A molecular switch in RCK2 triggers sodium-dependent activation of KNa1.1 (KCNT1) potassium channels.

The Na+-activated K+ channel KNa1.1, encoded by the KCNT1 gene, is an important regulator of neuronal excitability. How intracellular Na+ ions bind and increase channel activity is not well understood. Analysis of KNa1.1 channel structures indicate that there is a large twisting of the ßN-αQ loop in the intracellular RCK2 domain between the inactive and Na+-activated conformations, with a lysine (K885, human subunit numbering) close enough to potentially form a salt bridge with an aspartate (D839) in ßL in the Na+-activated state. Concurrently, an aspartate (D884) adjacent in the same loop adopts a position within a pocket formed by the ßO strand. In carrying out mutagenesis and electrophysiology with human KNa1.1, we found that alanine substitution of selected residues in these regions resulted in almost negligible currents in the presence of up to 40 mM intracellular Na+. The exception was D884A, which resulted in constitutively active channels in both the presence and absence of intracellular Na+. Further mutagenesis of this site revealed an amino acid size-dependent effect. Substitutions at this site by an amino acid smaller than aspartate (D884V) also yielded constitutively active KNa1.1, and D884I had Na+ dependence similar to wild-type KNa1.1, while increasing the side-chain size larger than aspartate (D884E or D884F) yielded channels that could not be activated by up to 40 mM intracellular Na+. We conclude that Na+ binding results in a conformational change that accommodates D884 in the ßO pocket, which triggers further conformational changes in the RCK domains and channel activation.

Identification of Sodium- and Chloride-Sensitive Sites in the Slack Channel.

The Slack channel (KCNT1, Slo2.2) is a sodium-activated and chloride-activated potassium channel that regulates heart rate and maintains the normal excitability of the nervous system. Despite intense interest in the sodium gating mechanism, a comprehensive investigation to identify the sodium-sensitive and chloride-sensitive sites has been missing. In the present study, we identified two potential sodium-binding sites in the C-terminal domain of the rat Slack channel by conducting electrophysical recordings and systematic mutagenesis of cytosolic acidic residues in the rat Slack channel C terminus. In particular, by taking advantage of the M335A mutant, which results in the opening of the Slack channel in the absence of cytosolic sodium, we found that among the 92 screened negatively charged amino acids, E373 mutants could completely remove sodium sensitivity of the Slack channel. In contrast, several other mutants showed dramatic decreases in sodium sensitivity but did not abolish it altogether. Furthermore, molecular dynamics (MD) simulations performed at the hundreds of nanoseconds timescale revealed one or two sodium ions at the E373 position or an acidic pocket composed of several negatively charged residues. Moreover, the MD simulations predicted possible chloride interaction sites. By screening predicted positively charged residues, we identified R379 as a chloride interaction site. Thus, we conclude that the E373 site and the D863/E865 pocket are two potential sodium-sensitive sites, while R379 is a chloride interaction site in the Slack channel.SIGNIFICANCE STATEMENT The research presented here identified two distinct sodium and one chloride interaction sites located in the intracellular C-terminal domain of the Slack (Slo2.2, KCNT1) channel. Identification of the sites responsible for the sodium and chloride activation of the Slack channel sets its gating property apart from other potassium channels in the BK channel family. This finding sets the stage for future functional and pharmacological studies of this channel.

KCNT2-Related Disorders: Phenotypes, Functional, and Pharmacological Properties.

OBJECTIVE: Pathogenic variants in KCNT2 are rare causes of developmental epileptic encephalopathy (DEE). We herein describe the phenotypic and genetic features of patients with KCNT2-related DEE, and the in vitro functional and pharmacological properties of KCNT2 channels carrying 14 novel or previously untested variants. METHODS: Twenty-five patients harboring KCNT2 variants were investigated: 12 were identified through an international collaborative network, 13 were retrieved from the literature. Clinical data were collected and included in a standardized phenotyping sheet. Novel variants were detected using exome sequencing and classified using ACMG criteria. Functional and pharmacological studies were performed by whole-cell electrophysiology in HEK-293 and SH-SY5Y cells. RESULTS: The phenotypic spectrum encompassed: (a) intellectual disability/developmental delay (21/22 individuals with available information), ranging from mild to severe/profound; (b) epilepsy (15/25); (c) neurological impairment, with altered muscle tone (14/22); (d) dysmorphisms (13/20). Nineteen pathogenic KCNT2 variants were found (9 new, 10 reported previously): 16 missense, 1 in-frame deletion of a single amino acid, 1 nonsense, and 1 frameshift. Among tested variants, 8 showed gain-of-function (GoF), and 6 loss-of-function (LoF) features when expressed heterologously in vitro. Quinidine and fluoxetine blocked all GoF variants, whereas loxapine and riluzole activated some LoF variants while blocking others. INTERPRETATION: We expanded the phenotypic and genotypic spectrum of KCNT2-related disorders, highlighting novel genotype-phenotype associations. Pathogenic KCNT2 variants cause GoF or LoF in vitro phenotypes, and each shows a unique pharmacological profile, suggesting the need for in vitro functional and pharmacological investigation to enable targeted therapies based on the molecular phenotype. ANN NEUROL 2023;94:332-349.

Life-threatening pulmonary haemorrhage treated with coil embolisation followed by lobectomy in a patient with KCNT1 mutation.

KCNT1 mutations are associated with childhood epilepsy, developmental delay, and vascular malformations. We report a child with a likely pathogenic KCNT1 mutation (c.1885A>C, p.Lys629Glu) with recurrent pulmonary haemorrhage due to aortopulmonary collaterals successfully managed with coil embolisation followed by right upper lobectomy.

Structure-Activity Relationship Studies in a Series of Xanthine Inhibitors of SLACK Potassium Channels.

Gain-of-function mutations in the KCNT1 gene, which encodes the sodium-activated potassium channel known as SLACK, are associated with the rare but devastating developmental and epileptic encephalopathy known as epilepsy of infancy with migrating focal seizures (EIMFS). The design of small molecule inhibitors of SLACK channels represents a potential therapeutic approach to the treatment of EIMFS, other childhood epilepsies, and developmental disorders. Herein, we describe a hit optimization effort centered on a xanthine SLACK inhibitor (8) discovered via a high-throughput screen. Across three distinct regions of the chemotype, we synthesized 58 new analogs and tested each one in a whole-cell automated patch-clamp assay to develop structure-activity relationships for inhibition of SLACK channels. We further evaluated selected analogs for their selectivity versus a variety of other ion channels and for their activity versus clinically relevant SLACK mutants. Selectivity within the series was quite good, including versus hERG. Analog 80 (VU0948578) was a potent inhibitor of WT, A934T, and G288S SLACK, with IC50 values between 0.59 and 0.71 µM across these variants. VU0948578 represents a useful in vitro tool compound from a chemotype that is distinct from previously reported small molecule inhibitors of SLACK channels.

KCNT1 Channel Blockers: A Medicinal Chemistry Perspective.

Potassium channels have recently emerged as suitable target for the treatment of epileptic diseases. Among potassium channels, KCNT1 channels are the most widely characterized as responsible for several epileptic and developmental encephalopathies. Nevertheless, the medicinal chemistry of KCNT1 blockers is underdeveloped so far. In the present review, we describe and analyse the papers addressing the issue of KCNT1 blockers' development and identification, also evidencing the pros and the cons of the scientific approaches therein described. After a short introduction describing the epileptic diseases and the structure-function of potassium channels, we provide an extensive overview of the chemotypes described so far as KCNT1 blockers, and the scientific approaches used for their identification.

The Slack Channel Regulates Anxiety-Like Behaviors via Basolateral Amygdala Glutamatergic Projections to Ventral Hippocampus.

Anxiety disorders are a series of mental disorders characterized by anxiety and fear, but the molecular basis of these disorders remains unclear. In the present study, we find that the global Slack KO male mice exhibit anxious behaviors, whereas the Slack Y777H male mice manifest anxiolytic behaviors. The expression of Slack channels is rich in basolateral amygdala (BLA) glutamatergic neurons and downregulated in chronic corticosterone-treated mice. In addition, electrophysiological data show enhanced excitability of BLA glutamatergic neurons in the Slack KO mice and decreased excitability of these neurons in the Slack Y777H mice. Furthermore, the Slack channel deletion in BLA glutamatergic neurons is sufficient to result in enhanced avoidance behaviors, whereas Kcnt1 gene expression in the BLA or BLA-ventral hippocampus (vHPC) glutamatergic projections reverses anxious behaviors of the Slack KO mice. Our study identifies the role of the Slack channel in controlling anxious behaviors by decreasing the excitability of BLA-vHPC glutamatergic projections, providing a potential target for anxiolytic therapies.SIGNIFICANCE STATEMENT Anxiety disorders are a series of mental disorders characterized by anxiety and fear, but the molecular basis of these disorders remains unclear. Here, we examined the behaviors of loss- and gain-of-function of Slack channel mice in elevated plus maze and open field tests and found the anxiolytic role of the Slack channel. By altering the Slack channel expression in the specific neuronal circuit, we demonstrated that the Slack channel played its anxiolytic role by decreasing the excitability of BLA-vHPC glutamatergic projections. Our data reveal the role of the Slack channel in the regulation of anxiety, which may provide a potential molecular target for anxiolytic therapies.

Unusually Slow Spike Frequency Adaptation in Deep Cerebellar Nuclei Neurons Preserves Linear Transformations on the Subsecond Timescale.

Purkinje cells (PCs) are spontaneously active neurons of the cerebellar cortex that inhibit glutamatergic projection neurons within the deep cerebellar nuclei (DCN) that provide the primary cerebellar output. Brief reductions of PC firing rapidly increase DCN neuron firing. However, prolonged reductions of PC inhibition, as seen in some disease states, certain types of transgenic mice, during optogenetic suppression of PC firing, and in acute slices of the cerebellum, do not lead to large, sustained increases in DCN firing. Here we test whether DCN neurons undergo spike frequency adaptation that could account for these properties. We perform current-clamp recordings at near physiological temperature in acute brain slices from mice of both sexes to examine how DCN neurons respond to prolonged depolarizations. DCN neuron adaptation is exceptionally slow and bidirectional. A depolarizing current step evokes large initial increases in firing that decay to ∼20% of the initial increase within ∼10 s. We find that spike frequency adaptation in DCN neurons is mediated by a novel mechanism that is independent of the most promising candidates, including calcium entry and Na+-activated potassium channels mediated by Slo2.1 and Slo2.2 Slow adaptation allows DCN neurons to gradually and bidirectionally adapt to prolonged currents but to respond linearly to current injection on rapid timescales. This suggests that an important consequence of slow adaptation is that DCN neurons respond linearly to the rate of PC firing on rapid timescales but adapt to slow firing rate changes of PCs on long timescales.SIGNIFICANCE STATEMENT Excitatory neurons in the cerebellar nuclei provide the primary output from the cerebellum. This study finds that these neurons exhibit very slow bidirectional spike frequency adaptation that has important implications for cerebellar function. This mechanism allows neurons in the cerebellar nuclei to adapt to long-lasting changes in synaptic drive while also remaining responsive to short-term changes in excitatory or inhibitory drive.

Detection of mosaic variants using genome sequencing in a large pediatric cohort.

The increased use of next-generation sequencing has expanded our understanding of the involvement and prevalence of mosaicism in genetic disorders. We describe a total of eleven cases: nine in which mosaic variants detected by genome sequencing (GS) and/or targeted gene panels (TGPs) were considered to be causative for the proband's phenotype, and two of apparent parental mosaicism. Variants were identified in the following genes: PHACTR1, SCN8A, KCNT1, CDKL5, NEXMIF, CUX1, TSC2, GABRB2, and SMARCB1. In addition, we identified one large duplication including three genes, UBE3A, GABRB3, and MAGEL2, and one large deletion including deletion of ARFGAP1, EEF1A2, CHRNA4, and KCNQ2. All patients were enrolled in the NYCKidSeq study, a research program studying the communication of genomic information in clinical care, as well as the clinical utility and diagnostic yield of GS for children with suspected genetic disorders in diverse populations in New York City. We observed variability in the correlation between reported variant allele fraction and the severity of the patient's phenotype, although we were not able to determine the mosaicism percentage in clinically relevant tissue(s). Although our study was not sufficiently powered to assess differences in mosaicism detection between the two testing modalities, we saw a trend toward better detection by GS as compared with TGP testing. This case series supports the importance of mosaicism in childhood-onset genetic conditions and informs guidelines for laboratory and clinical interpretation of mosaic variants detected by GS.

Functional evaluation of epilepsy-associated KCNT1 variants in multiple cellular systems reveals a predominant gain of function impact on channel properties.

OBJECTIVE: Gain of function variants in the sodium-activated potassium channel KCNT1 have been associated with pediatric epilepsy disorders. Here, we systematically examine a spectrum of KCNT1 variants and establish their impact on channel function in multiple cellular systems. METHODS: KCNT1 variants identified from published reports and genetic screening of pediatric epilepsy patients were expressed in Xenopus oocytes and HEK cell lines. Variant impact on current magnitude, current-voltage relationships, and sodium ion modulation were examined. RESULTS: We determined basic properties of KCNT1 in Xenopus oocyte and HEK systems, including the role of extra- and intracellular sodium in regulating KCNT1 activity. The most common six KCNT1 variants demonstrated strong gain of function (GOF) effects on one or more channel properties. Analysis of 36 total variants identified phenotypic heterogeneity but a strong tendency for pathogenic variants to exert GOF effects on channel properties. By controlling intracellular sodium, we demonstrate that multiple pathogenic KCNT1 variants modulate channel voltage dependence by altering the sensitivity to sodium ions. SIGNIFICANCE: This study represents the largest systematic functional examination of KCNT1 variants to date. We both confirm previously reported GOF channel phenotypes and expand the number of variants with in vitro GOF effects. Our data provide further evidence that novel KCNT1 variants identified in epilepsy patients lead to disease through generalizable GOF mechanisms including increases in current magnitude and/or current-voltage relationships.

In Vitro Effects of Acitretin on Human Neuronal SH-SY5Y Cells.

Acitretin is an oral drug approved by the Food and Drug Administration that is commonly used to treat psoriasis. In recent years, acitretin has been identified as a candidate drug for the treatment of Alzheimer's disease, but its role in neuronal development is still unclear. In this study, the human neuroblastoma cell line SH-SY5Y was used as a model to study neuronal differentiation. We found that acitretin effectively promoted the differentiation of SH-SY5Y cells into neuronal cells and upregulated the expression of the neuronal marker ß-III tubulin and the mature neuronal marker NFH. Differentially expressed genes were identified by RNA sequencing and analyzed by bioinformatics approaches. The results showed that genes associated with neuron development-related pathways, such as SSPO and KCNT1, had significant changes in expression. Analysis showed that PRKCA and CAMK2B may play important roles in the process by which acitretin promotes neurodevelopment. Through whole-cell patch clamping and a microelectrode array assay, we found that acitretin-treated neurons generated electrical spikes similar to those generated by mature neurons. This study provided evidence to support an accessible and safe model of neuron-like cells and verified that acitretin can promote the differentiation of neurons and has the potential to treat brain tumors and neurodevelopmental and neurodegenerative diseases.

Design, synthesis, and biological evaluation of a novel series of 1,2,4-oxadiazole inhibitors of SLACK potassium channels: Identification of in vitro tool VU0935685.

Malignant migrating partial seizure of infancy (MMPSI) is a devastating and pharmacoresistant form of infantile epilepsy. MMPSI has been linked to multiple gain-of-function (GOF) mutations in the KCNT1 gene, which encodes for a potassium channel often referred to as SLACK. SLACK channels are sodium-activated potassium channels distributed throughout the central nervous system (CNS) and the periphery. The investigation described here aims to discover SLACK channel inhibitor tool compounds and profile their pharmacokinetic and pharmacodynamic properties. A SLACK channel inhibitor VU0531245 (VU245) was identified via a high-throughput screen (HTS) campaign. Structure-activity relationship (SAR) studies were conducted in five distinct regions of the hit VU245. VU245 analogs were evaluated for their ability to affect SLACK channel activity using a thallium flux assay in HEK-293 cells stably expressing wild-type (WT) human SLACK. Selected analogs were tested for metabolic stability in mouse liver microsomes and plasma-protein binding in mouse plasma. The same set of analogs was tested via thallium flux for activity versus human A934T SLACK and other structurally related potassium channels, including SLICK and Maxi-K. In addition, potencies for selected VU245 analogs were obtained using whole-cell electrophysiology (EP) assays in CHO cells stably expressing WT human SLACK through an automated patch clamp system. Results revealed that this scaffold tolerates structural changes in some regions, with some analogs demonstrating improved SLACK inhibitory activity, good selectivity against the other channels tested, and modest improvements in metabolic clearance. Analog VU0935685 represents a new, structurally distinct small-molecule inhibitor of SLACK channels that can serve as an in vitro tool for studying this target.

New use for an old drug: quinidine in KCNT1-related epilepsy therapy.

KCNT1 has been known to encode a subunit of the tetrameric sodium activated potassium channel (KNa1.1). Pathogenic variants of KCNT1, especially gain-of-function (GOF) variants, are associated with multiple epileptic disorders which are often refractory to conventional anti-seizure medications and summarized as KCNT1-related epilepsy. Although the detailed pathogenic mechanisms of KCNT1-related epilepsy remain unknown, increasing studies attempt to find effective medications for those patients by utilizing quinidine to inhibit hyperexcitable KNa1.1. However, it has been shown that controversial outcomes among studies and partial success in some individuals may be due to multiple factors, such as poor blood-brain barrier (BBB) penetration, mutation-dependent manner, phenotype-genotype associations, and rational therapeutic schedule. In recent years, with higher resolution of KNa1.1 structure in different activation states and advanced synthetic techniques, it improves the process performance of therapy targeting at KNa1.1 channel to achieve more effective outcomes. Here, we systematically reviewed the study history of quinidine on KCNT1-related epilepsy and its corresponding therapeutic effects. Then, we analyzed and summarized the possible causes behind the different outcomes of the application of quinidine. Finally, we outlooked the recent advances in precision medicine treatment for KCNT1-related epilepsy.

Lhermitte-Duclos disease with concomitant KCNT2 gene mutation: report of an extremely rare combination.

Lhermitte-Duclos disease (LDD) refers to cerebellar dysplastic gangliocytoma, a slow-growing tumor. Pathogenic variants of voltage-gated potassium channels have been associated with epilepsy of variable severity. These include the sodium-activated potassium channel subfamily T member 2 (KCNT2) gene, which encodes for pore-forming alpha subunits. KCNT2 gene mutations have been recently described to cause developmental and epileptic encephalopathies (DEEs). The purpose of the present article is to describe an extremely rare case of a young child who has both LDD and KCNT2 mutation. Our patient is an 11-year-old boy who presented with an absence episode, and his investigations revealed electroencephalography (EEG) abnormalities, LDD, and a heterozygous KCNT2 mutation. Regarding LDD patients, epileptic seizures have been reported in very few cases. Reports of patients with mutated KCNT2 variants are also extremely rare. It is for sure that LDD and KCNT2 mutation is an extremely rare combination. Although further follow-up is mandatory in order to draw safe conclusions for our case, the available data support that our patient is either the first reported case of a subclinical KCNT2 mutation or the first case of its clinical expression in late childhood so far.

Potassium Channel Subfamily T Member 1(KCNT1) Pathological Variant Causing Epilepsy Of Infancy With Migrating Focal Seizures: A Case Report.

Pathological mutation of potassium channel subfamily T member 1 (KCNT1) gene causes an autosomal dominant disorder characterised by secondarily generalised seizures/migratory focal seizure, cyanosis, and dysmorphic features. We report the case of a five-month old male with pathological KCNT1 variant who presented with focal clonic seizures, Mongol spots, and grade two systolic murmur at the left lower sternal border and loud P2. The seizures were refractory to most anti-epileptic drugs but showed some response to Valproic acid. This case demonstrated that EIMFS is a grave infantile epileptic encephalopathy which is refractory to anti epileptic drugs and can present with a wide spectrum of neurogenic and cardiogenic symptoms.

Slo2/KNa Channels in Drosophila Protect against Spontaneous and Induced Seizure-like Behavior Associated with an Increased Persistent Na+ Current.

Na+ sensitivity is a unique feature of Na+-activated K+ (KNa) channels, making them naturally suited to counter a sudden influx in Na+ ions. As such, it has long been suggested that KNa channels may serve a protective function against excessive excitation associated with neuronal injury and disease. This hypothesis, however, has remained largely untested. Here, we examine KNa channels encoded by the Drosophila Slo2 (dSlo2) gene in males and females. We show that dSlo2/KNa channels are selectively expressed in cholinergic neurons in the adult brain, as well as in glutamatergic motor neurons, where dampening excitation may function to inhibit global hyperactivity and seizure-like behavior. Indeed, we show that effects of feeding Drosophila a cholinergic agonist are exacerbated by the loss of dSlo2/KNa channels. Similar to mammalian Slo2/KNa channels, we show that dSlo2/KNa channels encode a TTX-sensitive K+ conductance, indicating that dSlo2/KNa channels can be activated by Na+ carried by voltage-dependent Na+ channels. We then tested the role of dSlo2/KNa channels in established genetic seizure models in which the voltage-dependent persistent Na+ current (INap) is elevated. We show that the absence of dSlo2/KNa channels increased susceptibility to mechanically induced seizure-like behavior. Similar results were observed in WT flies treated with veratridine, an enhancer of INap Finally, we show that loss of dSlo2/KNa channels in both genetic and pharmacologically primed seizure models resulted in the appearance of spontaneous seizures. Together, our results support a model in which dSlo2/KNa channels, activated by neuronal overexcitation, contribute to a protective threshold to suppress the induction of seizure-like activity.SIGNIFICANCE STATEMENT Slo2/KNa channels are unique in that they constitute a repolarizing K+ pore that is activated by the depolarizing Na+ ion, making them naturally suited to function as a protective "brake" against overexcitation and Na+ overload. Here, we test this hypothesis in vivo by examining how a null mutation of the Drosophila Slo2 (dSlo2)/KNa gene affects seizure-like behavior in genetic and pharmacological models of epilepsy. We show that indeed the loss of dSlo2/KNa channels results in increased incidence and severity of induced seizure behavior, as well as the appearance of spontaneous seizure activity. Our results advance our understanding of neuronal excitability and protective mechanisms that preserve normal physiology and the suppression of seizure susceptibility.

KNa1.1 gain-of-function preferentially dampens excitability of murine parvalbumin-positive interneurons.

KCNT1 encodes the sodium-activated potassium channel KNa1.1, expressed preferentially in the frontal cortex, hippocampus, cerebellum, and brainstem. Pathogenic missense variants in KCNT1 are associated with intractable epilepsy, namely epilepsy of infancy with migrating focal seizures (EIMFS), and sleep-related hypermotor epilepsy (SHE). In vitro studies of pathogenic KCNT1 variants support predominantly a gain-of-function molecular mechanism, but how these variants behave in a neuron or ultimately drive formation of an epileptogenic circuit is an important and timely question. Using CRISPR/Cas9 gene editing, we introduced a gain-of-function variant into the endogenous mouse Kcnt1 gene. Compared to wild-type (WT) littermates, heterozygous and homozygous knock-in mice displayed greater seizure susceptibility to the chemoconvulsants kainate and pentylenetetrazole (PTZ), but not to flurothyl. Using acute slice electrophysiology in heterozygous and homozygous Kcnt1 knock-in and WT littermates, we demonstrated that CA1 hippocampal pyramidal neurons exhibit greater amplitude of miniature inhibitory postsynaptic currents in mutant mice with no difference in frequency, suggesting greater inhibitory tone associated with the Kcnt1 mutation. To address alterations in GABAergic signaling, we bred Kcnt1 knock-in mice to a parvalbumin-tdTomato reporter line, and found that parvalbumin-expressing (PV+) interneurons failed to fire repetitively with large amplitude current injections and were more prone to depolarization block. These alterations in firing can be recapitulated by direct application of the KNa1.1 channel activator loxapine in WT but are occluded in knock-in littermates, supporting a direct channel gain-of-function mechanism. Taken together, these results suggest that KNa1.1 gain-of-function dampens interneuron excitability to a greater extent than it impacts pyramidal neuron excitability, driving seizure susceptibility in a mouse model of KCNT1-associated epilepsy.

Slack K+ channels attenuate NMDA-induced excitotoxic brain damage and neuronal cell death.

The neuronal Na+ -activated K+ channel Slack (aka Slo2.2, KNa 1.1, or Kcnt1) has been implicated in setting and maintaining the resting membrane potential and defining excitability and firing patterns, as well as in the generation of the slow afterhyperpolarization following bursts of action potentials. Slack activity increases significantly under conditions of high intracellular Na+ levels, suggesting this channel may exert important pathophysiological functions. To address these putative roles, we studied whether Slack K+ channels contribute to pathological changes and excitotoxic cell death caused by glutamatergic overstimulation of Ca2+ - and Na+ -permeable N-methyl-D-aspartic acid receptors (NMDAR). Slack-deficient (Slack KO) and wild-type (WT) mice were subjected to intrastriatal microinjections of the NMDAR agonist NMDA. NMDA-induced brain lesions were significantly increased in Slack KO vs WT mice, suggesting that the lack of Slack renders neurons particularly susceptible to excitotoxicity. Accordingly, excessive neuronal cell death was seen in Slack-deficient primary cerebellar granule cell (CGC) cultures exposed to glutamate and NMDA. Differences in neuronal survival between WT and Slack KO CGCs were largely abolished by the NMDAR antagonist MK-801, but not by NBQX, a potent and highly selective competitive antagonist of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type ionotropic glutamate receptors. Interestingly, NMDAR-evoked Ca2+ signals did not differ with regard to Slack genotype in CGCs. However, real-time monitoring of K+ following NMDAR activation revealed a significant contribution of this channel to the intracellular drop in K+ . Finally, TrkB and TrkC neurotrophin receptor transcript levels were elevated in NMDA-exposed Slack-proficient CGCs, suggesting a mechanism by which this K+ channel contributes to the activation of the extracellular-signal-regulated kinase (Erk) pathway and thereby to neuroprotection. Combined, our findings suggest that Slack-dependent K+ signals oppose the NMDAR-mediated excitotoxic neuronal injury by promoting pro-survival signaling via the BDNF/TrkB and Erk axis.

Neuroblastoma SH-SY5Y Cell Differentiation to Mature Neuron by AM580 Treatment.

Neuroblastoma is a type of developmental childhood cancer that arises from the neural crest. It is the most common pediatric solid tumor in the world. AM580 is a powerful cyto-differentiating molecule on acute promyelocytic leukemia cells and induced pluripotent stem cells, but its effect on neuroblastoma is still unknown. In this study, the neuronal differentiation impact of AM580 was investigated using the human neuroblastoma cell line SH-SY5Y as a model. AM580 successfully stimulated the SH-SY5Y cells to develop into neuron-like cells. Functional enrichment analysis of RNAseq data revealed that differentially expressed genes (DEGs) were substantially enriched for GO keywords and KEGG pathways linked to neuron development. Some potassium ion channel genes associated with neuronal excitation, such as KCNT1, were shown to be upregulated. Through the MEA tests, we found the AM580-induced neurons possessed electrical spikes as mature neurons. AM580 also induced the neuronal marker ß-tubulin III and mature neurons marker Neurofilament H. Our study proved that AM580 can promote the differentiation of neurons and has the potential to treat neuroblastoma, neurodevelopmental and neurodegenerative diseases.

Precision therapy with quinidine of KCNT1-related epileptic disorders: A systematic review.

AIMS: Despite numerous studies on quinidine therapies for epilepsies associated with KCNT1 gene mutations, there is no consensus on its clinical utility. Thus, we reviewed studies evaluating the efficacy and safety of quinidine in KCNT1-related epileptic disorders. METHODS: Electronic databases were queried for in vivo and in vitro studies on quinidine therapy in KCNT1-related epilepsies published on or before 1 May 2022. The evaluation of evidence was done as per the American Academy of Neurology's classification scheme. Identification of significant factors that possibly influenced therapeutic effects of quinidine were performed using χ2 tests. RESULTS: Twenty-seven studies containing 82 patient records were reviewed. Records of 80 patients with 33 KCNT1 mutations were analysed, of which 20 patients had gained ≥50% seizure reduction due to quinidine therapy. However, quinidine therapy often had different effects on patients with the same KCNT1 mutation. Age, genotypes of KCNT1 mutations, seizure types and brain MRI did not significantly influence the therapeutic effect of quinidine. Prolonged QTc was the most common among all adverse events with quinidine. Notably, results of in vitro quinidine tests did not correspond with in vivo tests. CONCLUSIONS: Therapeutic effects of quinidine on KCNT1-related epilepsies remained indefinite as contradictory results were detected in similar patients. Age, seizure types, genotypes of KCNT1 mutations and brain MRI did not influence the therapeutic effects of quinidine. Insensitivity to quinidine by a certain Kcnt1 genotype in molecular tests is predictive of its inefficacy in human populations of the respective mutation.