Sodium Channel Β1 Subunit Research Articles

Role of protein domains in trafficking and localization of the voltage-gated sodium channel β2 subunit

The voltage-gated sodium (NaV) channel is critical for cardiomyocyte function since it is responsible for action potential initiation and its propagation throughout the cell. It consists of a protein complex made of a pore forming α subunit and associated β subunits, which regulate α subunit function and subcellular localization. We previously showed the implication of N-linked glycosylation and S-acylation of β2 in its polarized trafficking. Here, we present evidence of β2 dimerization. Moreover, we demonstrate the implication of the cytoplasmic tail, extracellular loop, and transmembrane domain on proper β2 folding and export to the cell surface of polarized Madin-Darby canine kidney cells. Substantial alteration, or lack of any of these domains, leads to accumulation of β2 in the endoplasmic reticulum, along with impaired complex N-glycosylation, which is needed for its efficient surface delivery. We also show that these alterations to β2 affected to certain extent NaV1.5 surface localization. Conversely, however, NaV1.5 had little or no influence on β2 trafficking, its localization to the surface, or homodimer formation. Altogether, our data link the architecture of the β2 domains to the establishment of its proper subcellular localization. These findings could provide valuable insights to gain a deeper comprehension of the elusive biology of β subunits in excitable cells, such as neurons and cardiomyocytes.

MicroRNA-6954-3p Downregulation Contributes to Orofacial Neuropathic Pain in Mice Via Targeting Voltage-Gated Sodium Channel β2 Subunit Protein

The voltage-gated sodium channel β2 subunit protein (SCN2B) plays a crucial role in neuropathic pain. However, the role and mechanisms of SCN2B in orofacial neuropathic pain are still unclear. This study aimed to investigate the upstream regulatory mechanisms of SCN2B in the trigeminal ganglion (TG) underlying orofacial neuropathic pain. Chronic constriction injury of the infraorbital nerve (CCI-ION) of mice was performed to establish the model of orofacial neuropathic pain. Von Frey filament test was performed to detect the head withdrawal threshold (HWT) of mice. Quantitative reverse transcription-polymerase chain, western blotting (WB), fluorescence in situ hybridization, and immunofluorescence (IF) staining were used to detect the expression and distribution of SCN2B and miR-6954-3p in the TG of mice. A luciferase activity assay was carried out to prove the binding between SCN2B messenger ribonucleic acid (mRNA) and miR-6954-3p. After the CCI-ION surgery, the levels of Scn2b mRNA and protein significantly increased and miR-6954-3p decreased in the TG of mice with decreasing HWT. IF staining revealed that SCN2B was expressed specifically in the TG neurons. Silencing SCN2B in the TG of CCI-ION mice significantly increased the HWT. Importantly, the 3′-untranslated region of Scn2b mRNA was proved to bind with miR-6954-3p. Fluorescence in situ hybridization and IF staining demonstrated that miR-6954-3p was expressed in TG neurons and co-expressed with SCN2B. Furthermore, intraganglionic injection of miR-6954-3p agomir into the TG of CCI-ION mice resulted in the downregulation of SCN2B and increased the HWT. These findings suggest that the downregulation of miR-6954-3p in the TG promotes orofacial neuropathic pain by promoting SCN2B expression following trigeminal nerve injury. PerspectiveThis study points to the important role of SCN2B in orofacial neuropathic pain. Furthermore, miR-6954-3p is proven to regulate the expression of SCN2B by binding to the 3′-untranslated region of Scn2b mRNA. These findings indicate that SCN2B and miR-6954-3p are potential therapeutic targets for the treatment of orofacial neuropathic pain.

LRRC37B is a human modifier of voltage-gated sodium channels and axon excitability in cortical neurons

The enhanced cognitive abilities characterizing the human species result from specialized features of neurons and circuits. Here, we report that the hominid-specific gene LRRC37B encodes a receptor expressed in human cortical pyramidal neurons (CPNs) and selectively localized to the axon initial segment (AIS), the subcellular compartment triggering action potentials. Ectopic expression of LRRC37B in mouse CPNs invivo leads to reduced intrinsic excitability, a distinctive feature of some classes of human CPNs. Molecularly, LRRC37B binds to the secreted ligand FGF13A and to the voltage-gated sodium channel (Nav) β-subunit SCN1B. LRRC37B concentrates inhibitory effects of FGF13A on Nav channel function, thereby reducing excitability, specifically at the AIS level. Electrophysiological recordings in adult human cortical slices reveal lower neuronal excitability in human CPNs expressing LRRC37B. LRRC37B thus acts as a species-specific modifier of human neuron excitability, linking human genome and cell evolution, with important implications for human brain function and diseases.

Epilepsy and sudden unexpected death in epilepsy in a mouse model of human SCN1B-linked developmental and epileptic encephalopathy.

Voltage-gated sodium channel β1 subunits are essential proteins that regulate excitability. They modulate sodium and potassium currents, function as cell adhesion molecules and regulate gene transcription following regulated intramembrane proteolysis. Biallelic pathogenic variants in SCN1B, encoding β1, are linked to developmental and epileptic encephalopathy 52, with clinical features overlapping Dravet syndrome. A recessive variant, SCN1B-c.265C>T, predicting SCN1B-p.R89C, was homozygous in two children of a non-consanguineous family. One child was diagnosed with Dravet syndrome, while the other had a milder phenotype. We identified an unrelated biallelic SCN1B-c.265C>T patient with a clinically more severe phenotype than Dravet syndrome. We used CRISPR/Cas9 to knock-in SCN1B-p.R89C to the mouse Scn1b locus (Scn1bR89/C89). We then rederived the line on the C57BL/6J background to allow comparisons between Scn1bR89/R89 and Scn1bC89/C89 littermates with Scn1b+/+ and Scn1b-/- mice, which are congenic on C57BL/6J, to determine whether the SCN1B-c.265C>T variant results in loss-of-function. Scn1bC89/C89 mice have normal body weights and ∼20% premature mortality, compared with severely reduced body weight and 100% mortality in Scn1b-/- mice. β1-p.R89C polypeptides are expressed in brain at comparable levels to wild type. In heterologous cells, β1-p.R89C localizes to the plasma membrane and undergoes regulated intramembrane proteolysis similar to wild type. Heterologous expression of β1-p.R89C results in sodium channel α subunit subtype specific effects on sodium current. mRNA abundance of Scn2a, Scn3a, Scn5a and Scn1b was increased in Scn1bC89/C89 somatosensory cortex, with no changes in Scn1a. In contrast, Scn1b-/- mouse somatosensory cortex is haploinsufficient for Scn1a, suggesting an additive mechanism for the severity of the null model via disrupted regulation of another Dravet syndrome gene. Scn1bC89/C89 mice are more susceptible to hyperthermia-induced seizures at post-natal Day 15 compared with Scn1bR89/R89 littermates. EEG recordings detected epileptic discharges in young adult Scn1bC89/C89 mice that coincided with convulsive seizures and myoclonic jerks. We compared seizure frequency and duration in a subset of adult Scn1bC89/C89 mice that had been exposed to hyperthermia at post-natal Day 15 versus a subset that were not hyperthermia exposed. No differences in spontaneous seizures were detected between groups. For both groups, the spontaneous seizure pattern was diurnal, occurring with higher frequency during the dark cycle. This work suggests that the SCN1B-c.265C>T variant does not result in complete loss-of-function. Scn1bC89/C89 mice more accurately model SCN1B-linked variants with incomplete loss-of-function compared with Scn1b-/- mice, which model complete loss-of-function, and thus add to our understanding of disease mechanisms as well as our ability to develop new therapeutic strategies.

Therapeutic Potential of Targeting Regulated Intramembrane Proteolysis Mechanisms of Voltage-Gated Ion Channel Subunits and Cell Adhesion Molecules.

Several integral membrane proteins undergo regulated intramembrane proteolysis (RIP), a tightly controlled process through which cells transmit information across and between intracellular compartments. RIP generates biologically active peptides by a series of proteolytic cleavage events carried out by two primary groups of enzymes: sheddases and intramembrane-cleaving proteases (iCLiPs). Following RIP, fragments of both pore-forming and non-pore-forming ion channel subunits, as well as immunoglobulin super family (IgSF) members, have been shown to translocate to the nucleus to function in transcriptional regulation. As an example, the voltage-gated sodium channel β1 subunit, which is also an IgSF-cell adhesion molecule (CAM), is a substrate for RIP. β1 RIP results in generation of a soluble intracellular domain, which can regulate gene expression in the nucleus. In this review, we discuss the proposed RIP mechanisms of voltage-gated sodium, potassium, and calcium channel subunits as well as the roles of their generated proteolytic products in the nucleus. We also discuss other RIP substrates that are cleaved by similar sheddases and iCLiPs, such as IgSF macromolecules, including CAMs, whose proteolytically generated fragments function in the nucleus. Importantly, dysfunctional RIP mechanisms are linked to human disease. Thus, we will also review how understanding RIP events and subsequent signaling processes involving ion channel subunits and IgSF proteins may lead to the discovery of novel therapeutic targets. SIGNIFICANCE STATEMENT: Several ion channel subunits and immunoglobulin superfamily molecules have been identified as substrates of regulated intramembrane proteolysis (RIP). This signal transduction mechanism, which generates polypeptide fragments that translocate to the nucleus, is an important regulator of gene transcription. RIP may impact diseases of excitability, including epilepsy, cardiac arrhythmia, and sudden death syndromes. A thorough understanding of the role of RIP in gene regulation is critical as it may reveal novel therapeutic strategies for the treatment of previously intractable diseases.

Scn1b expression in the adult mouse heart modulates Na+ influx in myocytes and reveals a mechanistic link between Na+ entry and diastolic function.

Voltage-gated sodium channels (VGSCs) are macromolecular assemblies composed of a number of proteins regulating channel conductance and properties. VGSCs generate Na+ current (INa) in myocytes and play fundamental roles in excitability and impulse conduction in the heart. Moreover, VGSCs condition mechanical properties of the myocardium, a process that appears to involve the late component of INa. Variants in the gene SCN1B, encoding the VGSC β1- and β1B-subunits, result in inherited neurological disorders and cardiac arrhythmias. But the precise contributions of β1/β1B-subunits and VGSC integrity to the overall function of the adult heart remain to be clarified. For this purpose, adult mice with cardiac-restricted, inducible deletion of Scn1b (conditional knockout, cKO) were studied. Myocytes from cKO mice had increased densities of fast (+20%)- and slow (+140%)-inactivating components of INa, with respect to control cells. By echocardiography and invasive hemodynamics, systolic function was preserved in cKO mice, but diastolic properties and ventricular compliance were compromised, with respect to control animals. Importantly, inhibition of late INa with GS967 normalized left ventricular filling pattern and isovolumic relaxation time in cKO mice. At the cellular level, cKO myocytes presented delayed kinetics of Ca2+ transients and cell mechanics, defects that were corrected by inhibition of INa. Collectively, these results document that VGSC β1/β1B-subunits modulate electrical and mechanical function of the heart by regulating, at least in part, Na+ influx in cardiomyocytes.NEW & NOTEWORTHY We have investigated the consequences of deletion of Scn1b, the gene encoding voltage-gated sodium channel β1-subunits, on myocyte and cardiac function. Our findings support the notion that Scn1b expression controls properties of Na+ influx and Ca2+ cycling in cardiomyocytes affecting the modality of cell contraction and relaxation. These effects at the cellular level condition electrical recovery and diastolic function in vivo, substantiating the multifunctional role of β1-subunits in the physiology of the heart.

A novel gain-of-function sodium channel β2 subunit mutation in idiopathic small fiber neuropathy.

Small fiber neuropathy (SFN) is a common condition affecting thinly myelinated Aδ and unmyelinated C fibers, often resulting in excruciating pain and dysautonomia. SFN has been associated with several conditions, but a significant number of cases have no discernible cause. Recent genetic studies have identified potentially pathogenic gain-of-function mutations in several pore-forming voltage-gated sodium channel α subunits (NaV) in a subset of patients with SFN, but the auxiliary sodium channel β subunits have been less implicated in the development of the disease. β subunits modulate NaV trafficking and gating, and several mutations have been linked to epilepsy and cardiac dysfunction. Recently, we provided the first evidence for the contribution of a mutation in the β2 subunit to pain in human painful diabetic neuropathy. Here, we provide the first evidence for the involvement of a sodium channel β subunit mutation in the pathogenesis of SFN with no other known causes. We show, through current-clamp analysis, that the newly identified Y69H variant of the β2 subunit induces neuronal hyperexcitability in dorsal root ganglion neurons, lowering the threshold for action potential firing and allowing for increased repetitive action potential spiking. Underlying the hyperexcitability induced by the β2-Y69H variant, we demonstrate an upregulation in tetrodotoxin-sensitive, but not tetrodotoxin-resistant sodium currents. This provides the first evidence for the involvement of β2 subunits in SFN and strengthens the link between sodium channel β subunits and the development of neuropathic pain in humans.NEW & NOTEWORTHY Small fiber neuropathy (SFN) often has no discernible cause, although mutations in the voltage-gated sodium channel α subunits have been implicated in some cases. We identify a patient suffering from SFN with a mutation in the auxiliary β2 subunit and no other discernible causes for SFN. Functional assessment confirms this mutation renders dorsal root ganglion neurons hyperexcitable and upregulates tetrodotoxin-sensitive sodium currents. This study strengthens a newly emerging link between sodium channel β2 subunit mutations and human pain disorders.

The voltage-gated sodium channel β2 subunit associates with lipid rafts by S-palmitoylation.

The voltage-gated sodium channel is critical for cardiomyocyte function. It consists of a protein complex comprising a pore-forming α subunit and associated β subunits. In polarized Madin-Darby canine kidney cells, we show evidence by acyl-biotin exchange that β2 is S-acylated at Cys-182. Interestingly, we found that palmitoylation increases β2 association with detergent-resistant membranes. β2 localizes exclusively to the apical surface. However, depletion of plasma membrane cholesterol, or blocking intracellular cholesterol transport, caused mislocalization of β2, as well as of the non-palmitoylable C182S mutant, to the basolateral domain. Apical β2 did not undergo endocytosis and displayed limited diffusion within the plane of the membrane; such behavior suggests that, at least in part, it is cytoskeleton anchored. Upon acute cholesterol depletion, its mobility was greatly reduced, and a slight reduction was also measured as a result of lack of palmitoylation, supporting β2 association with cholesterol-rich lipid rafts. Indeed, lipid raft labeling confirmed a partial overlap with apical β2. Although β2 palmitoylation was not required to promote surface localization of the α subunit, our data suggest that it is likely implicated in lipid raft association and the polarized localization of β2.

Sodium channel β1 subunits participate in regulated intramembrane proteolysis-excitation coupling.

Loss-of-function (LOF) variants in SCN1B, encoding voltage-gated sodium channel β1 subunits, are linked to human diseases with high risk of sudden death, including developmental and epileptic encephalopathy and cardiac arrhythmia. β1 Subunits modulate the cell-surface localization, gating, and kinetics of sodium channel pore-forming α subunits. They also participate in cell-cell and cell-matrix adhesion, resulting in intracellular signal transduction, promotion of cell migration, calcium handling, and regulation of cell morphology. Here, we investigated regulated intramembrane proteolysis (RIP) of β1 by BACE1 and γ-secretase and show that β1 subunits are substrates for sequential RIP by BACE1 and γ-secretase, resulting in the generation of a soluble intracellular domain (ICD) that is translocated to the nucleus. Using RNA sequencing, we identified a subset of genes that are downregulated by β1-ICD overexpression in heterologous cells but upregulated in Scn1b-null cardiac tissue, which lacks β1-ICD signaling, suggesting that the β1-ICD may normally function as a molecular brake on gene transcription in vivo. We propose that human disease variants resulting in SCN1B LOF cause transcriptional dysregulation that contributes to altered excitability. Moreover, these results provide important insights into the mechanism of SCN1B-linked channelopathies, adding RIP-excitation coupling to the multifunctionality of sodium channel β1 subunits.

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 β4-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.

The Nanoscale Basis of Atrial Fibrillation: Functional Impact of Disrupting NaV1.5-rich Intercalated Disk Nanodomains.

Background:Atrial fibrillation (AF), which is characterized by chaotic patterns of electrical activation of the atria, affects over 4 million people in the US alone. We previously identified nanoscale structural abnormalities in the hearts of AF patients. Specifically, they displayed swelling of gap junction (GJ) –adjacent perinexi, specialized nanodomains rich in cardiac sodium channels (NaV1.5) and located within intercalated disks (IDs; sites of electromechanical contact between adjacent cells). However, the functional consequences of these nanoscale structural changes remain unclear.Objective:We assessed the structural and functional impacts of selectively disrupting different NaV1.5-rich ID nanodomains.Methods and Results:We utilized peptide mimetics of adhesion domains to selectively inhibit adhesion within different ID nanodomains: 1) Nadp1 (target: N-cadherin), 2) dadp1 (target: Desmoglein-2), and 3) βadp1 (target: sodium channel β1 subunit [SCN1b]). Each active peptide was compared against a corresponding inactive control peptide (Nadp1-c, dadp1-c, βadp1-scr). Sub-diffraction confocal imaging revealed ID enrichment of active peptides, but not inactive controls. Furthermore, each active peptide was preferentially localized in ID regions rich in its corresponding protein target. Peptide treatment (100 μM; 60 minutes) of ex vivo mouse hearts revealed profound widening of perinexi by βadp1 and of mechanical junctions by Nadp1. Dadp1 also induced widening of mechanical junctions albeit to a lower degree. STORM single molecule localization microscopy identified about 50&per; of ID-localized NaV1.5 within GJ-adjacent perinexi, while an additional ∼35&per; was located within N-cad-rich ID sites. Nadp1 and βadp1 induced redistribution of ID localized NaV1.5 away from perinexi and mechanical junctions respectively. Dadp1, again, had similar but milder effects compared to Nadp1. Western blot revealed the expression levels of NaV1.5, connexin 43 (Cx43), connexin 40 (Cx40), β1 in peptide treated hearts to be within 10&per; of levels in untreated controls. Optical mapping revealed atrial conduction slowing in hearts treated with Nadp1 (17cm/s, 70.83&per; of control) and βadp1 (13 cm/s, 54.17&per; of control), but not inactive control peptides (24 cm/s). Volume-conducted electrocardiograms (ECG) revealed P wave prolongation in active peptide treated hearts (Nadp1: 26.5ms, βadp1: 31ms), consistent with conduction slowing compared to the inactive control peptides (16ms). Importantly, burst pacing elicited atrial arrhythmias in all hearts treated with Nadp1 and βadp1. Arrhythmia burden (duration, number of arrhythmias) was highest with βadp1.Conclusions:These results suggest that disruption of NaV1.5-rich ID nanodomains impairs electrical impulse propagation and promotes arrhythmias in the atria. Furthermore, the magnitude of functional impacts are likely determined by the amount of sodium channels contained within the nanodomains disrupted.

M6A RNA Methylation Regulators Contribute to Eutopic Endometrium and Myometrium Dysfunction in Adenomyosis.

Adenomyosis is a prevalent, estrogen-dependent uterine disorder wherein endometrial cells are abnormally present in the myometrium and are surrounded by hyperplastic/hypertrophic smooth muscle. Its etiology is unclear, although endometrial cell invasion into the myometrium has been postulated. RNA methylation, particularly N6-methyladenosine (m6A), plays an important role in regulating various physiological processes and invasive disorders. The goal of this in silico and lab-based experimental study was to explore a possible role for m6A in adenomyosis. Gene expression profiles of both the endometrium and myometrium of women with adenomyosis (cases) and without disease (controls) were obtained from the publicly available Gene Expression Omnibus (GEO) database. In the endometrium, STRING database analysis revealed that METTL3 functions as a “hub” gene of m6A RNA methylation regulators, and the genes involved in m6A regulation, including METTL3, FTO, ZC3H13, and YTHDC1 expression, were significantly decreased in cases versus controls. Functional, co-expression, and correlational analyses of endometrium from cases versus controls revealed decreased total m6A levels, induced by METTL3, and the downstream elevated insulin−like growth factor−1(IGF1) and D-Dopachrome Tautomerase (DDT), with the latter two having known functions in epithelial proliferation and cell migration, which are important processes in the pathogenesis of adenomyosis in endometrium. m6A RNA methylation regulators, including RBM15/15B, ALKBH5, FTO, YTHDF1/2, KIAA1429, HNRNPC, METTL3, ZC3H13, and YTHDC2, were also differentially expressed in the myometrium from cases versus controls. We validated decreased total m6A levels and differential expression of m6A RNA methylation regulators in the myometrium of patients with adenomyosis using qRT-PCR, immunohistochemistry and tissues available from our biorepository. Possible target genes, including cadherin 3(CDH3), sodium channelβ-subunit 4 (SCN4B), and placenta-specific protein 8 (PLAC8), which are involved in cell adhesion, muscle contraction and immune response in the myometrium of adenomyosis patients were also validated. Thus, through extensive public database mining and validation of select genes, this study, for the first time, implicates m6A and its methylation regulators in the pathogenesis of adenomyosis. Follow on functional studies are anticipated to elucidate mechanisms involving m6A and its regulators and down-stream effectors in the pathogenesis of this enigmatic reproductive disorder and potentially identify druggable targets to control its associated symptoms.

Sodium channel β1 subunits are post-translationally modified by tyrosine phosphorylation, S-palmitoylation, and regulated intramembrane proteolysis

Voltage-gated sodium channel (VGSC) β1 subunits are multifunctional proteins that modulate the biophysical properties and cell-surface localization of VGSC α subunits and participate in cell-cell and cell-matrix adhesion, all with important implications for intracellular signal transduction, cell migration, and differentiation. Human loss-of-function variants in SCN1B, the gene encoding the VGSC β1 subunits, are linked to severe diseases with high risk for sudden death, including epileptic encephalopathy and cardiac arrhythmia. We showed previously that β1 subunits are post-translationally modified by tyrosine phosphorylation. We also showed that β1 subunits undergo regulated intramembrane proteolysis via the activity of β-secretase 1 and γ-secretase, resulting in the generation of a soluble intracellular domain, β1-ICD, which modulates transcription. Here, we report that β1 subunits are phosphorylated by FYN kinase. Moreover, we show that β1 subunits are S-palmitoylated. Substitution of a single residue in β1, Cys-162, to alanine prevented palmitoylation, reduced the level of β1 polypeptides at the plasma membrane, and reduced the extent of β1-regulated intramembrane proteolysis, suggesting that the plasma membrane is the site of β1 proteolytic processing. Treatment with the clathrin-mediated endocytosis inhibitor, Dyngo-4a, re-stored the plasma membrane association of β1-p.C162A to WT levels. Despite these observations, palmitoylation-null β1-p.C162A modulated sodium current and sorted to detergent-resistant membrane fractions normally. This is the first demonstration of S-palmitoylation of a VGSC β subunit, establishing precedence for this post-translational modification as a regulatory mechanism in this protein family.

β1 and β3 subunits amplify mechanosensitivity of the cardiac voltage-gated sodium channel Nav1.5

In cardiomyocytes, electrical activity is coupled to cellular contraction, thus exposing all proteins expressed in the sarcolemma to mechanical stress. The voltage-gated sodium channel Nav1.5 is the main contributor to the rising phase of the action potential in the heart. There is growing evidence that gating and kinetics of Nav1.5 are modulated by mechanical forces and pathogenic variants that affect mechanosensitivity have been linked to arrhythmias. Recently, the sodium channel β1 subunit has been described to stabilise gating against mechanical stress of Nav1.7 expressed in neurons. Here, we tested the effect of β1 and β3 subunits on mechanosensitivity of the cardiac Nav1.5. β1 amplifies stress-induced shifts of V1/2 of steady-state fast inactivation to hyperpolarised potentials (ΔV1/2: 6.2 mV without and 10.7 mV with β1 co-expression). β3, on the other hand, almost doubles stress-induced speeding of time to sodium current transient peak (Δtime to peak at - 30 mV: 0.19 ms without and 0.37 ms with β3 co-expression). Our findings may indicate that in cardiomyocytes, the interdependence of electrical activity and contraction is used as a means of fine tuning cardiac sodium channel function, allowing quicker but more strongly inactivating sodium currents under conditions of increased mechanical stress. This regulation may help to shorten action potential duration during tachycardia, to prevent re-entry phenomena and thus arrhythmias.

N-Glycosylation of the voltage-gated sodium channel β2 subunit is required for efficient trafficking of NaV1.5/β2 to the plasma membrane

The voltage-gated sodium channel is critical for cardiomyocyte function and consists of a protein complex comprising a pore-forming α subunit and two associated β subunits. It has been shown previously that the associated β2 subunits promote cell surface expression of the α subunit. The major α isoform in the adult human heart is NaV1.5, and germline mutations in the NaV1.5-encoding gene, sodium voltage-gated channel α subunit 5 (SCN5A), often cause inherited arrhythmias. Here, we investigated the mechanisms that regulate β2 trafficking and how they may determine proper NaV1.5 cell surface localization. Using heterologous expression in polarized Madin-Darby canine kidney cells, we show that β2 is N-glycosylated in vivo and in vitro at residues 42, 66, and 74, becoming sialylated only at Asn-42. We found that fully nonglycosylated β2 was mostly retained in the endoplasmic reticulum, indicating that N-linked glycosylation is required for efficient β2 trafficking to the apical plasma membrane. The nonglycosylated variant reached the cell surface by bypassing the Golgi compartment at a rate of only approximately one-third of that of WT β2. YFP-tagged, nonglycosylated β2 displayed mobility kinetics in the plane of the membrane similar to that of WT β2. However, it was defective in promoting surface localization of NaV1.5. Interestingly, β2 with a single intact glycosylation site was as effective as the WT in promoting NaV1.5 surface localization. In conclusion, our results indicate that N-linked glycosylation of β2 is required for surface localization of NaV1.5, a property that is often defective in inherited cardiac arrhythmias.

Trafficking and Function of the Voltage-Gated Sodium Channel β2 Subunit.

The voltage-gated sodium channel is vital for cardiomyocyte function, and consists of a protein complex containing a pore-forming α subunit and two associated β subunits. A fundamental, yet unsolved, question is to define the precise function of β subunits. While their location in vivo remains unclear, large evidence shows that they regulate localization of α and the biophysical properties of the channel. The current data support that one of these subunits, β2, promotes cell surface expression of α. The main α isoform in an adult heart is NaV1.5, and mutations in SCN5A, the gene encoding NaV1.5, often lead to hereditary arrhythmias and sudden death. The association of β2 with cardiac arrhythmias has also been described, which could be due to alterations in trafficking, anchoring, and localization of NaV1.5 at the cardiomyocyte surface. Here, we will discuss research dealing with mechanisms that regulate β2 trafficking, and how β2 could be pivotal for the correct localization of NaV1.5, which influences cellular excitability and electrical coupling of the heart. Moreover, β2 may have yet to be discovered roles on cell adhesion and signaling, implying that diverse defects leading to human disease may arise due to β2 mutations.

NaV1.6 and NaV1.7 channels are major endogenous voltage-gated sodium channels in ND7/23 cells

ND7/23 cells are gaining traction as a host model to express peripheral sodium channels such as NaV1.8 and NaV1.9 that have been difficult to express in widely utilized heterologous cells, like CHO and HEK293. Use of ND7/23 as a model cell to characterize the properties of sodium channels requires clear understanding of the endogenous ion channels. To define the nature of the background sodium currents in ND7/23 cells, we aimed to comprehensively profile the voltage-gated sodium channel subunits by endpoint and quantitative reverse transcription-PCR and by whole-cell patch clamp electrophysiology. We found that untransfected ND7/23 cells express endogenous peak sodium currents that average –2.12nA (n = 15) and with kinetics typical of fast sodium currents having activation and inactivation completed within few milliseconds. Furthermore, sodium currents were reduced to virtually nil upon exposure to 100nM tetrodotoxin, indicating that ND7/23 cells have essentially null background for tetrodotoxin-resistant (TTX-R) currents. qRT-PCR profiling indicated a major expression of TTX-sensitive (TTX-S) NaV1.6 and NaV1.7 at similar levels and very low expression of TTX-R NaV1.9 transcripts. There was no expression of TTX-R NaV1.8 in ND7/23 cells. There was low expression of NaV1.1, NaV1.2, NaV1.3 and no expression of cardiac or skeletal muscle sodium channels. As for the sodium channel auxiliary subunits, β1 and β3 subunits were expressed, but not the β2 and β4 subunits that covalently associate with the α-subunits. In addition, our results also showed that only the mouse forms of NaV1.6, NaV1.7 and NaV1.9 sodium channels were expressed in ND7/23 cells that was originally generated as a hybridoma of rat embryonic DRG and mouse neuroblastoma cell-line. By molecular profiling of auxiliary β- and principal α-subunits of the voltage gated sodium channel complex, our results define the background sodium channels expressed in ND7/23 cells, and confirm their utility for detailed functional studies of emerging pain channelopathies ascribed to mutations of the TTX-R sodium channels of sensory neurons.

MiR-34a is differentially expressed in dorsal root ganglia in a rat model of chronic neuropathic pain

IntroductionRecent evidence shows that numerous microRNAs (miRNAs) regulate pain-related genes in chronic pain. The aim of the present study was to further explore the regulation of miRNAs and their effect on the expression of pain-associated target genes in experimental neuropathic pain. MethodsMale Wistar rats underwent chronic constriction injury (CCI) of the sciatic nerve or Sham procedure. After assessment of mechanical allodynia, the ipsilateral dorsal root ganglia (DRG) were harvested. MiRNA expression levels were analysed with Agilent microRNA microarrays and real time quantitative PCR. An interaction between miRNAs and pain-relevant genes was confirmed by luciferase assays. Western Blot analysis and ELISA were performed to evaluate protein expression, respectively. ResultsMechanical allodynia developed within 6 days after CCI. MiRNA-arrays revealed the differential expression of 49 miRNAs after 4 h, of 3 miRNAs after 1 d, of 26 miRNAs after 6 d and of 28 miRNAs after 12 d in the CCI group versus Sham. Time-dependent down regulation of miR-34a was verified by qPCR. Bioinformatic prediction revealed an interaction with several pain-relevant targets including voltage-gated sodium channel β2 subunit (SCN2B) and vesicle-associated membrane protein 2 (VAMP-2), both of which were subsequently confirmed by luciferase assay. VAMP-2 expression was statistically significantly increased 12 d after CCI. A non-significant upregulation of SCN2B in the DRG after CCI was confirmed by ELISA. DiscussionPeripheral mononeuropathic pain in rats was associated with distinct alterations of miRNA expression in the ipsilateral DRG. Notably, miR-34a was time-dependently down regulated. We validated SCN2B and VAMP-2 as new targets of miR-34a. While SCN2B expression was only marginally altered, VAMP-2 expression was increased. The present study underlines that the induction and maintenance of neuropathic pain is accompanied by expression changes of miRNAs in the peripheral nervous system, adding several previously unreported miRNAs, including miR-34a.

Identification of rare variants in cardiac sodium channel β4-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 β-subunit, Navβ4, 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 Navβ4. 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.

A Novel Model for Studying Voltage-Gated Ion Channel Gene Expression during Reversible Ischemic Stroke.

The dysfunction of voltage-gated ion channels contributes to the pathology of ischemic stroke. In this study, we developed rat models of transient ischemic attack (TIA) and reversible ischemic neurological deficit (RIND) that was induced via the injection of artificial embolic particles during full consciousness, that allow us to monitor the neurologic deficit and positron emission tomography (PET) scans in real-time. We then evaluated the infarction volume of brain tissue was confirmed by 2,3,5-triphenyl tetrazolium chloride (TTC) staining, and gene expressions were evaluated by quantitative real-time PCR (qPCR). We found that rats with TIA or RIND exhibited neurological deficits as determined by negative TTC and PET findings. However, the expression of voltage-gated sodium channels in the hippocampus was significantly up-regulated in the qPCR array study. Furthermore, an altered expression of sodium channel β-subunits and potassium channels, were observed in RIND compared to TIA groups. In conclusion, to our knowledge, this is the first report of the successful evaluation of voltage-gated ion channel gene expression in TIA and RIND animal models. This model will aid future studies in investigating pathophysiological mechanisms, and in developing new therapeutic compounds for the treatment of TIA and RIND.