CircFNTA promotes tumorigenesis and progression of gastric cancer via miR-604/miR-647/SCN8A axis.

Gastric cancer (GC) is a major contributor to cancer-related deaths and is characterized by high heterogeneity in epidemiology and histopathology worldwide. Increasing evidence indicates that circular RNAs (circRNAs) play multifaceted roles in cellular processes in human cancers. Here, we demonstrated that circFNTA high expression increases the proliferation, metastasis, and epithelial-mesenchymal transition process and tumorigenicity of GC cells. First, we found that circFNTA was upregulated in GC cells and tissues, and the high circFNTA levels were positively associated with the poor prognosis in GC patients. Using luciferase reporter and RNA-pull down assays, we elucidated that circFNTA sponged two microRNAs, miR-604 and miR-647. In addition, the proliferation and metastatic ability of GC cell reduction caused by silencing circFNTA was hindered by inhibitors of miR-604 and miR-647. Moreover, SCN8A was predicted by miRDB as a common target gene of miR-604 and miR-647, which was then verified by the luciferase reporter assay. Knockdown of circFNTA causes messenger RNA and protein levels in SCN8A to be downregulated in GC cells. However, this effect was overturned by cotransfection miR-604 and miR-647. Also, we identified that SCN8A was downregulated in GC tissues, which was positively correlated with circFNTA expression. In rescue experiments, the attenuated cell proliferation and metastatic ability caused by circFNTA knockdown was reversed by miR-604 and miR-647 inhibitors and SCN8A overexpression. Collectively, our findings suggest an oncogenic role of circFNTA in GC progression and elucidate that circFNTA exerts its function by modulating the miR-604/miR-647/SCN8A axis.

In vitro effects of eslicarbazepine (S-licarbazepine) as a potential precision therapy on SCN8A variants causing neuropsychiatric disorders.

BACKGROUND AND PURPOSE: Variants in SCN8A, the NaV 1.6 channel's coding gene, are characterized by a variety of symptoms, including intractable epileptic seizures, psychomotor delay, progressive cognitive decline, autistic features, ataxia or dystonia. Standard anticonvulsant treatment has a limited impact on the course of disease. EXPERIMENTAL APPROACH: We investigated the therapeutic potential of eslicarbazepine (S-licarbazepine; S-lic), an enhancer of slow inactivation of voltage gated sodium channels, on two variants with biophysical and neuronal gain-of-function (G1475R and M1760I) and one variant with biophysical gain-of-function but neuronal loss-of-function (A1622D) in neuroblastoma cells and in murine primary hippocampal neuron cultures. These three variants cover the broad spectrum of NaV 1.6-associated disease and are linked to representative phenotypes of mild to moderate epilepsy (G1475R), developmental and epileptic encephalopathy (M1760I) and intellectual disability without epilepsy (A1622D). KEY RESULTS: Similar to known effects on NaV 1.6 wildtype channels, S-lic predominantly enhances slow inactivation on all tested variants, irrespective of their particular biophysical mechanisms. Beyond that, S-lic exhibits variant-specific effects including a partial reversal of pathologically slowed fast inactivation dynamics (A1622D and M1760I) and a trend to reduce enhanced persistent Na+ current by A1622D variant channels. Furthermore, our data in primary transfected neurons reveal that not only variant-associated hyperexcitability (M1760I and G1475R) but also hypoexcitability (A1622D) can be modulated by S-lic. CONCLUSIONS AND IMPLICATIONS: S-lic has not only substance-specific effects but also variant-specific effects. Personalized treatment regimens optimized to achieve such variant-specific pharmacological modulation may help to reduce adverse side effects and improve the overall therapeutic outcome of SCN8A-related disease.

Neuraxial block anesthetic technique in a patient with SCN8A encephalopathy: case report

Abstract Mutations in SCN8A gene lead to changes in sodium channels in the brain, which are correlated with severe epileptic syndrome. Due to the rarity, there are few studies that support anesthesia in that population. The present study aims to report alternatives to inhalation anesthesia at epileptic encephalopathy. Case report: Male, 4 years old, with SCN8A encephalopathy with surgical indication of orchidopexy. Neuroaxis block was performed and dexmedetomidine was used as a pre-anesthetic and sedation. The anestheticsurgical act was uneventful. Conclusion: The association of neuraxial block and dexmedetomidine proved to be a viable alternative for surgery in patients with SCN8A encephalopathy.

Neuraxial block anesthetic technique in a patient with SCN8A encephalopathy: case report.

Mutations in SCN8A gene lead to changes in sodium channels in the brain, which are correlated with severe epileptic syndrome. Due to the rarity, there are few studies that support anesthesia in that population. The present study aims to report alternatives to inhalation anesthesia at epileptic encephalopathy. CASE REPORT: Male, 4 years old, with SCN8A encephalopathy with surgical indication of orchidopexy. Neuroaxis block was performed and dexmedetomidine was used as a pre-anesthetic and sedation. The anestheticsurgical act was uneventful. CONCLUSION: The association of neuraxial block and dexmedetomidine proved to be a viable alternative for surgery in patients with SCN8A encephalopathy.

Upregulation of Nav1.6 Mediated by the p38 MAPK Pathway in the Dorsal Root Ganglia Contributes to Cancer-Induced Bone Pain in Rats.

Cancer-induced bone pain (CIBP) occurs frequently among advanced cancer patients. Voltage-gated sodium channels (VGSCs) have been associated with chronic pain, but how VGSCs function in CIBP is poorly understood. Here, we aimed to investigate the specific role of VGSCs in the dorsal root ganglia (DRGs) in CIBP. A CIBP rat model was generated by the intratibial inoculation of MRMT-1 breast carcinoma cells. Transcriptome sequencing was conducted to assess the gene expression profiles. The expression levels of key genes and differentiated genes related to activated pathways were measured by Western blotting and qPCR. We implanted a catheter intrathecally for the administration of lentivirus and drugs. Then, the changes in the mechanical withdrawal threshold (MWT) were measured. We identified 149 differentially expressed mRNAs (DEmRNAs) in the DRGs of CIBP model rats. The expression of Nav1.6, which was among these DEmRNAs, was significantly upregulated. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the DEmRNAs showed that they were mainly enriched in the mitogen-activated protein kinase (MAPK) pathway. The decrease in MWT induced by bone cancer was attenuated by Nav1.6 knockdown. Western blot analysis revealed that a p38 inhibitor decreased the expression of Nav1.6 and attenuated pain behavior. Our study shows that the upregulation of Nav1.6 expression by p38 MAPK in the DRGs of rats contributes to CIBP.

Genotype-phenotype correlations in SCN8A-related disorders reveal prognostic and therapeutic implications.

We report detailed functional analyses and genotype-phenotype correlations in 392 individuals carrying disease-causing variants in SCN8A, encoding the voltage-gated Na+ channel Nav1.6, with the aim of describing clinical phenotypes related to functional effects. Six different clinical subgroups were identified: Group 1, benign familial infantile epilepsy (n = 15, normal cognition, treatable seizures); Group 2, intermediate epilepsy (n = 33, mild intellectual disability, partially pharmaco-responsive); Group 3, developmental and epileptic encephalopathy (n = 177, severe intellectual disability, majority pharmaco-resistant); Group 4, generalized epilepsy (n = 20, mild to moderate intellectual disability, frequently with absence seizures); Group 5, unclassifiable epilepsy (n = 127); and Group 6, neurodevelopmental disorder without epilepsy (n = 20, mild to moderate intellectual disability). Those in Groups 1-3 presented with focal or multifocal seizures (median age of onset: 4 months) and focal epileptiform discharges, whereas the onset of seizures in patients with generalized epilepsy was later (median: 42 months) with generalized epileptiform discharges. We performed functional studies expressing missense variants in ND7/23 neuroblastoma cells and primary neuronal cultures using recombinant tetrodotoxin-insensitive human Nav1.6 channels and whole-cell patch-clamping. Two variants causing developmental and epileptic encephalopathy showed a strong gain-of-function (hyperpolarizing shift of steady-state activation, strongly increased neuronal firing rate) and one variant causing benign familial infantile epilepsy or intermediate epilepsy showed a mild gain-of-function (defective fast inactivation, less increased firing). In contrast, all three variants causing generalized epilepsy induced a loss-of-function (reduced current amplitudes, depolarizing shift of steady-state activation, reduced neuronal firing). Functional effects were known for 170 individuals. All 136 individuals carrying a functionally tested gain-of-function variant had either focal (n = 97, Groups 1-3) or unclassifiable (n = 39) epilepsy, whereas 34 individuals with a loss-of-function variant had either generalized (n = 14), no (n = 11) or unclassifiable (n = 6) epilepsy; only three had developmental and epileptic encephalopathy. Computational modelling in the gain-of-function group revealed a significant correlation between the severity of the electrophysiological and clinical phenotypes. Gain-of-function variant carriers responded significantly better to sodium channel blockers than to other anti-seizure medications, and the same applied for all individuals in Groups 1-3. In conclusion, our data reveal clear genotype-phenotype correlations between age at seizure onset, type of epilepsy and gain- or loss-of-function effects of SCN8A variants. Generalized epilepsy with absence seizures is the main epilepsy phenotype of loss-of-function variant carriers and the extent of the electrophysiological dysfunction of the gain-of-function variants is a main determinant of the severity of the clinical phenotype in focal epilepsies. Our pharmacological data indicate that sodium channel blockers present a treatment option in SCN8A-related focal epilepsy with onset in the first year of life.

Perioperative Management and Considerations for Patients With Voltage-Gated Sodium Channel Mutations: A Pediatric Case Report.

A 13-year-old girl with a voltage-gated sodium channel mutation (SCN8A)-associated intractable epilepsy presented for bilateral mastectomy for painful juvenile fibroadenomatosis. Sodium channel mutations are more frequently diagnosed with continued advances in genetic testing. Understanding the effects of sodium channel mutations is important to provide safe anesthetic care to these patients. In this article, we discuss what is known regarding the physiology of SCN8A channels and the anesthetic considerations when caring for patients with an SCN8A mutation.

A SCN8A variant associated with severe early onset epilepsy and developmental delay: Loss- or gain-of-function?

SCN8A, encoding the voltage-gated sodium channel subunit NaV1.6, has been associated with a wide spectrum of neuropsychiatric disorders. Missense variants in SCN8A which increase the channel activity can cause a severe developmental and epileptic encephalopathy (DEE). One DEE variant (p.(Arg223Gly)) was described to cause a predominant loss-of-function (LOF) mechanism when expressed in neuroblastoma cells, which is not consistent with the genotype-phenotype correlations in this gene. To resolve this discrepancy and understand the pathophysiological mechanism of this variant, we performed comprehensive electrophysiological studies in both neuroblastoma cells and primary hippocampal neuronal cultures. Although we also found that p.(Arg223Gly) significantly decreased Na+ current density and enhanced fast inactivation compared to the wild type (WT) channel in transfected neuroblastoma cells (both LOF mechanisms), it also caused a strong hyperpolarizing shift of steady-state activation and accelerated the recovery from fast inactivation (both gain-of-function (GOF) mechanisms). In cultured neurons transfected with mutant vs. WT NaV1.6 channels, we found more depolarized resting membrane potentials and a decreased rheobase leading to enhanced action potential firing. We conclude that SCN8A p.(Arg223Gly) leads to a net GOF resulting in neuronal hyperexcitability and a higher firing rate, fitting with the central role of GOF mechanisms in DEE.

Nav1.6 promotes inflammation and neuronal degeneration in a mouse model of multiple sclerosis.

BACKGROUND: In multiple sclerosis (MS) and in the experimental autoimmune encephalomyelitis (EAE) model of MS, the Nav1.6 voltage-gated sodium (Nav) channel isoform has been implicated as a primary contributor to axonal degeneration. Following demyelination Nav1.6, which is normally co-localized with the Na+/Ca2+ exchanger (NCX) at the nodes of Ranvier, associates with ß-APP, a marker of neural injury. The persistent influx of sodium through Nav1.6 is believed to reverse the function of NCX, resulting in an increased influx of damaging Ca2+ ions. However, direct evidence for the role of Nav1.6 in axonal degeneration is lacking. METHODS: In mice floxed for Scn8a, the gene that encodes the α subunit of Nav1.6, subjected to EAE we examined the effect of eliminating Nav1.6 from retinal ganglion cells (RGC) in one eye using an AAV vector harboring Cre and GFP, while using the contralateral either injected with AAV vector harboring GFP alone or non-targeted eye as control. RESULTS: In retinas, the expression of Rbpms, a marker for retinal ganglion cells, was found to be inversely correlated to the expression of Scn8a. Furthermore, the gene expression of the pro-inflammatory cytokines Il6 (IL-6) and Ifng (IFN-γ), and of the reactive gliosis marker Gfap (GFAP) were found to be reduced in targeted retinas. Optic nerves from targeted eyes were shown to have reduced macrophage infiltration and improved axonal health. CONCLUSION: Taken together, our results are consistent with Nav1.6 promoting inflammation and contributing to axonal degeneration following demyelination.

Generalized tonic seizures with autonomic signs are the hallmark of SCN8A developmental and epileptic encephalopathy.

Developmental and epileptic encephalopathy (DEE) due to SCN8A gene variants is characterized by drug-resistant early onset epilepsy associated with severe intellectual disability. Different seizure types have been reported, and a sequence of autonomic manifestations such as brady-/tachycardia, irregular breathing, and cyanosis. Nevertheless, an exhaustive video-polygraphic documentation is still lacking. In this study, we reviewed the ictal electroencephalograms (EEGs) of five patients with SCN8A-DEE followed-up at the Neuroscience Department at Bambino Gesù Children's Hospital in Rome. We identified generalized tonic seizure as the major seizure type at epilepsy onset. Seizure severity could vary from subtle to marked clinical manifestations, depending from the extent and groups of muscles involved and association with autonomic modifications. We found autonomic signs in 80% of seizures in our cases, and we were able to identify a stereotyped sequence of ictal events for most of seizures. Autonomic signs occurred in rapid sequence: flushing of the face, sometimes associated with sialorrhea, bradycardia, and hypopnea appeared within the first 1-2 s. Tachycardia, polypnea, perioral cyanosis, and pallor occurred later in the course of the seizure. Generalized tonic seizures are rarely described in other genetic epileptic conditions of early infancy because of ion channel mutations, such as in DEE due to KCNQ2 or SCN2A gene mutations, where seizures are most frequently reported as focal to bilateral tonic. Therefore, generalized symmetric tonic seizures with autonomic signs can be considered a clinical hallmark for diagnosis of SCN8A-related DEE and relevant for therapeutic implications.

Curcumin's antiepileptic effect, and alterations in Nav1.1 and Nav1.6 expression in iron-induced epilepsy.

The present study was carried out to evaluate: the antiepileptic effect of dietary curcumin, and the effect of epileptic state and curcumin on the molecular expression of voltage-activated Na+ channel subtypes Nav1.1 and Nav1.6 in the iron-induced experimental epilepsy in the rat. Rats were divided into four groups; Group I (control rats), Group II (epileptic rats), Group III (curcumin-fed epileptic rats), and Group IV (curcumin-fed rats). Curcumin was fed chronically to rats approximately at the dose of 100 mg/kg body wt. The animals were made epileptic by intracortical injection of FeCl3. The mRNA and protein expressions of Nav1.1 and Nav1.6 were examined by RT-PCR analysis and immuno-histochemistry. Results showed a significant increase (upregulation) in the expression of both Nav1.1 and Nav1.6 with seizure activity in the cortex and hippocampus of epileptic rats. Epileptic rats fed with curcumin showed a marked decrease in epileptiform activity, and reduced mRNA and protein levels of Nav1.1. It appears that the antiepileptic action of curcumin may be associated with the downregulation of Nav1.1 in the cortex.

Neuronal mechanisms of mutations in SCN8A causing epilepsy or intellectual disability.

Ion channel mutations can cause distinct neuropsychiatric diseases. We first studied the biophysical and neurophysiological consequences of four mutations in the human Na+ channel gene SCN8A causing either mild (E1483K) or severe epilepsy (R1872W), or intellectual disability and autism without epilepsy (R1620L, A1622D). Only combined electrophysiological recordings of transfected wild-type or mutant channels in both neuroblastoma cells and primary cultured neurons revealed clear genotype-phenotype correlations. The E1483K mutation causing mild epilepsy showed no significant biophysical changes, whereas the R1872W mutation causing severe epilepsy induced clear gain-of-function biophysical changes in neuroblastoma cells. However, both mutations increased neuronal firing in primary neuronal cultures. In contrast, the R1620L mutation associated with intellectual disability and autism-but not epilepsy-reduced Na+ current density in neuroblastoma cells and expectedly decreased neuronal firing. Interestingly, for the fourth mutation, A1622D, causing severe intellectual disability and autism without epilepsy, we observed a dramatic slowing of fast inactivation in neuroblastoma cells, which induced a depolarization block in neurons with a reduction of neuronal firing. This latter finding was corroborated by computational modelling. In a second series of experiments, we recorded three more mutations (G1475R, M1760I, G964R, causing intermediate or severe epilepsy, or intellectual disability without epilepsy, respectively) that revealed similar results confirming clear genotype-phenotype relationships. We found intermediate or severe gain-of-function biophysical changes and increases in neuronal firing for the two epilepsy-causing mutations and decreased firing for the loss-of-function mutation causing intellectual disability. We conclude that studies in neurons are crucial to understand disease mechanisms, which here indicate that increased or decreased neuronal firing is responsible for distinct clinical phenotypes.

The invasiveness of human cervical cancer associated to the function of NaV1.6 channels is mediated by MMP-2 activity.

Voltage-gated sodium (NaV) channels have been related with cell migration and invasiveness in human cancers. We previously reported the contribution of NaV1.6 channels activity with the invasion capacity of cervical cancer (CeCa) positive to Human Papilloma Virus type 16 (HPV16), which accounts for 50% of all CeCa cases. Here, we show that NaV1.6 gene (SCN8A) overexpression is a general characteristic of CeCa, regardless of the HPV type. In contrast, no differences were observed in NaV1.6 channel expression between samples of non-cancerous and cervical intraepithelial neoplasia. Additionally, we found that CeCa cell lines, C33A, SiHa, CaSki and HeLa, express mainly the splice variant of SCN8A that lacks exon 18, shown to encode for an intracellularly localized NaV1.6 channel, whereas the full-length adult form was present in CeCa biopsies. Correlatively, patch-clamp experiments showed no evidence of whole-cell sodium currents (INa) in CeCa cell lines. Heterologous expression of full-length NaV1.6 isoform in C33A cells produced INa, which were sufficient to significantly increase invasion capacity and matrix metalloproteinase type 2 (MMP-2) activity. These data suggest that upregulation of NaV1.6 channel expression occurs when cervical epithelium have been transformed into cancer cells, and that NaV1.6-mediated invasiveness of CeCa cells involves MMP-2 activity. Thus, our findings support the notion about using NaV channels as therapeutic targets against cancer metastasis.

Conditional knockout of NaV1.6 in adult mice ameliorates neuropathic pain.

Voltage-gated sodium channels NaV1.7, NaV1.8 and NaV1.9 have been the focus for pain studies because their mutations are associated with human pain disorders, but the role of NaV1.6 in pain is less understood. In this study, we selectively knocked out NaV1.6 in dorsal root ganglion (DRG) neurons, using NaV1.8-Cre directed or adeno-associated virus (AAV)-Cre mediated approaches, and examined the specific contribution of NaV1.6 to the tetrodotoxin-sensitive (TTX-S) current in these neurons and its role in neuropathic pain. We report here that NaV1.6 contributes up to 60% of the TTX-S current in large, and 34% in small DRG neurons. We also show NaV1.6 accumulates at nodes of Ranvier within the neuroma following spared nerve injury (SNI). Although NaV1.8-Cre driven NaV1.6 knockout does not alter acute, inflammatory or neuropathic pain behaviors, AAV-Cre mediated NaV1.6 knockout in adult mice partially attenuates SNI-induced mechanical allodynia. Additionally, AAV-Cre mediated NaV1.6 knockout, mostly in large DRG neurons, significantly attenuates excitability of these neurons after SNI and reduces NaV1.6 accumulation at nodes of Ranvier at the neuroma. Together, NaV1.6 in NaV1.8-positive neurons does not influence pain thresholds under normal or pathological conditions, but NaV1.6 in large NaV1.8-negative DRG neurons plays an important role in neuropathic pain.

Intraspecific variation of Centruroides sculpturatus scorpion venom from two regions of Arizona.

This study investigated geographic variability in the venom of Centruroides sculpturatus scorpions from different biotopes. Venom from scorpions collected from two different regions in Arizona; Santa Rita Foothills (SR) and Yarnell (Yar) were analyzed. We found differences between venoms, mainly in the two most abundant peptides; SR (CsEv2e and CsEv1f) and Yar (CsEv2 and CsEv1c) identified as natural variants of CsEv1 and CsEv2. Sequence analyses of these peptides revealed conservative amino acid changes between variants, which may underlie biological activity against arthropods. A third peptide (CsEv6) was highly abundant in the Yar venom compared to the SR venom. CsEv6 is a 67 amino acid peptide with 8 cysteines. CsEv6 did not exhibit toxicity to the three animal models tested. However, both venoms shared similarities in peptides that are predicted to deter predators. For example, both venoms expressed CsEI (lethal to chick) in similar abundance, while CsEd and CsEM1a (toxic to mammals) displayed only moderate variation in their abundance. Electrophysiological evaluation of CsEd and CsEM1a showed that both toxins act on the human sodium-channel subtype 1.6 (hNav 1.6). Complete sequencing revealed that both toxins are structurally similar to beta-toxins isolated from different Centruroides species that also target hNav 1.6.

Gain-of-function FHF1 mutation causes early-onset epileptic encephalopathy with cerebellar atrophy.

OBJECTIVE: Voltage-gated sodium channel (Nav)-encoding genes are among early-onset epileptic encephalopathies (EOEE) targets, suggesting that other genes encoding Nav-binding proteins, such as fibroblast growth factor homologous factors (FHFs), may also play roles in these disorders. METHODS: To identify additional genes for EOEE, we performed whole-exome sequencing in a family quintet with 2 siblings with a lethal disease characterized by EOEE and cerebellar atrophy. The pathogenic nature and functional consequences of the identified sequence alteration were determined by electrophysiologic studies in vitro and in vivo. RESULTS: A de novo heterozygous missense mutation was identified in the FHF1 gene (FHF1AR114H, FHF1BR52H) in the 2 affected siblings. The mutant FHF1 proteins had a strong gain-of-function phenotype in transfected Neuro2A cells, enhancing the depolarizing shifts in Nav1.6 voltage-dependent fast inactivation, predicting increased neuronal excitability. Surprisingly, the gain-of-function effect is predicted to result from weaker interaction of mutant FHF1 with the Nav cytoplasmic tail. Transgenic overexpression of mutant FHF1B in zebrafish larvae enhanced epileptiform discharges, demonstrating the epileptic potential of this FHF1 mutation in the affected children. CONCLUSIONS: Our data demonstrate that gain-of-function FHF mutations can cause neurologic disorder, and expand the repertoire of genetic causes (FHF1) and mechanisms (altered Nav gating) underlying EOEE and cerebellar atrophy.

Human Nav1.6 Channels Generate Larger Resurgent Currents than Human Nav1.1 Channels, but the Navß4 Peptide Does Not Protect Either Isoform from Use-Dependent Reduction.

Voltage-gated sodium channels are responsible for the initiation and propagation of action potentials (APs). Two brain isoforms, Nav1.1 and Nav1.6, have very distinct cellular and subcellular expression. Specifically, Nav1.1 is predominantly expressed in the soma and proximal axon initial segment of fast-spiking GABAergic neurons, while Nav1.6 is found at the distal axon initial segment and nodes of Ranvier of both fast-spiking GABAergic and excitatory neurons. Interestingly, an auxiliary voltage-gated sodium channel subunit, Navß4, is also enriched in the axon initial segment of fast-spiking GABAergic neurons. The C-terminal tail of Navß4 is thought to mediate resurgent sodium current, an atypical current that occurs immediately following the action potential and is predicted to enhance excitability. To better understand the contribution of Nav1.1, Nav1.6 and Navß4 to high frequency firing, we compared the properties of these two channel isoforms in the presence and absence of a peptide corresponding to part of the C-terminal tail of Navß4. We used whole-cell patch clamp recordings to examine the biophysical properties of these two channel isoforms in HEK293T cells and found several differences between human Nav1.1 and Nav1.6 currents. Nav1.1 channels exhibited slower closed-state inactivation but faster open-state inactivation than Nav1.6 channels. We also observed a greater propensity of Nav1.6 to generate resurgent currents, most likely due to its slower kinetics of open-state inactivation, compared to Nav1.1. These two isoforms also showed differential responses to slow and fast AP waveforms, which were altered by the Navß4 peptide. Although the Navß4 peptide substantially increased the rate of recovery from apparent inactivation, Navß4 peptide did not protect either channel isoform from undergoing use-dependent reduction with 10 Hz step-pulse stimulation or trains of slow or fast AP waveforms. Overall, these two channels have distinct biophysical properties that may differentially contribute to regulating neuronal excitability.

Preferential targeting of Nav1.6 voltage-gated Na+ Channels to the axon initial segment during development.

During axonal maturation, voltage-gated sodium (Nav) channels accumulate at the axon initial segment (AIS) at high concentrations. This localization is necessary for the efficient initiation of action potentials. The mechanisms underlying channel trafficking to the AIS during axonal development have remained elusive due to a lack of Nav reagents suitable for high resolution imaging of channels located specifically on the cell surface. Using an optical pulse-chase approach in combination with a novel Nav1.6 construct containing an extracellular biotinylation domain we demonstrate that Nav1.6 channels are preferentially inserted into the AIS membrane during neuronal development via direct vesicular trafficking. Single-molecule tracking illustrates that axonal channels are immediately immobilized following delivery, while channels delivered to the soma are often mobile. Neither a Nav1.6 channel lacking the ankyrin-binding motif nor a chimeric Kv2.1 channel containing the Nav ankyrinG-binding domain show preferential AIS insertion. Together these data support a model where ankyrinG-binding is required for preferential Nav1.6 insertion into the AIS plasma membrane. In contrast, ankyrinG-binding alone does not confer the preferential delivery of proteins to the AIS.

SCN8A mutations in Chinese children with early onset epilepsy and intellectual disability.

OBJECTIVE: Mutations in SCN8A, a voltage-gated sodium-channel type VIII alpha subunit gene, have recently been recognized as one of the pathogenic mechanisms leading to epilepsy and intellectual/developmental disabilities (IDDs). The aim of this study was to detect SCN8A mutations in Chinese patients with epilepsy of unknown etiology and ID/DD. METHODS: We used targeted next-generation sequencing to identify SCN8A mutations in Chinese patients with epilepsy of unknown etiology and IDDs. A filter process was performed to prioritize rare variants of potential functional significance. Sanger sequencing confirmed the variants and determined the parental origin. We followed all patients with SCN8A mutations in our cohort and analyzed their clinical data. RESULTS: Five de novo SCN8A mutations were identified, including four novel mutations (p.Ala890Thr, p.Leu407Phe, p.Arg850Gln, and p.Ser1596Cys) and one reported (p.Arg1617Gln). Polyphen2 and SIFT software predicted that all five mutations probably damaged Nav1.6 protein function; Mutation Taster indicated that all mutations were disease-causing. Three of these five patients were controlled well by sodium channel blockers (SCBs). Two of these three patients remained seizure free for 6 and 1.5 months, respectively. One patient had sudden unexpected death in epilepsy (SUDEP) at the age of 1 year and 4 months. SIGNIFICANCE: Five SCN8A mutations were first reported in Chinese patients with epilepsy and ID/DD, expanding the phenotype and mutation spectrum of SCN8A mutations. Although three of these patients were controlled well by SCBs in our study, the effectiveness of SCBs should be validated in more patients with epilepsy caused by SCN8A mutations in the future. One of our five patients had sudden unexpected death in epilepsy SUDEP, suggesting that we should pay more attention to SUDEP in epileptic patients with SCN8A mutations.