and intermolecular hydrophobic and hydrogen-bonding interactions of the N-terminal segments (Ser30-Val35). Under reducing conditions, an N-terminally deleted ß4 mutant exhibited decreased cell adhesion compared with the wild type, indicating that the ß4 cis dimer contributes to the trans homophilic interaction of ß4 in cell-cell adhesion. Furthermore, this mutant exhibited increased association with the α subunit, indicating that the cis dimerization of ß4 affects α-ß4 complex formation. These observations provide the structural basis for the parallel dimer formation of ß4 in VGSCs and reveal its mechanism in cell-cell adhesion.

Transcriptome organization for chronic alcohol abuse in human brain.

Alcohol dependence is a heterogeneous psychiatric disorder characterized by high genetic heritability and neuroadaptations occurring from repeated drug exposure. Through an integrated systems approach we observed consistent differences in transcriptome organization within postmortem human brain tissue associated with the lifetime consumption of alcohol. Molecular networks, determined using high-throughput RNA sequencing, for drinking behavior were dominated by neurophysiological targets and signaling mechanisms of alcohol. The systematic structure of gene sets demonstrates a novel alliance of multiple ion channels, and related processes, underlying lifetime alcohol consumption. Coordinate expression of these transcripts was enriched for genome-wide association signals in alcohol dependence and a meta-analysis of alcohol self-administration in mice. Further dissection of genes within alcohol consumption networks revealed the potential interaction of alternatively spliced transcripts. For example, expression of a human-specific isoform of the voltage-gated sodium channel subunit SCN4B was significantly correlated to lifetime alcohol consumption. Overall, our work demonstrates novel convergent evidence for biological networks related to excessive alcohol consumption, which may prove fundamentally important in the development of pharmacotherapies for alcohol dependence.

Crystallographic insights into sodium-channel modulation by the ß4 subunit.

Voltage-gated sodium (Nav) channels are embedded in a multicomponent membrane signaling complex that plays a crucial role in cellular excitability. Although the mechanism remains unclear, ß-subunits modify Nav channel function and cause debilitating disorders when mutated. While investigating whether ß-subunits also influence ligand interactions, we found that ß4 dramatically alters toxin binding to Nav1.2. To explore these observations further, we solved the crystal structure of the extracellular ß4 domain and identified (58)Cys as an exposed residue that, when mutated, eliminates the influence of ß4 on toxin pharmacology. Moreover, our results suggest the presence of a docking site that is maintained by a cysteine bridge buried within the hydrophobic core of ß4. Disrupting this bridge by introducing a ß1 mutation implicated in epilepsy repositions the (58)Cys-containing loop and disrupts ß4 modulation of Nav1.2. Overall, the principles emerging from this work (i) help explain tissue-dependent variations in Nav channel pharmacology; (ii) enable the mechanistic interpretation of ß-subunit-related disorders; and (iii) provide insights in designing molecules capable of correcting aberrant ß-subunit behavior.

Navß4 regulates fast resurgent sodium currents and excitability in sensory neurons.

BACKGROUND: Increased electrical activity in peripheral sensory neurons including dorsal root ganglia (DRG) and trigeminal ganglia neurons is an important mechanism underlying pain. Voltage gated sodium channels (VGSC) contribute to the excitability of sensory neurons and are essential for the upstroke of action potentials. A unique type of VGSC current, resurgent current (INaR), generates an inward current at repolarizing voltages through an alternate mechanism of inactivation referred to as open-channel block. INaRs are proposed to enable high frequency firing and increased INaRs in sensory neurons are associated with pain pathologies. While Nav1.6 has been identified as the main carrier of fast INaR, our understanding of the mechanisms that contribute to INaR generation is limited. Specifically, the open-channel blocker in sensory neurons has not been identified. Previous studies suggest Navß4 subunit mediates INaR in central nervous system neurons. The goal of this study was to determine whether Navß4 regulates INaR in DRG sensory neurons. RESULTS: Our immunocytochemistry studies show that Navß4 expression is highly correlated with Nav1.6 expression predominantly in medium-large diameter rat DRG neurons. Navß4 knockdown decreased endogenous fast INaR in medium-large diameter neurons as measured with whole-cell voltage clamp. Using a reduced expression system in DRG neurons, we isolated recombinant human Nav1.6 sodium currents in rat DRG neurons and found that overexpression of Navß4 enhanced Nav1.6 INaR generation. By contrast neither overexpression of Navß2 nor overexpression of a Navß4-mutant, predicted to be an inactive form of Navß4, enhanced Nav1.6 INaR generation. DRG neurons transfected with wild-type Navß4 exhibited increased excitability with increases in both spontaneous activity and evoked activity. Thus, Navß4 overexpression enhanced INaR and excitability, whereas knockdown or expression of mutant Navß4 decreased INaR generation. CONCLUSION: INaRs are associated with inherited and acquired pain disorders. However, our ability to selectively target and study this current has been hindered due to limited understanding of how it is generated in sensory neurons. This study identified Navß4 as an important regulator of INaR and excitability in sensory neurons. As such, Navß4 is a potential target for the manipulation of pain sensations.

Na+ channel-dependent recruitment of Navß4 to axon initial segments and nodes of Ranvier.

The axon initial segment (AIS) and nodes of Ranvier are the sites of action potential initiation and regeneration in axons. Although the basic molecular architectures of AIS and nodes, characterized by dense clusters of Na(+) and K(+) channels, are similar, firing patterns vary among cell types. Neuronal firing patterns are established by the collective activity of voltage-gated ion channels and can be modulated through interaction with auxiliary subunits. Here, we report the neuronal expression pattern and subcellular localization of Navß4, the modulatory Na(+) channel subunit thought to underlie resurgent Na(+) current. Immunostaining of rat tissues revealed that Navß4 is strongly enriched at the AIS of a select set of neuron types, including many characterized by high-frequency firing, and at nodes of Ranvier in the PNS and some nodes in the CNS. By introducing full-length and mutant GFP-tagged Navß4 into cultured neurons, we determined that the AIS and nodal localization of Navß4 depends on its direct interaction with Na(+) channel α subunits through an extracellular disulfide bond. Based on these results, we propose that differences in the specific composition of the Na(+) channel complexes enriched at the AIS and nodes contribute to the diverse physiologies observed among cell types.

Antagonism of lidocaine inhibition by open-channel blockers that generate resurgent Na current.

Na channels that generate resurgent current express an intracellular endogenous open-channel blocking protein, whose rapid binding upon depolarization and unbinding upon repolarization minimizes fast and slow inactivation. Na channels also bind exogenous compounds, such as lidocaine, which functionally stabilize inactivation. Like the endogenous blocking protein, these use-dependent inhibitors bind most effectively at depolarized potentials, raising the question of how lidocaine-like compounds affect neurons with resurgent Na current. We therefore recorded lidocaine inhibition of voltage-clamped, tetrodotoxin-sensitive Na currents in mouse Purkinje neurons, which express a native blocking protein, and in mouse hippocampal CA3 pyramidal neurons with and without a peptide from the cytoplasmic tail of NaVß4 (the ß4 peptide), which mimics endogenous open-channel block. To control channel states during drug exposure, lidocaine was applied with rapid-solution exchange techniques during steps to specific voltages. Inhibition of Na currents by lidocaine was diminished by either the ß4 peptide or the native blocking protein. In peptide-free CA3 cells, prolonging channel opening with a site-3 toxin, anemone toxin II, reduced lidocaine inhibition; this effect was largely occluded by open-channel blockers, suggesting that lidocaine binding is favored by inactivation but prevented by open-channel block. In constant 100 µm lidocaine, current-clamped Purkinje cells continued to fire spontaneously. Similarly, the ß4 peptide reduced lidocaine-dependent suppression of spiking in CA3 neurons in slices. Thus, the open-channel blocking protein responsible for resurgent current acts as a natural antagonist of lidocaine. Neurons with resurgent current may therefore be less susceptible to use-dependent Na channel inhibitors used as local anesthetic, antiarrhythmic, and anticonvulsant drugs.

Control of transient, resurgent, and persistent current by open-channel block by Na channel beta4 in cultured cerebellar granule neurons.

Voltage-gated Na channels in several classes of neurons, including cells of the cerebellum, are subject to an open-channel block and unblock by an endogenous protein. The Na(V)beta4 (Scn4b) subunit is a candidate blocking protein because a free peptide from its cytoplasmic tail, the beta4 peptide, can block open Na channels and induce resurgent current as channels unblock upon repolarization. In heterologous expression systems, however, Na(V)beta4 fails to produce resurgent current. We therefore tested the necessity of this subunit in generating resurgent current, as well as its influence on Na channel gating and action potential firing, by studying cultured cerebellar granule neurons treated with siRNA targeted against Scn4b. Knockdown of Scn4b, confirmed with quantitative RT-PCR, led to five electrophysiological phenotypes: a loss of resurgent current, a reduction of persistent current, a hyperpolarized half-inactivation voltage of transient current, a higher rheobase, and a decrease in repetitive firing. All disruptions of Na currents and firing were rescued by the beta4 peptide. The simplest interpretation is that Na(V)beta4 itself blocks Na channels of granule cells, making this subunit the first blocking protein that is responsible for resurgent current. The results also demonstrate that a known open-channel blocking peptide not only permits a rapid recovery from nonconducting states upon repolarization from positive voltages but also increases Na channel availability at negative potentials by antagonizing fast inactivation. Thus, Na(V)beta4 expression determines multiple aspects of Na channel gating, thereby regulating excitability in cultured cerebellar granule cells.

SCN4B inhibits the progression of lung adenocarcinoma and is associated with better prognosis.

INTRODUCTION: Lung adenocarcinoma (LUAD) is the major type of non-small cell lung cancer with low a survival rate caused by metastasis. SCN4B encoding voltage-gated sodium channel ß subunit is regarded as a metastasis-suppressor gene. We aim to explore how SCN4B influences the progression and prognosis of LUAD. METHODS: The gene expression profiles of 585 LUAD samples in TCGA and GSE31210, GSE116959, and GSE72094 datasets from the GEO database were downloaded for analysis. Differentially expressed genes were obtained through the "limma" package. The "clusterProfiler" package was used to conduct GSEA. Survival analysis was conducted via "survival" and "survminer" packages. Transcription factors regulating SCN4B expression were screened by correlation analysis and further predicted by FIMO. Infiltration of immune cells was analyzed by CIBERSORT. ESTIMATE algorithm was used to evaluate the immune-related scores. RESULTS: SCN4B expressed higher in normal samples than in LUAD samples and higher in female samples than male samples. One hundred and twenty-six pathways were significantly enriched between high and low SCN4B expression groups. Six transcription factors' expressions were positively related to SCN4B expression, and ChIP-seq data from "Cistrome" verified that TAL1 and ERG might bind to the upstream sequence of SCN4B. SCN4B expression was significantly correlated with activated memory CD4 T cells, resting mast cells, and monocytes. TMB status, three scores based on ESTIMATE algorithm, and expression of three immune checkpoints showed significant differences between SCN4B high- and low-expression groups. SCN4B could be considered as an independent prognostic signature of LUAD patients that higher expression represents a better prognosis. CONCLUSION: SCN4B expresses higher in normal samples, and SCN4B is able to be an independent prognostic signature for LUAD patients. TAL1 and ERG may regulate the expression of SCN4B by binding its upstream sequences. Our research is valuable in improving the effectiveness of treatment in LUAD.

Cross-species conservation of open-channel block by Na channel ß4 peptides reveals structural features required for resurgent Na current.

Voltage-gated Na channels in many neurons, including several in the cerebellum and brainstem, are specialized to allow rapid firing of action potentials. Repetitive firing is facilitated by resurgent Na current, which flows upon repolarization as Na channels recover through open states from block by an endogenous protein. The best candidate blocking protein to date is Na(V)ß4. The sequence of this protein diverges among species, however, while high-frequency firing is maintained, raising the question of whether the proposed blocking action of the Na(V)ß4 cytoplasmic tail has been conserved. Here, we find that, despite differences in the Na(V)ß4 sequence, Purkinje cells isolated from embryonic chick have resurgent currents with kinetics and amplitudes indistinguishable from those in mouse Purkinje cells. Furthermore, synthetic peptides derived from the divergent Na(V)ß4 cytoplasmic tails from five species have the capacity to induce resurgent current in mouse hippocampal neurons, which lack a functional endogenous blocking protein. These data further support a blocking role for Na(V)ß4 and also indicate the relative importance of different residues in inducing open-channel block. To investigate the contribution of the few highly conserved residues to open-channel block, we synthesized several mutant peptides in which the identities and relative orientations of a phenylalanine and two lysines were disrupted. These mutant peptides produced currents with vastly different kinetics than did the species-derived peptides, suggesting that these residues are required for an open-channel block that approximates physiological resurgent Na current. Thus, if other blocking proteins exist, they may share these structural elements with the Na(V)ß4 cytoplasmic tail.

Intrinsic mechanisms in the gating of resurgent Na+ currents.

The resurgent component of the voltage-gated sodium current (INaR) is a depolarizing conductance, revealed on membrane hyperpolarizations following brief depolarizing voltage steps, which has been shown to contribute to regulating the firing properties of numerous neuronal cell types throughout the central and peripheral nervous systems. Although mediated by the same voltage-gated sodium (Nav) channels that underlie the transient and persistent Nav current components, the gating mechanisms that contribute to the generation of INaR remain unclear. Here, we characterized Nav currents in mouse cerebellar Purkinje neurons, and used tailored voltage-clamp protocols to define how the voltage and the duration of the initial membrane depolarization affect the amplitudes and kinetics of INaR. Using the acquired voltage-clamp data, we developed a novel Markov kinetic state model with parallel (fast and slow) inactivation pathways and, we show that this model reproduces the properties of the resurgent, as well as the transient and persistent, Nav currents recorded in (mouse) cerebellar Purkinje neurons. Based on the acquired experimental data and the simulations, we propose that resurgent Na+ influx occurs as a result of fast inactivating Nav channels transitioning into an open/conducting state on membrane hyperpolarization, and that the decay of INaR reflects the slow accumulation of recovered/opened Nav channels into a second, alternative and more slowly populated, inactivated state. Additional simulations reveal that extrinsic factors that affect the kinetics of fast or slow Nav channel inactivation and/or impact the relative distribution of Nav channels in the fast- and slow-inactivated states, such as the accessory Navß4 channel subunit, can modulate the amplitude of INaR.

Inhibition of Navß4 peptide-mediated resurgent sodium currents in Nav1.7 channels by carbamazepine, riluzole, and anandamide.

Paroxysmal extreme pain disorder (PEPD) and inherited erythromelalgia (IEM) are inherited pain syndromes arising from different sets of gain-of-function mutations in the sensory neuronal sodium channel isoform Nav1.7. Mutations associated with PEPD, but not IEM, result in destabilized inactivation of Nav1.7 and enhanced resurgent sodium currents. Resurgent currents arise after relief of ultra-fast open-channel block mediated by an endogenous blocking particle and are thought to influence neuronal excitability. As such, enhancement of resurgent currents may constitute a pathological mechanism contributing to sensory neuron hyperexcitability and pain hypersensitivity associated with PEPD. Furthermore, pain associated with PEPD, but not IEM, is alleviated by the sodium channel inhibitor carbamazepine. We speculated that selective attenuation of PEPD-enhanced resurgent currents might contribute to this therapeutic effect. Here we examined whether carbamazepine and two other sodium channel inhibitors, riluzole and anandamide, exhibit differential inhibition of resurgent currents. To gain further insight into the potential mechanism(s) of resurgent currents, we examined whether these inhibitors produced correlative changes in other properties of sodium channel inactivation. Using stably transfected human embryonic kidney 293 cells expressing wild-type Nav1.7 and the PEPD mutants T1464I and M1627K, we examined the effects of the three drugs on Navß4 peptide-mediated resurgent currents. We observed a correlation between resurgent current inhibition and a drug-mediated increase in the rate of inactivation and inhibition of persistent sodium currents. Furthermore, although carbamazepine did not selectively target resurgent currents, anandamide strongly inhibited resurgent currents with minimal effects on the peak transient current amplitude, demonstrating that resurgent currents can be selectively targeted.

Nav1.7 mutations associated with paroxysmal extreme pain disorder, but not erythromelalgia, enhance Navbeta4 peptide-mediated resurgent sodium currents.

Abnormal pain sensitivity associated with inherited and acquired pain disorders occurs through increased excitability of peripheral sensory neurons in part due to changes in the properties of voltage-gated sodium channels (Navs). Resurgent sodium currents (I(NaR)) are atypical currents believed to be associated with increased excitability of neurons and may have implications in pain. Mutations in Nav1.7 (peripheral Nav isoform) associated with two genetic pain disorders, inherited erythromelalgia (IEM) and paroxysmal extreme pain disorder (PEPD), enhance Nav1.7 function via distinct mechanisms. We show that changes in Nav1.7 function due to mutations associated with PEPD, but not IEM, are important in I(NaR) generation, suggesting that I(NaR) may play a role in pain associated with PEPD. This knowledge provides us with a better understanding of the mechanism of I(NaR) generation and may lead to the development of specialized treatment for pain disorders associated with I(NaR).

ß-Site APP-cleaving enzyme 1 (BACE1) cleaves cerebellar Na+ channel ß4-subunit and promotes Purkinje cell firing by slowing the decay of resurgent Na+ current.

In cerebellar Purkinje cells, the ß4-subunit of voltage-dependent Na(+) channels has been proposed to serve as an open-channel blocker giving rise to a "resurgent" Na(+) current (I (NaR)) upon membrane repolarization. Notably, the ß4-subunit was recently identified as a novel substrate of the ß-secretase, BACE1, a key enzyme of the amyloidogenic pathway in Alzheimer's disease. Here, we asked whether BACE1-mediated cleavage of ß4-subunit has an impact on I (NaR) and, consequently, on the firing properties of Purkinje cells. In cerebellar tissue of BACE1-/- mice, mRNA levels of Na(+) channel α-subunits 1.1, 1.2, and 1.6 and of ß-subunits 1-4 remained unchanged, but processing of ß4 peptide was profoundly altered. Patch-clamp recordings from acutely isolated Purkinje cells of BACE1-/- and WT mice did not reveal any differences in steady-state properties and in current densities of transient, persistent, and resurgent Na(+) currents. However, I (NaR) was found to decay significantly faster in BACE1-deficient Purkinje cells than in WT cells. In modeling studies, the altered time course of I (NaR) decay could be replicated when we decreased the efficiency of open-channel block. In current-clamp recordings, BACE1-/- Purkinje cells displayed lower spontaneous firing rate than normal cells. Computer simulations supported the hypothesis that the accelerated decay kinetics of I (NaR) are responsible for the slower firing rate. Our study elucidates a novel function of BACE1 in the regulation of neuronal excitability that serves to tune the firing pattern of Purkinje cells and presumably other neurons endowed with I (NaR).

Genetically determined differences in sodium current characteristics modulate conduction disease severity in mice with cardiac sodium channelopathy.

Conduction slowing of the electric impulse that drives the heartbeat may evoke lethal cardiac arrhythmias. Mutations in SCN5A, which encodes the pore-forming cardiac sodium channel alpha subunit, are associated with familial arrhythmia syndromes based on conduction slowing. However, disease severity among mutation carriers is highly variable. We hypothesized that genetic modifiers underlie the variability in conduction slowing and disease severity. With the aim of identifying such modifiers, we studied the Scn5a(1798insD/+) mutation in 2 distinct mouse strains, FVB/N and 129P2. In 129P2 mice, the mutation resulted in more severe conduction slowing particularly in the right ventricle (RV) compared to FVB/N. Pan-genomic mRNA expression profiling in the 2 mouse strains uncovered a drastic reduction in mRNA encoding the sodium channel auxiliary subunit beta4 (Scn4b) in 129P2 mice compared to FVB/N. This corresponded to low to undetectable beta4 protein levels in 129P2 ventricular tissue, whereas abundant beta4 protein was detected in FVB/N. Sodium current measurements in isolated myocytes from the 2 mouse strains indicated that sodium channel activation in myocytes from 129P2 mice occurred at more positive potentials compared to FVB/N. Using computer simulations, this difference in activation kinetics was predicted to explain the observed differences in conduction disease severity between the 2 strains. In conclusion, genetically determined differences in sodium current characteristics on the myocyte level modulate disease severity in cardiac sodium channelopathies. In particular, the sodium channel subunit beta4 (SCN4B) may constitute a potential genetic modifier of conduction and cardiac sodium channel disease.

Silencing of microRNA-3175 represses cell proliferation and invasion in prostate cancer by targeting the potential tumor-suppressor SCN4B.

MicroRNA-3175 (miR-3175) expression is upregulated in prostate cancer, but its roles and the underlying mechanisms in prostate cancer cell growth and invasion need to be elucidated. This study aimed to uncover the roles of miR-3175 in regulating cell growth and migration, as well as the expression of its predicted target gene cardiac sodium channel ß4-subunit gene (SCN4B). Real-time quantitative PCR (RT-qPCR) and/or western blotting techniques were used to measure miR-3175 and SCN4B expression levels in prostate cancer cells. Inhibitor or mimics transfections were used to overexpress or silence miR-3175 in prostate cancer cells. MTT and Edu assays were applied to assess cell viability. Scratch assay and transwell chambers were used to examine cell migration and invasion abilities. The interaction between miR-3175 and SCN4B was determined by means of luciferase gene reporter, RT-qPCR, and western blotting assays. The results showed that miR-3175 expression was increased and SCN4B expression was decreased in prostate cancer cell lines as compared with normal human prostatic epithelial cells. Compared with the control group, knockdown of miR-3175 resulted in strong inhibitions of cell growth, migration, invasion, and N-cadherin expression, together with an increase in E-cadherin expression. In addition, knockdown of miR-3175 dramatically increased the luciferase activity of the luciferase vector of SCN4B, and increased SCN4B expression. Together, this study illustrated that downregulation of miR-3175 repressed the proliferation and invasion of prostate cancer cells, which might be induced by SCN4B downregulation.