[Analysis of the Divalent Cation Blocking in Ion Channels by Crystal Structure and Molecular Dynamics Simulations].

Neural activity generates essential responses, such as thinking, memory formation, and muscle contraction. It is controlled by the well-coordinated activity of various cation-selective channels of the cell membrane. The divalent cation block plays an essential role in various tetrameric ion channels. For example, N-methyl-D-aspartic acid receptors, which are tetrameric ion channels involved in memory formation, are inhibited by magnesium ions. Divalent cations are thought to bind in the ion pathway of the ion channel and as a consequence block the channel current, however, direct observation of such a block has not been reported yet. As a consequence, the behavior of these blocking divalent cations remains poorly understood. NavAb, a similar tetrameric sodium channel cloned from Arcobacter butzleri, is one of the most structurally analyzed tetrameric channels that is not inhibited by divalent cations. In this study, we elucidated the molecular mechanism of the divalent cation block by reproducing the divalent cation block in NavAb. The X-ray crystal structure of divalent-cation-block mutants show electron density in the ion transmission pathway of the divalent cation blocked mutants, indicating that the mutations increasing the hydrophilicity of the inner vestibule of the pore domain enable a divalent cation to stack into the ion pathway. In molecular dynamics simulations, the stacked calcium ion repels the sodium ions near the channel lumen's entrance at the selective filter's bottom. These results suggest the primary process of the divalent cation block mechanism in tetrameric cation channels and suggest a process of functional acquisition in ion channel evolution.

Similar excitability through different sodium channels and implications for the analgesic efficacy of selective drugs.

Nociceptive sensory neurons convey pain-related signals to the CNS using action potentials. Loss-of-function mutations in the voltage-gated sodium channel NaV1.7 cause insensitivity to pain (presumably by reducing nociceptor excitability) but clinical trials seeking to treat pain by inhibiting NaV1.7 pharmacologically have struggled. This may reflect the variable contribution of NaV1.7 to nociceptor excitability. Contrary to claims that NaV1.7 is necessary for nociceptors to initiate action potentials, we show that nociceptors can achieve similar excitability using different combinations of NaV1.3, NaV1.7, and NaV1.8. Selectively blocking one of those NaV subtypes reduces nociceptor excitability only if the other subtypes are weakly expressed. For example, excitability relies on NaV1.8 in acutely dissociated nociceptors but responsibility shifts to NaV1.7 and NaV1.3 by the fourth day in culture. A similar shift in NaV dependence occurs in vivo after inflammation, impacting ability of the NaV1.7-selective inhibitor PF-05089771 to reduce pain in behavioral tests. Flexible use of different NaV subtypes exemplifies degeneracy - achieving similar function using different components - and compromises reliable modulation of nociceptor excitability by subtype-selective inhibitors. Identifying the dominant NaV subtype to predict drug efficacy is not trivial. Degeneracy at the cellular level must be considered when choosing drug targets at the molecular level.

Schisandrin B from Schisandra chinensis alleviated pain via glycine receptors, Nav1.7 channels and Cav2.2 channels.

ETHNOPHARMACOLOGICAL RELEVANCE: Schisandra chinensis, the dried and ripe fruit of the magnolia family plant Schisandra chinensis (Turcz.) Baill, was commonly used in traditional analgesic prescription. Studies have shown that the extract of Schisandra chinensis (SC) displayed analgesic activity. However, the analgesic active component and the exact mechanisms have yet to be revealed. AIM OF THE STUDY: The present study was to investigate the anti-nociceptive constituent of Schisandra chinensis, assess its analgesic effect, and explore the potential molecular mechanisms. MATERIALS AND METHODS: The effects of a series of well-recognized compounds from SC on glycine receptors were investigated. The analgesic effect of the identified compound was evaluated in three pain models. Mechanistic studies were performed using patch clamp technique on various targets expressed in recombinant cells. These targets included glycine receptors, Nav1.7 sodium channels, Cav2.2 calcium channels et al. Meanwhile, primary cultured spinal dorsal horn (SDH) neurons and dorsal root ganglion (DRG) neurons were also utilized. RESULTS: Schisandrin B (SchB) was a positive allosteric modulator of glycine receptors in spinal dorsal horn neurons. The EC50 of SchB on glycine receptors in spinal dorsal horn neurons was 2.94 ± 0.28 µM. In three pain models, the analgesic effect of SchB was comparable to that of indomethacin at the same dose. Besides, SchB rescued PGE2-induced suppression of α3 GlyR activity and alleviated persistent pain. Notably, SchB could also potently decrease the frequency of action potentials and inhibit sodium and calcium channels in DRG neurons. Consistent with the data from DRG neurons, SchB was also found to significantly block Nav1.7 sodium channels and Cav2.2 channels in recombinant cells. CONCLUSION: Our results demonstrated that, Schisandrin B, the primary lignan component of Schisandra chinensis, may exert its analgesic effect by acting on multiple ion channels, including glycine receptors, Nav1.7 channels, and Cav2.2 channels.

Ecologically mediated differences in electric organ discharge drive evolution in a sodium channel gene in South American electric fishes.

Active electroreception-the ability to detect objects and communicate with conspecifics via the detection and generation of electric organ discharges (EODs)-has evolved convergently in several fish lineages. South American electric fishes (Gymnotiformes) are a highly species-rich group, possibly in part due to evolution of an electric organ (EO) that can produce diverse EODs. Neofunctionalization of a voltage-gated sodium channel gene accompanied the evolution of electrogenic tissue from muscle and resulted in a novel gene (scn4aa) uniquely expressed in the EO. Here, we investigate the link between variation in scn4aa and differences in EOD waveform. We combine gymnotiform scn4aa sequences encoding the C-terminus of the Nav1.4a protein, with biogeographic data and EOD recordings to test whether physiological transitions among EOD types accompany differential selection pressures on scn4aa. We found positive selection on scn4aa coincided with shifts in EOD types. Species that evolved in the absence of predators, which likely selected for reduced EOD complexity, exhibited increased scn4aa evolutionary rates. We model mutations in the protein that may underlie changes in protein function and discuss our findings in the context of gymnotiform signalling ecology. Together, this work sheds light on the selective forces underpinning major evolutionary transitions in electric signal production.

Diversity and distribution of sodium channel mutations in Aedes albopictus (Diptera: Culicidae).

There is growing interest in insecticide resistance in the mosquito, Aedes albopictus (Skuse), as its potential for spreading diseases is increasing as urbanization and control efforts intensify. Here we review the presence and diversity of mutations in the voltage-sensitive sodium channel (Vssc) gene associated with pyrethroid resistance and report on additional surveys of these mutations in new populations with an analysis of their spread. The known diversity of these mutations has increased in recent years including the identification of 26 non-synonymous mutations, although phenotypic data associating mutations with resistance remain limited. We provide data on mutations in several new locations including those in Timor Leste, Indonesia, and Vanuatu. We use population genomic data from ddRAD analyses of target populations with the 1534C mutation to identify single nucleotide polymorphisms (SNPs) associated with the mutant to test for clustering of SNPs based on the presence of the 1534C mutation rather than population origin. Our findings suggest spread of resistance alleles via genetic invasion, which is further supported by patterns from a genome-wide principal components analysis. These data point to movement of resistance alleles across wide areas with likely impacts on local control options.

Evolutionary origin and distribution of amino acid mutations associated with resistance to sodium channel modulators in onion thrips, Thrips tabaci.

In onion thrips Thrips tabaci, reduced sensitivity of the sodium channel caused by several sodium channel mutations have been correlated with pyrethroid resistance. For this study, using mitochondrial cytochrome c oxidase subunit I gene sequences, we examined the phylogenetic relation among a total of 52 thelytokous and arrhenotokous strains with different genotypes of the sodium channel mutations. Then, we used flow cytometry to estimate their ploidy. Results showed that the strains are divisible into three groups: diploid thelytoky, triploid thelytoky, and diploid arrhenotoky. Using 23 whole genome resequencing data obtained from 20 strains out of 52, we examined their genetic relation further using principal component analysis, admixture analysis, and a fixation index. Results showed that diploid and triploid thelytokous groups are further classifiable into two based on the sodium channel mutations harbored by the respective group members (strains). The greatest genetic divergence was observed between thelytokous and arrhenotokous groups with a pair of T929I and K1774N. Nevertheless, they shared a genomic region with virtually no polymorphism around the sodium channel gene loci, suggesting a hard selective sweep. Based on these findings, we discuss the evolutionary origin and distribution of the sodium channel mutations in T. tabaci.

Long-term effectiveness and tolerability of ketogenic diet therapy in patients with genetic developmental and epileptic encephalopathy onset within the first 6 months of life.

OBJECTIVE: To investigate the effectiveness and tolerability of ketogenic diet therapy (KDT) in patients with developmental and epileptic encephalopathy (DEE) associated with genetic etiology which onset within the first 6 months of life, and to explore the association between response to KDT and genotype/clinical parameters. METHODS: We retrospectively reviewed data from patients with genetic DEE who started KDT at Beijing Children's Hospital between January 1, 2016, and December 31, 2021. RESULTS: A total of 32 patients were included, involving 14 pathogenic or likely pathogenic single genes, and 16 (50.0%) patients had sodium/potassium channel gene variants. The median age at onset of epilepsy was 1.0 (IQR: 0.1, 3.0) months. The median age at initiation of KDT was 10.0 (IQR: 5.3, 13.8) months and the median duration of maintenance was 14.0 (IQR: 7.0, 26.5) months, with a mean blood ß-hydroxybutyrate of 2.49 ± 0.62 mmol/L. During the maintenance period of KDT, 26 (81.3%) patients had a ≥50% reduction of seizure frequency, of which 12 (37.5%) patients achieved seizure freedom. Better responses were observed in patients with STXBP1 variants, with four out of five patients achieving seizure freedom. There were no statistically differences in the age of onset, duration of epilepsy before KDT, blood ketone values, or the presence of ion channel gene variants between the seizure-free patients and the others. The most common adverse effects were gastrointestinal side effects, which occurred in 21 patients (65.6%), but all were mild and easily corrected. Only one patient discontinued KDT due to nephrolithiasis. SIGNIFICANCE: KDT is effective in treating early onset genetic DEE, and no statistically significant relationship has been found between genotype and effectiveness in this study. KDT is well tolerated in most young patients, with mild and reversible gastrointestinal side effects being the most common, but usually not the reason to discontinue KDT. PLAIN LANGUAGE SUMMARY: This study evaluated the response and side effects of ketogenic diet therapy (KDT) in patients who had seizures within the first 6 months of life, and were diagnosed with genetic developmental and epileptic encephalopathy (DEE), a type of severe epilepsy with developmental delay caused by gene variants. Thirty-two patients involving 14 gene variants who started KDT at Beijing Children's Hospital between were included. KDT was effective in treating early onset genetic DEE in this cohort, and patients with STXBP1 variants responded better; however, no statistically significant relationship was found between gene variant and response. Most young patients tolerated KDT well, with mild and reversible gastrointestinal side effects being the most common.

Sodium Channel Gene Variants in Fetuses with Abnormal Sonographic Findings: Expanding the Prenatal Phenotypic Spectrum of Sodium Channelopathies.

Voltage-gated sodium channels (VGSCs) are responsible for the initiation and propagation of action potentials in the brain and muscle. Pathogenic variants in genes encoding VGSCs have been associated with severe disorders including epileptic encephalopathies and congenital myopathies. In this study, we identified pathogenic variants in genes encoding the α subunit of VGSCs in the fetuses of two unrelated families with the use of trio-based whole exome sequencing, as part of a larger cohort study. Sanger sequencing was performed for variant confirmation as well as parental phasing. The fetus of the first family carried a known de novo heterozygous missense variant in the SCN2A gene (NM_001040143.2:c.751G>A p.(Val251Ile)) and presented intrauterine growth retardation, hand clenching and ventriculomegaly. Neonatally, the proband also exhibited refractory epilepsy, spasms and MRI abnormalities. The fetus of the second family was a compound heterozygote for two parentally inherited novel missense variants in the SCN4A gene (NM_000334.4:c.4340T>C, p.(Phe1447Ser), NM_000334.4:c.3798G>C, p.(Glu1266Asp)) and presented a severe prenatal phenotype including talipes, fetal hypokinesia, hypoplastic lungs, polyhydramnios, ear abnormalities and others. Both probands died soon after birth. In a subsequent pregnancy of the latter family, the fetus was also a compound heterozygote for the same parentally inherited variants. This pregnancy was terminated due to multiple ultrasound abnormalities similar to the first pregnancy. Our results suggest a potentially crucial role of the VGSC gene family in fetal development and early lethality.

Development and application of a multiple reaction monitoring method for the simultaneous quantification of sodium channels Nav 1.1, Nav 1.2, and Nav 1.6 in solubilized membrane proteins from stable HEK293 cell lines, rodents, and human brain tissues.

RATIONALE: Nav 1.1, 1.2, and 1.6 are transmembrane proteins acting as voltage-gated sodium channels implicated in various forms of epilepsy. There is a need for knowing their actual concentration in target tissues during drug development. METHODS: Unique peptides for Nav 1.1, Nav 1.2, and Nav 1.6 were selected as quantotropic peptides for each protein and used for their quantification in membranes from stably transfected HEK293 cells and rodent and human brain samples using ultra-high-performance liquid chromatography-electrospray ionization tandem mass spectrometry. RESULTS: Nav 1.1, 1.2, and 1.6 protein expressions in three stably individually transfected HEK293 cell lines were found to be 2.1 ± 0.2, 6.4 ± 1.2, and 4.0 ± 0.6 fmol/µg membrane protein, respectively. In brains, Nav 1.2 showed the highest expression, with approximately three times higher (P < 0.003) in rodents than in humans at 3.05 ± 0.57, with 3.35 ± 0.56 in mouse and rat brains and 1.09 ± 0.27 fmol/µg in human brain. Both Nav 1.1 and 1.6 expressions were much lower in the brains, with approximately 40% less expression in human Nav 1.1 than rodent Nav 1.1 at 0.49 ± 0.1 (mouse), 0.43 ± 0.3 (rat), and 0.28 ± 0.04 (humans); whereas Nav 1.6 had approximately 60% less expression in humans than rodents at 0.27 ± 0.09 (mouse), 0.26 ± 0.06 (rat), and 0.11 ± 0.02 (humans) fmol/µg membrane proteins. CONCLUSIONS: Multiple reaction monitoring was used to quantify sodium channels Nav 1.1, 1.2, and 1.6 expressed in stably transfected HEK293 cells and brain tissues from mice, rats, and humans. We found significant differences in the expression of these channels in mouse, rat, and human brains. Nav expression ranking among the three species was Nav 1.2 ≫ Nav 1.1 > Nav 1.6, with the human brain expressing much lower concentrations overall compared to rodent brain.

Mannitol and hyponatremia regulate cardiac ventricular conduction in the context of sodium channel loss of function.

Scn5a heterozygous null (Scn5a+/-) mice have historically been used to investigate arrhythmogenic mechanisms of diseases such as Brugada syndrome (BrS) and Lev's disease. Previously, we demonstrated that reducing ephaptic coupling (EpC) in ex vivo hearts exacerbates pharmacological voltage-gated sodium channel (Nav)1.5 loss of function (LOF). Whether this effect is consistent in a genetic Nav1.5 LOF model is yet to be determined. We hypothesized that loss of EpC would result in greater reduction in conduction velocity (CV) for the Scn5a+/- mouse relative to wild type (WT). In vivo ECGs and ex vivo optical maps were recorded from Langendorff-perfused Scn5a+/- and WT mouse hearts. EpC was reduced with perfusion of a hyponatremic solution, the clinically relevant osmotic agent mannitol, or a combination of the two. Neither in vivo QRS duration nor ex vivo CV during normonatremia was significantly different between the two genotypes. In agreement with our hypothesis, we found that hyponatremia severely slowed CV and disrupted conduction for 4/5 Scn5a+/- mice, but 0/6 WT mice. In addition, treatment with mannitol slowed CV to a greater extent in Scn5a+/- relative to WT hearts. Unexpectedly, treatment with mannitol during hyponatremia did not further slow CV in either genotype, but resolved the disrupted conduction observed in Scn5a+/- hearts. Similar results in guinea pig hearts suggest the effects of mannitol and hyponatremia are not species specific. In conclusion, loss of EpC through either hyponatremia or mannitol alone results in slowed or disrupted conduction in a genetic model of Nav1.5 LOF. However, the combination of these interventions attenuates conduction slowing.NEW & NOTEWORTHY Cardiac sodium channel loss of function (LOF) diseases such as Brugada syndrome (BrS) are often concealed. We optically mapped mouse hearts with reduced sodium channel expression (Scn5a+/-) to evaluate whether reduced ephaptic coupling (EpC) can unmask conduction deficits. Data suggest that conduction deficits in the Scn5a+/- mouse may be unmasked by treatment with hyponatremia and perinexal widening via mannitol. These data support further investigation of hyponatremia and mannitol as novel diagnostics for sodium channel loss of function diseases.

The Ablation of Sensory Neurons Expressing the Nav1.8 Sodium Channel Improves Glucose Homeostasis and Amplifies the GLP-1 Signaling in Obese Female Mice.

SCOPE: Sensory neurons expressing the sodium channel Nav1.8 contain a repertoire of receptors for nutrient, hormonal, and inflammatory ligands. However, their function in key regulators of energy homeostasis control is not well understood and is completely unexplored in females. METHODS AND RESULTS: Mice lacking neurons expressing the sodium channel Nav1.8 were generated using an ablation strategy based on cre recombinase-mediated expression of diphtheria toxin fragment A (DTA) (Nav1.8-cre/DTA mice) to investigate whether these neurons modulate body weight, food intake, gut hormone secretion, gastrointestinal transit, and glucose tolerance in response to nutrient challenges in a sex-dependent manner. Male Nav1.8-cre/DTA mice show resistance to gain weight in response to high-fat high-sugar diet (HFHSD), whereas females lacking Nav1.8+ neurons have improved oral glucose tolerance accompanied by higher insulin levels and attenuated glucagon secretion after an oral glucose load. Female Nav1.8-cre/DTA mice also show higher fasting and postprandial glucagon like peptide-1 (GLP-1) levels with an increased number of GLP-1-positive cells. Finally, ablation of Nav1.8-expressing neurons accelerates the gastrointestinal transit in female mice under HFHSD. CONCLUSION: This data demonstrates sex-dependent differences in the Nav1.8-mediated regulation of energy metabolism, and provides new insights that may help in the design of sex-specific neuromodulation therapies for metabolic disorders induced by diets rich in fats and simple sugars.

Reduced cone photoreceptor function and subtle systemic manifestations in two siblings with loss of SCLT1.

BACKGROUND: The sodium channel and clathrin linker 1 gene (SCLT1) has been involved in the pathogenesis of various ciliopathy disorders such as Bardet-Biedl syndrome, orofaciodigital syndrome type IX, and Senior-Løken syndrome. Detailed exams are warranted to outline all clinical features. Here, we present a family with a milder phenotype of SCLT1-related disease. MATERIAL AND METHODS: Comprehensive eye examination including fundus images, OCT, color vision, visual fields and electroretinography were performed. Affected individuals were assessed by a pediatrician and a medical geneticist for systemic features of ciliopathy. Investigations included echocardiography, abdominal ultrasonography, blood work-up for diabetes, liver and kidney function. Genetic testing included NGS retinal dystrophy panel, segregation analysis and transcriptome sequencing. RESULTS: Two male children, age 10 and 8 years, were affected with attention deficit hyperactivity disorder (ADHD), obesity and mild photophobia. The ophthalmic exam revealed reduced best-corrected visual acuity (BCVA), strabismus, hyperopia, astigmatism and moderate red-green defects. Milder changes suggesting photoreceptors disease were found on retinal imaging. Electroretinogram confirmed cone photoreceptors dysfunction. Genetic testing revealed a homozygous likely pathogenic, splice-site variant in SCLT1 gene NM_144643.3: c.1439 + 1del in the proband and in the affected brother. The unaffected parents were heterozygous for the SCLT1 variant. Transcriptome sequencing showed retention of intron 16 in the proband. CONCLUSIONS: In this report, we highlight the importance of further extensive diagnostics in patients with unexplained reduced vision, strabismus, refractive errors and ADHD spectrum disorders. SCLT1-related retinal degeneration is very rare and isolated reduced function of cone photoreceptors has not previously been observed.

Anti-PD-1 treatment protects against seizure by suppressing sodium channel function.

AIMS: Although programmed cell death protein 1 (PD-1) typically serves as a target for immunotherapies, a few recent studies have found that PD-1 is expressed in the nervous system and that neuronal PD-1 might play a crucial role in regulating neuronal excitability. However, whether brain-localized PD-1 is involved in seizures and epileptogenesis is still unknown and worthy of in-depth exploration. METHODS: The existence of PD-1 in human neurons was confirmed by immunohistochemistry, and PD-1 expression levels were measured by real-time quantitative PCR (RT-qPCR) and western blotting. Chemoconvulsants, pentylenetetrazol (PTZ) and cyclothiazide (CTZ), were applied for the establishment of in vivo (rodents) and in vitro (primary hippocampal neurons) models of seizure, respectively. SHR-1210 (a PD-1 monoclonal antibody) and sodium stibogluconate (SSG, a validated inhibitor of SH2-containing protein tyrosine phosphatase-1 [SHP-1]) were administrated to investigate the impact of PD-1 pathway blockade on epileptic behaviors of rodents and epileptiform discharges of neurons. A miRNA strategy was applied to determine the impact of PD-1 knockdown on neuronal excitability. The electrical activities and sodium channel function of neurons were determined by whole-cell patch-clamp recordings. The interaction between PD-1 and α-6 subunit of human voltage-gated sodium channel (Nav1.6) was validated by performing co-immunostaining and co-immunoprecipitation (co-IP) experiments. RESULTS: Our results reveal that PD-1 protein and mRNA levels were upregulated in lesion cores compared with perifocal tissues of surgically resected specimens from patients with intractable epilepsy. Furthermore, we show that anti-PD-1 treatment has anti-seizure effects both in vivo and in vitro. Then, we reveal that PD-1 blockade can alter the electrophysiological properties of sodium channels. Moreover, we reveal that PD-1 acts together with downstream SHP-1 to regulate sodium channel function and hence neuronal excitability. Further investigation suggests that there is a direct interaction between neuronal PD-1 and Nav1.6. CONCLUSION: Our study reveals that neuronal PD-1 plays an important role in epilepsy and that anti-PD-1 treatment protects against seizures by suppressing sodium channel function, identifying anti-PD-1 treatment as a novel therapeutic strategy for epilepsy.

Targeted blockade of aberrant sodium current in a stem cell-derived neuron model of SCN3A encephalopathy.

Missense variants in SCN3A encoding the voltage-gated sodium (Na+) channel α subunit Nav1.3 are associated with SCN3A-related neurodevelopmental disorder (SCN3A-NDD), a spectrum of disease that includes epilepsy and malformation of cortical development. How genetic variation in SCN3A leads to pathology remains unclear, as prior electrophysiological work on disease-associated variants has been performed exclusively in heterologous cell systems. To further investigate the mechanisms of SCN3A-NDD pathogenesis, we used CRISPR/Cas9 gene editing to modify a control human induced pluripotent stem cell (iPSC) line to express the recurrent de novo missense variant SCN3A c.2624T>C (p.Ile875Thr). With the established Ngn2 rapid induction protocol, we generated glutamatergic forebrain-like neurons (iNeurons), which we showed to express SCN3A mRNA and Nav1.3-mediated Na+ currents. We performed detailed whole-cell patch clamp recordings to determine the effect of the SCN3A-p.Ile875Thr variant on endogenous Na+ currents in, and intrinsic excitability of, human neurons. Compared to control iNeurons, variant-expressing iNeurons exhibit markedly increased slowly-inactivating/persistent Na+ current, abnormal firing patterns with paroxysmal bursting and plateau-like potentials with action potential failure, and a hyperpolarized voltage threshold for action potential generation. We then validated these findings using a separate iPSC line generated from a patient harbouring the SCN3A-p.Ile875Thr variant compared to a corresponding CRISPR-corrected isogenic control line. Finally, we found that application of the Nav1.3-selective blocker ICA-121431 normalizes action potential threshold and aberrant firing patterns in SCN3A-p.Ile1875Thr iNeurons; in contrast, consistent with action as a Na+ channel blocker, ICA-121431 decreases excitability of control iNeurons. Our findings demonstrate that iNeurons can model the effects of genetic variation in SCN3A yet reveal a complex relationship between gain-of-function at the level of the ion channel versus impact on neuronal excitability. Given the transient expression of SCN3A in the developing human nervous system, selective blockade or suppression of Nav1.3-containing Na+ channels could represent a therapeutic approach towards SCN3A-NDD.

Novel insights into voltage-gated ion channels: Translational breakthroughs in medical oncology.

Preclinical evidence suggests that voltage gradients can act as a kind of top-down master regulator during embryogenesis and orchestrate downstream molecular-genetic pathways during organ regeneration or repair. Moreover, electrical stimulation shifts response to injury toward regeneration instead of healing or scarring. Cancer and embryogenesis not only share common phenotypical features but also commonly upregulated molecular pathways. Voltage-gated ion channel activity is directly or indirectly linked to the pathogenesis of cancer hallmarks, while experimental and clinical studies suggest that their modulation, e.g., by anesthetic agents, may exert antitumor effects. A large recent clinical trial served as a proof-of-principle for the benefit of preoperative use of topical sodium channel blockade as a potential anticancer strategy against early human breast cancers. Regardless of whether ion channel aberrations are primary or secondary cancer drivers, understanding the functional consequences of these events may guide us toward the development of novel therapeutic approaches.

Long-Term Downregulation of the Sodium Channel Gene Scn8a Is Therapeutic in Mouse Models of SCN8A Epilepsy.

OBJECTIVE: De novo mutations of the voltage-gated sodium channel gene SCN8A cause developmental and epileptic encephalopathy (DEE). Most pathogenic variants result in gain-of-function changes in activity of the sodium channel Nav1.6, poorly controlled seizures, and significant comorbidities. In previous work, an antisense oligonucleotide (ASO) reduced Scn8a transcripts and increased lifespan after neonatal administration to a mouse model. Here, we tested long-term ASO treatment initiated after seizure onset, as required for clinical application. METHODS: ASO treatment was initiated after observation of a convulsive seizure and repeated at 4 to 6 week intervals for 1 year. We also tested the long-term efficacy of an AAV10-short hairpin RNA (shRNA) virus administered on P1. RESULTS: Repeated treatment with the Scn8a ASO initiated after seizure onset provided long-term survival and reduced seizure frequency during a 12 month observation period. A single treatment with viral shRNA was also protective during 12 months of observation. INTERPRETATION: Downregulation of Scn8a expression that is initiated after the onset of seizures is effective for long-term treatment in a model of SCN8A-DEE. Repeated ASO administration or a single dose of viral shRNA prevented seizures and extended survival through 12 months of observation. ANN NEUROL 2024;95:754-759.

New insights into the physiology and pathophysiology of the atypical sodium leak channel NALCN.

Cell excitability and its modulation by hormones and neurotransmitters involve the concerted action of a large repertoire of membrane proteins, especially ion channels. Unique complements of coexpressed ion channels are exquisitely balanced against each other in different excitable cell types, establishing distinct electrical properties that are tailored for diverse physiological contributions, and dysfunction of any component may induce a disease state. A crucial parameter controlling cell excitability is the resting membrane potential (RMP) set by extra- and intracellular concentrations of ions, mainly Na+, K+, and Cl-, and their passive permeation across the cell membrane through leak ion channels. Indeed, dysregulation of RMP causes significant effects on cellular excitability. This review describes the molecular and physiological properties of the Na+ leak channel NALCN, which associates with its accessory subunits UNC-79, UNC-80, and NLF-1/FAM155 to conduct depolarizing background Na+ currents in various excitable cell types, especially neurons. Studies of animal models clearly demonstrate that NALCN contributes to fundamental physiological processes in the nervous system including the control of respiratory rhythm, circadian rhythm, sleep, and locomotor behavior. Furthermore, dysfunction of NALCN and its subunits is associated with severe pathological states in humans. The critical involvement of NALCN in physiology is now well established, but its study has been hampered by the lack of specific drugs that can block or agonize NALCN currents in vitro and in vivo. Molecular tools and animal models are now available to accelerate our understanding of how NALCN contributes to key physiological functions and the development of novel therapies for NALCN channelopathies.

Ibuprofen modulates tetrodotoxin-resistant persistent Na+ currents at acidic pH in rat trigeminal ganglion neurons.

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used to relieve various symptoms such as headache, arthralgia, and dental pain. While the primary mechanism of NSAID-based pain relief is the inhibition of cyclooxygenase-2, several NSAIDs also modulate other molecular targets related to nociceptive transmission such as voltage-gated Na+ channels. In the present study, we examined the effects of NSAIDs on persistent Na+ current (INaP) mediated by tetrodotoxin-resistant (TTX-R) Na+ channels in small-to medium-sized trigeminal ganglion neurons using a whole-cell patch-clamp technique. At clinically relevant concentrations, all propionic acid derivatives tested (ibuprofen, naproxen, fenoprofen, and flurbiprofen) preferentially inhibited the TTX-R INaP. The inhibition was more potent at acidic extracellular pH (pH 6.5) than at normal pH (pH 7.4). Other NSAIDs, such as ketorolac, piroxicam, and aspirin, had a negligible effect on the TTX-R INaP. Ibuprofen both accelerated the onset of inactivation and retarded the recovery from inactivation of TTX-R Na+ channels at acidic extracellular pH. However, all NSAIDs tested in this study had minor effects on voltage-gated K+ currents, as well as hyperpolarization-activated and cyclic nucleotide-gated cation currents, at both acidic and normal extracellular pH. Under current-clamp conditions, ibuprofen decreased the number of action potentials elicited by depolarizing current stimuli at acidic (pH 6.5) extracellular pH. Considering that extracellular pH falls as low as 5.5 in inflamed tissues, TTX-R INaP inhibition could be a mechanism by which ibuprofen and propionic acid derivative NSAIDs modulate inflammatory pain.