The KCNT1 gene encodes the sodium-activated potassium channel Slack (KCNT1, KNa1.1), a regulator of neuronal excitability. Gain-of-function mutations in humans cause cortical network hyperexcitability, seizures, and severe intellectual disability. Using a mouse model expressing the Slack-R455H mutation, we find that Na+-dependent K+ (KNa) and voltage-dependent sodium (NaV) currents are increased in both excitatory and inhibitory cortical neurons. These increased currents, however, enhance the firing of excitability neurons but suppress that of inhibitory neurons. We further show that the expression of NaV channel subunits, particularly that of NaV1.6, is upregulated and that the length of the axon initial segment and of axonal NaV immunostaining is increased in both neuron types. Our study on the coordinate regulation of KNa currents and the expression of NaV channels may provide an avenue for understanding and treating epilepsies and other neurological disorders.

BACKGROUND: Sudden unexpected death in epilepsy (SUDEP) is a fatal complication experienced by otherwise healthy epilepsy patients. Dravet syndrome (DS) is an inherited epileptic disorder resulting from loss of function of the voltage-gated sodium channel, NaV 1.1, and is associated with particularly high SUDEP risk. Evidence is mounting that NaVs abundant in the brain also occur in the heart, suggesting that the very molecular mechanisms underlying epilepsy could also precipitate cardiac arrhythmias and sudden death. Despite marked reduction of NaV 1.1 functional expression in DS, pathogenic late sodium current (INa,L) is paradoxically increased in DS hearts. However, the mechanisms by which DS directly impacts the heart to promote sudden death remain unclear.
OBJECTIVE: In this study, the authors sought to provide evidence implicating remodeling of Na+ - and Ca2+ -handling machinery, including NaV 1.6 and Na+/Ca2+exchanger (NCX) within transverse (T)-tubules in DS-associated arrhythmias.
METHODS: The authors undertook scanning ion conductance microscopy (SICM)-guided patch clamp, super-resolution microscopy, confocal Ca2+ imaging, and in vivo electrocardiography studies in Scn1a haploinsufficient murine model of DS.
RESULTS: DS promotes INa,L in T-tubular nanodomains, but not in other subcellular regions. Consistent with increased NaV activity in these regions, super-resolution microscopy revealed increased NaV 1.6 density near Ca2+release channels, the ryanodine receptors (RyR2) and NCX in DS relative to WT hearts. The resulting INa,L in these regions promoted aberrant Ca2+ release, leading to ventricular arrhythmias in vivo. Cardiac-specific deletion of NaV 1.6 protects adult DS mice from increased T-tubular late NaV activity and the resulting arrhythmias, as well as sudden death.
CONCLUSIONS: These data demonstrate that NaV 1.6 undergoes remodeling within T-tubules of adult DS hearts serving as a substrate for Ca2+ -mediated cardiac arrhythmias and may be a druggable target for the prevention of SUDEP in adult DS subjects.

The voltage-gated sodium channel Nav1.6 is associated with more than 300 cases of epileptic encephalopathy. Nav1.6 epilepsy-causing mutations are spread over the entire channel\'s structure and only 10% of mutations have been characterized at the molecular level, with most of them being gain of function mutations. In this study, we analyzed three previously uncharacterized Nav1.6 epilepsy-causing mutations: G214D, N215D and V216D, located within a mutation hot-spot at the S3-S4 extracellular loop of Domain1. Voltage clamp experiments showed a 6-16 mV hyperpolarizing shift in the activation mid-point for all three mutants. V216D presented the largest shift along with decreased current amplitude, enhanced inactivation and a lack of persistent current. Recordings at hyperpolarized potentials indicated that all three mutants presented gating pore currents. Furthermore, trafficking experiments performed in cultured hippocampal neurons demonstrated that the mutants trafficked properly to the cell surface, with no significant differences regarding surface expression within the axon initial segment or soma compared to wild-type. These trafficking data suggest that the disease-causing consequences are due to only changes in the biophysical properties of the channel. Interestingly, the patient carrying the V216D mutation, which is the mutant with the greatest electrophysiological changes as compared to wild-type, exhibited the most severe phenotype. These results emphasize that these mutations will mandate unique treatment approaches, for normal sodium channel blockers may not work given that the studied mutations present gating pore currents. This study emphasizes the importance of molecular characterization of disease-causing mutations in order to improve the pharmacological treatment of patients.

Oxaliplatin is a third-generation derivative of platinum that is effective in the treatment of multiple solid tumors. However, it can cause peripheral neuropathic pain, and the molecular mechanisms of this effect remain unknown. We induced a model of peripheral neuropathic pain in rats by intraperitoneally injecting them with oxaliplatin twice a week for 4.5 weeks. We found that both the mRNA and protein expression levels of Nav1.6 (encoded by the gene Scn8a) increased while the miR-30b-5p (shorthand for miR-30b) expression decreased in the dorsal root ganglion (DRG) of treated rats. Using TargetScan and miRanda predictive software, we discovered that Scn8a was a major target of miR-30b. Moreover, we found that miR-30b negatively regulated Scn8a by binding to the Scn8a 3\'UTR in PC12 cells. In addition, Nav1.6 and miR-30b were colocalized in the DRG neurons of naive rats. Overexpression of miR-30b using an miR-30b agomir attenuated neuropathic pain induced by oxaliplatin and inhibited both the mRNA and protein expression levels of Nav1.6 both in vitro and in vivo. Conversely, the inhibition of miR-30b with an miR-30b antagomir resulted in neuropathic pain and an increase in the expression of Nav1.6. More importantly, overexpression of miR-30b inhibited the proliferation of LS-174t cells (Colorectal cancer cells). These data suggest that miR-30b contributes to oxaliplatin-induced chronic neuropathic pain through Nav1.6 downregulation and could be a novel therapeutic target for the treatment of oxaliplatin-induced neuropathic pain as a side effect of chemotherapy in cancer patients.

Parkinson\'s disease (PD), the second most common age-related progressive neurodegenerative disorder, is characterized by dopamine depletion and the loss of dopaminergic (DA) neurons with accompanying neuroinflammation. Zonisamide is an-anti-convulsant drug that has recently been shown to improve clinical symptoms of PD through its inhibition of monoamine oxidase B (MAO-B). However, zonisamide has additional targets, including voltage-gated sodium channels (Nav), which may contribute to its reported neuroprotective role in preclinical models of PD. Here, we report that Nav1.6 is highly expressed in microglia of post-mortem PD brain and of mice treated with the parkinsonism-inducing neurotoxin MPTP. Administration of zonisamide (20 mg/kg, i.p. every 4 h × 3) following a single injection of MPTP (12.5 mg/kg, s.c.) reduced microglial Nav 1.6 and microglial activation in the striatum, as indicated by Iba-1 staining and mRNA expression of F4/80. MPTP increased the levels of the pro-inflammatory cytokine TNF-α and gp91phox, and this was significantly reduced by zonisamide. Together, these findings suggest that zonisamide may reduce neuroinflammation through the down-regulation of microglial Nav 1.6. Thus, in addition to its effects on parkinsonian symptoms through inhibition of MAO-B, zonisamide may have disease modifying potential through the inhibition of Nav 1.6 and neuroinflammation.

Temporal lobe epilepsy (TLE) is a common form of adult epilepsy involving the limbic structures of the temporal lobe. Subiculum neurons act to provide a major output from the hippocampus and consist of a large population of endogenously bursting excitatory neurons. In TLE, subiculum neurons are largely spared, become hyperexcitable and show spontaneous epileptiform activity. The basis for this hyperexcitability is unclear, but is likely to involve alterations in the expression levels and function of various ion channels. In this study, we sought to determine the importance of sodium channel currents in facilitating neuronal hyperexcitability of subiculum neurons in the continuous hippocampal stimulation (CHS) rat model of TLE. Subiculum neurons from TLE rats were hyperexcitable, firing a higher frequency of action potentials after somatic current injection and action potential (AP) bursts after synaptic stimulation. Voltage clamp recordings revealed increases in resurgent (INaR) and persistent (INaP) sodium channel currents and pro-excitatory shifts in sodium channel activation and inactivation parameters that would facilitate increases in AP generation. Attenuation of INaR and INaP currents with 4,9-anhydro-tetrodotoxin (4,9-ah TTX; 100nM), a toxin with increased potency against Nav1.6 channels, suppressed neuronal firing frequency and inhibited AP bursting induced by synaptic stimulation in TLE neurons. These findings support an important role of sodium channels, particularly Nav1.6, in facilitating subiculum neuron hyperexcitability in TLE and provide further support for the importance of INaR and INaP currents in establishing epileptiform activity of subiculum neurons.

Mutations of the neuronal sodium channel gene SCN8A are associated with lethal movement disorders in the mouse and with human epileptic encephalopathy. We describe a spontaneous mouse mutation, Scn8a(9J), that is associated with a chronic movement disorder with early onset tremor and adult onset dystonia. Scn8a(9J) homozygotes have a shortened lifespan, with only 50% of mutants surviving beyond 6 months of age. The 3 bp in-frame deletion removes 1 of the 3 adjacent isoleucine residues in transmembrane segment DIVS6 of Nav1.6 (p.Ile1750del). The altered helical orientation of the transmembrane segment displaces pore-lining amino acids with important roles in channel activation and inactivation. The predicted impact on channel activity was confirmed by analysis of cerebellar Purkinje neurons from mutant mice, which lack spontaneous and induced repetitive firing. In a heterologous expression system, the activity of the mutant channel was below the threshold for detection. Observations of decreased nerve conduction velocity and impaired behavior in an open field are also consistent with reduced activity of Nav1.6. The Nav1.6Δ1750 protein is only partially glycosylated. The abundance of mutant Nav1.6 is reduced at nodes of Ranvier and is not detectable at the axon initial segment. Despite a severe reduction in channel activity, the lifespan and motor function of Scn8a(9J/9J) mice are significantly better than null mutants lacking channel protein. The clinical phenotype of this severe hypomorphic mutant expands the spectrum of Scn8a disease to include a recessively inherited, chronic and progressive movement disorder.

We describe neurotransmitter abnormalities in two patients with drug-resistant epilepsy resulting from deleterious de novo mutations in sodium channel genes. Whole exome sequencing identified a de novo SCN2A splice-site mutation (c.2379+1G>A, p.Glu717Gly.fs*30) resulting in deletion of exon 14, in a 10-year old male with early onset global developmental delay, intermittent ataxia, autism, hypotonia, epileptic encephalopathy and cerebral/cerebellar atrophy. In the cerebrospinal fluid both homovanillic acid and 5-hydroxyindoleacetic acid were significantly decreased; extensive biochemical and genetic investigations ruled out primary neurotransmitter deficiencies and other known inborn errors of metabolism. In an 8-year old female with an early onset intractable epileptic encephalopathy, developmental regression, and progressive cerebellar atrophy, a previously unreported de novo missense mutation was identified in SCN8A (c.5615G>A; p.Arg1872Gln), affecting a highly conserved residue located in the C-terminal of the Nav1.6 protein. Aside from decreased homovanillic acid and 5-hydroxyindoleacetic acid, 5-methyltetrahydrofolate was also found to be low. We hypothesize that these channelopathies cause abnormal synaptic mono-amine metabolite secretion/uptake via impaired vesicular release and imbalance in electrochemical ion gradients, which in turn aggravate the seizures. Treatment with oral 5-hydroxytryptophan, l-Dopa/Carbidopa, and a dopa agonist resulted in mild improvement of seizure control in the male case, most likely via dopamine and serotonin receptor activated signal transduction and modulation of glutamatergic, GABA-ergic and glycinergic neurotransmission. Neurotransmitter analysis in other sodium channelopathy patients will help validate our findings, potentially yielding novel treatment opportunities.

Understanding the role of SCN8A in epilepsy and behavior is critical in light of recently identified human SCN8A epilepsy mutations. We have previously demonstrated that Scn8a(med) and Scn8a(med-jo) mice carrying mutations in the Scn8a gene display increased resistance to flurothyl and kainic acid-induced seizures; however, they also exhibit spontaneous absence seizures. To further investigate the relationship between altered SCN8A function and epilepsy, we introduced the SCN1A-R1648H mutation, identified in a family with generalized epilepsy with febrile seizures plus (GEFS+), into the corresponding position (R1627H) of the mouse Scn8a gene. Heterozygous R1627H mice exhibited increased resistance to some forms of pharmacologically and electrically induced seizures and the mutant Scn8a allele ameliorated the phenotype of Scn1a-R1648H mutants. Hippocampal slices from heterozygous R1627H mice displayed decreased bursting behavior compared to wild-type littermates. Paradoxically, at the homozygous level, R1627H mice did not display increased seizure resistance and were susceptible to audiogenic seizures. We furthermore observed increased hippocampal pyramidal cell excitability in heterozygous and homozygous Scn8a-R1627H mutants, and decreased interneuron excitability in heterozygous Scn8a-R1627H mutants. These results expand the phenotypes associated with disruption of the Scn8a gene and demonstrate that an Scn8a mutation can both confer seizure protection and increase seizure susceptibility.

SCN1A mutations are the main cause of the epilepsy disorders Dravet syndrome (DS) and genetic epilepsy with febrile seizures plus (GEFS+). Mutations that reduce the activity of the mouse Scn8a gene, in contrast, are found to confer seizure resistance and extend the lifespan of mouse models of DS and GEFS+. To investigate the mechanism by which reduced Scn8a expression confers seizure resistance, we induced interictal-like burst discharges in hippocampal slices of heterozygous Scn8a null mice (Scn8a(med/+)) with elevated extracellular potassium. Scn8a(med/+) mutants exhibited reduced epileptiform burst discharge activity after P20, indicating an age-dependent increased threshold for induction of epileptiform discharges. Scn8a deficiency also reduced the occurrence of burst discharges in a GEFS+ mouse model (Scn1a(R1648H/+)). There was no detectable change in the expression levels of Scn1a (Nav1.1) or Scn2a (Nav1.2) in the hippocampus of adult Scn8a(med/+) mutants. To determine whether the increased seizure resistance associated with reduced Scn8a expression was due to alterations that occurred during development, we examined the effect of deleting Scn8a in adult mice. Global Cre-mediated deletion of a heterozygous floxed Scn8a allele in adult mice was found to increase thresholds to chemically and electrically induced seizures. Finally, knockdown of Scn8a gene expression in the adult hippocampus via lentiviral Cre injection resulted in a reduction in the number of EEG-confirmed seizures following the administration of picrotoxin. Our results identify the hippocampus as an important structure in the mediation of Scn8a-dependent seizure protection and suggest that selective targeting of Scn8a activity might be efficacious in patients with epilepsy.