Expanded clinical phenotype spectrum correlates with variant function in SCN2A-related disorders.

SCN2A-related disorders secondary to altered function in the voltage-gated sodium channel NaV1.2 are rare with clinically heterogeneous expressions that include epilepsy, autism, and multiple severe to profound impairments and other conditions. To advance understanding of the clinical phenotypes and their relation to channel function, 81 patients (36, 44% female, median age 5.4 years) with 69 unique SCN2A variants were systematically phenotyped and their NaV1.2 channel function systematically assessed. Participants were recruited through the FamileSCN2A Foundation. Primary phenotype (epilepsy of neonatal-onset, N=27; infant onset, N=18; and later onset N=24; and autism without seizures, (N=12) was strongly correlated with a non-seizure severity index (p=0.002), which was based on presence of severe impairments in gross motor, fine motor, communication abilities, gastrostomy tube dependence, and diagnosis of cortical visual impairment and scoliosis. Non-seizure severity was greatest in the neonatal-onset group and least in the autism group (p=0.002). Children with the lowest severity indices were still severely impaired, as reflected by an average Vineland adaptive behavior composite score of 49.5 (>3 SD below the test's norm-referenced mean). Epileptic spasms were significantly more common in infant onset (67%) than in neonatal (22%) or later-onset (29%) epilepsy (p=0.007). Primary phenotype also strongly correlated with variant function (p<0.0001); gain of function and mixed function variants predominated in neonatal-onset epilepsy, shifting to moderate loss of function in infant-onset epilepsy, and severe and complete loss of function in later-onset epilepsy and autism groups. Exploratory cluster analysis identified five groups representing (1) primarily later-onset epilepsy with moderate loss of function variants and low severity indices, (2) mostly infant-onset epilepsy with moderate loss of function variants but higher severity indices, (3) late-onset and autism only with the lowest severity indices (mostly 0) and severe/complete loss of function variants. Two exclusively neonatal clusters were distinguished from each other largely on non-seizure severity scores and secondarily on variant function. The relation between primary phenotype and variant function emphasizes the role of developmental factors in the differential clinical expression of SCN2A variants based on their effects on NaV1.2 channel function. The non-seizure severity of SCN2A disorders depends on a combination of the age at seizure onset (primary phenotype) and variant function. As precision therapies for SCN2A-related disorders advance toward clinical trials, knowledge of the relationship between variant function and clinical disease expression will be valuable for identifying appropriate patients for these trials and in selecting efficient clinical outcomes.

Arrhythmia Variant Associations and Reclassifications in the eMERGE-III Sequencing Study.

BACKGROUND: Sequencing Mendelian arrhythmia genes in individuals without an indication for arrhythmia genetic testing can identify carriers of pathogenic or likely pathogenic (P/LP) variants. However, the extent to which these variants are associated with clinically meaningful phenotypes before or after return of variant results is unclear. In addition, the majority of discovered variants are currently classified as variants of uncertain significance, limiting clinical actionability. METHODS: The eMERGE-III study (Electronic Medical Records and Genomics Phase III) is a multicenter prospective cohort that included 21 846 participants without previous indication for cardiac genetic testing. Participants were sequenced for 109 Mendelian disease genes, including 10 linked to arrhythmia syndromes. Variant carriers were assessed with electronic health record-derived phenotypes and follow-up clinical examination. Selected variants of uncertain significance (n=50) were characterized in vitro with automated electrophysiology experiments in HEK293 cells. RESULTS: As previously reported, 3.0% of participants had P/LP variants in the 109 genes. Herein, we report 120 participants (0.6%) with P/LP arrhythmia variants. Compared with noncarriers, arrhythmia P/LP carriers had a significantly higher burden of arrhythmia phenotypes in their electronic health records. Fifty-four participants had variant results returned. Nineteen of these 54 participants had inherited arrhythmia syndrome diagnoses (primarily long-QT syndrome), and 12 of these 19 diagnoses were made only after variant results were returned (0.05%). After in vitro functional evaluation of 50 variants of uncertain significance, we reclassified 11 variants: 3 to likely benign and 8 to P/LP. CONCLUSIONS: Genome sequencing in a large population without indication for arrhythmia genetic testing identified phenotype-positive carriers of variants in congenital arrhythmia syndrome disease genes. As the genomes of large numbers of people are sequenced, the disease risk from rare variants in arrhythmia genes can be assessed by integrating genomic screening, electronic health record phenotypes, and in vitro functional studies. REGISTRATION: URL: https://www. CLINICALTRIALS: gov; Unique identifier; NCT03394859.

Targeting the Microtubule EB1-CLASP2 Complex Modulates NaV1.5 at Intercalated Discs.

[Figure: see text].

Predicting the functional impact of KCNQ1 variants with artificial neural networks.

Recent advances in experimental and computational protein structure determination have provided access to high-quality structures for most human proteins and mutants thereof. However, linking changes in structure in protein mutants to functional impact remains an active area of method development. If successful, such methods can ultimately assist physicians in taking appropriate treatment decisions. This work presents three artificial neural network (ANN)-based predictive models that classify four key functional parameters of KCNQ1 variants as normal or dysfunctional using PSSM-based evolutionary and/or biophysical descriptors. Recent advances in predicting protein structure and variant properties with artificial intelligence (AI) rely heavily on the availability of evolutionary features and thus fail to directly assess the biophysical underpinnings of a change in structure and/or function. The central goal of this work was to develop an ANN model based on structure and physiochemical properties of KCNQ1 potassium channels that performs comparably or better than algorithms using only on PSSM-based evolutionary features. These biophysical features highlight the structure-function relationships that govern protein stability, function, and regulation. The input sensitivity algorithm incorporates the roles of hydrophobicity, polarizability, and functional densities on key functional parameters of the KCNQ1 channel. Inclusion of the biophysical features outperforms exclusive use of PSSM-based evolutionary features in predicting activation voltage dependence and deactivation time. As AI is increasingly applied to problems in biology, biophysical understanding will be critical with respect to 'explainable AI', i.e., understanding the relation of sequence, structure, and function of proteins. Our model is available at www.kcnq1predict.org.

Disease-linked supertrafficking of a potassium channel.

Gain-of-function (GOF) mutations in the voltage-gated potassium channel subfamily Q member 1 (KCNQ1) can induce cardiac arrhythmia. In this study, it was tested whether any of the known human GOF disease mutations in KCNQ1 act by increasing the amount of KCNQ1 that reaches the cell surface-"supertrafficking." Seven of the 15 GOF mutants tested were seen to surface traffic more efficiently than the WT channel. Among these, we found that the levels of R231C KCNQ1 in the plasma membrane were fivefold higher than the WT channel. This was shown to arise from the combined effects of enhanced efficiency of translocon-mediated membrane integration of the S4 voltage-sensor helix and from enhanced post-translational folding/trafficking related to the energetic linkage of C231 with the V129 and F166 side chains. Whole-cell electrophysiology recordings confirmed that R231C KCNQ1 in complex with the voltage-gated potassium channel-regulatory subfamily E member 1 not only exhibited constitutive conductance but also revealed that the single-channel activity of this mutant is only 20% that of WT. The GOF phenotype associated with R231C therefore reflects the effects of supertrafficking and constitutive channel activation, which together offset reduced channel activity. These investigations show that membrane protein supertrafficking can contribute to human disease.

Peripheral myelin protein 22 modulates store-operated calcium channel activity, providing insights into Charcot-Marie-Tooth disease etiology.

Charcot-Marie-Tooth (CMT) disease is a peripheral neuropathy associated with gene duplication and point mutations in the peripheral myelin protein 22 (PMP22) gene. However, the role of PMP22 in Schwann cell physiology and the mechanisms by which PMP22 mutations cause CMT are not well-understood. On the basis of homology between PMP22 and proteins associated with modulation of ion channels, we hypothesized that PMP22 alters ion channel activity. Using whole-cell electrophysiology, we show here that heterologous PMP22 expression increases the amplitude of currents similar to those ascribed to store-operated calcium (SOC) channels, particularly those involving transient receptor canonical channel 1 (TrpC1). These channels help replenish Ca2+ in the endoplasmic reticulum (ER) following stimulus-induced depletion. Currents with similar properties were recorded in WT but not pmp22-/- mouse Schwann cells. Heterologous expression of the CMT-associated PMP22_L16P variant, which fails to reach the plasma membrane and localizes to the ER, led to larger currents than WT PMP22. Similarly, Schwann cells isolated from Trembler J (TrJ; PMP22_L16P) mice had larger currents than WT littermates. Calcium imaging in live nerves and cultured Schwann cells revealed elevated intracellular Ca2+ in TrJ mice compared with WT. Moreover, we found that PMP22 co-immunoprecipitated with stromal interaction molecule 1 (STIM1), the Ca2+ sensor SOC channel subunit in the ER. These results suggest that in the ER, PMP22 interacts with STIM1 and increases Ca2+ influx through SOC channels. Excess or mutant PMP22 in the ER may elevate intracellular Ca2+ levels, which could contribute to CMT pathology.

Spectrum of KV 2.1 Dysfunction in KCNB1-Associated Neurodevelopmental Disorders.

OBJECTIVE: Pathogenic variants in KCNB1, encoding the voltage-gated potassium channel KV 2.1, are associated with developmental and epileptic encephalopathy (DEE). Previous functional studies on a limited number of KCNB1 variants indicated a range of molecular mechanisms by which variants affect channel function, including loss of voltage sensitivity, loss of ion selectivity, and reduced cell-surface expression. METHODS: We evaluated a series of 17 KCNB1 variants associated with DEE or other neurodevelopmental disorders (NDDs) to rapidly ascertain channel dysfunction using high-throughput functional assays. Specifically, we investigated the biophysical properties and cell-surface expression of variant KV 2.1 channels expressed in heterologous cells using high-throughput automated electrophysiology and immunocytochemistry-flow cytometry. RESULTS: Pathogenic variants exhibited diverse functional defects, including altered current density and shifts in the voltage dependence of activation and/or inactivation, as homotetramers or when coexpressed with wild-type KV 2.1. Quantification of protein expression also identified variants with reduced total KV 2.1 expression or deficient cell-surface expression. INTERPRETATION: Our study establishes a platform for rapid screening of KV 2.1 functional defects caused by KCNB1 variants associated with DEE and other NDDs. This will aid in establishing KCNB1 variant pathogenicity and the mechanism of dysfunction, which will enable targeted strategies for therapeutic intervention based on molecular phenotype. ANN NEUROL 2019;86:899-912.

Long QT syndrome KCNH2 mutation with sequential fetal and maternal sudden death.

We report a case of a woman who experienced intrauterine fetal death at full term pregnancy, and then died suddenly soon after learning about the death of her fetus. At autopsy, previously undiagnosed neurofibromatosis and an adrenal gland pheochromocytoma were discovered in the mother. Genetic screening also revealed a novel KCNH2mutation in both fetus and mother indicating type 2 congenital long-QT syndrome (LQTS). A catecholamine surge was suspected as the precipitating event of fetal cardiac arrhythmia and sudden fetal death, while the addition of emotional stress provoked a lethal cardiac event in the mother. This case illustrates the potential for lethal interactions between two occult diseases (pheochromocytoma, LQTS).

SCN3A deficiency associated with increased seizure susceptibility.

Mutations in voltage-gated sodium channels expressed highly in the brain (SCN1A, SCN2A, SCN3A, and SCN8A) are responsible for an increasing number of epilepsy syndromes. In particular, mutations in the SCN3A gene, encoding the pore-forming Nav1.3 α subunit, have been identified in patients with focal epilepsy. Biophysical characterization of epilepsy-associated SCN3A variants suggests that both gain- and loss-of-function SCN3A mutations may lead to increased seizure susceptibility. In this report, we identified a novel SCN3A variant (L247P) by whole exome sequencing of a child with focal epilepsy, developmental delay, and autonomic nervous system dysfunction. Voltage clamp analysis showed no detectable sodium current in a heterologous expression system expressing the SCN3A-L247P variant. Furthermore, cell surface biotinylation demonstrated a reduction in the amount of SCN3A-L247P at the cell surface, suggesting the SCN3A-L247P variant is a trafficking-deficient mutant. To further explore the possible clinical consequences of reduced SCN3A activity, we investigated the effect of a hypomorphic Scn3a allele (Scn3aHyp) on seizure susceptibility and behavior using a gene trap mouse line. Heterozygous Scn3a mutant mice (Scn3a+/Hyp) did not exhibit spontaneous seizures nor were they susceptible to hyperthermia-induced seizures. However, they displayed increased susceptibility to electroconvulsive (6Hz) and chemiconvulsive (flurothyl and kainic acid) induced seizures. Scn3a+/Hyp mice also exhibited deficits in locomotor activity and motor learning. Taken together, these results provide evidence that loss-of-function of SCN3A caused by reduced protein expression or deficient trafficking to the plasma membrane may contribute to increased seizure susceptibility.

Allelic Complexity in Long QT Syndrome: A Family-Case Study.

Congenital long QT syndrome (LQTS) is associated with high genetic and allelic heterogeneity. In some cases, more than one genetic variant is identified in the same (compound heterozygosity) or different (digenic heterozygosity) genes, and subjects with multiple pathogenic mutations may have a more severe disease. Standard-of-care clinical genetic testing for this and other arrhythmia susceptibility syndromes improves the identification of complex genotypes. Therefore, it is important to distinguish between pathogenic mutations and benign rare variants. We identified four genetic variants (KCNQ1-p.R583H, KCNH2-p.C108Y, KCNH2-p.K897T, and KCNE1-p.G38S) in an LQTS family. On the basis of in silico analysis, clinical data from our family, and the evidence from previous studies, we analyzed two mutated channels, KCNQ1-p.R583H and KCNH2-p.C108Y, using the whole-cell patch clamp technique. We found that KCNQ1-p.R583H was not associated with a severe functional impairment, whereas KCNH2-p.C108Y, a novel variant, encoded a non-functional channel that exerts dominant-negative effects on the wild-type. Notably, the common variants KCNH2-p.K897T and KCNE1-p.G38S were previously reported to produce more severe phenotypes when combined with disease-causing alleles. Our results indicate that the novel KCNH2-C108Y variant can be a pathogenic LQTS mutation, whereas KCNQ1-p.R583H, KCNH2-p.K897T, and KCNE1-p.G38S could be LQTS modifiers.

Reactivation of Lysosomal Ca2+ Efflux Rescues Abnormal Lysosomal Storage in FIG4-Deficient Cells.

Loss of function of FIG4 leads to Charcot-Marie-Tooth disease Type 4J, Yunis-Varon syndrome, or an epilepsy syndrome. FIG4 is a phosphatase with its catalytic specificity toward 5'-phosphate of phosphatidylinositol-3,5-diphosphate (PI3,5P2). However, the loss of FIG4 decreases PI3,5P2 levels likely due to FIG4's dominant effect in scaffolding a PI3,5P2 synthetic protein complex. At the cellular level, all these diseases share similar pathology with abnormal lysosomal storage and neuronal degeneration. Mice with no FIG4 expression (Fig4(-/-)) recapitulate the pathology in humans with FIG4 deficiency. Using a flow cytometry technique that rapidly quantifies lysosome sizes, we detected an impaired lysosomal fission, but normal fusion, in Fig4(-/-) cells. The fission defect was associated with a robust increase of intralysosomal Ca(2+) in Fig4(-/-) cells, including FIG4-deficient neurons. This finding was consistent with a suppressed Ca(2+) efflux of lysosomes because the endogenous ligand of lysosomal Ca(2+) channel TRPML1 is PI3,5P2 that is deficient in Fig4(-/-) cells. We reactivated the TRPML1 channels by application of TRPML1 synthetic ligand, ML-SA1. This treatment reduced the intralysosomal Ca(2+) level and rescued abnormal lysosomal storage in Fig4(-/-) culture cells and ex vivo DRGs. Furthermore, we found that the suppressed Ca(2+) efflux in Fig4(-/-) culture cells and Fig4(-/-) mouse brains profoundly downregulated the expression/activity of dynamin-1, a GTPase known to scissor organelle membranes during fission. This downregulation made dynamin-1 unavailable for lysosomal fission. Together, our study revealed a novel mechanism explaining abnormal lysosomal storage in FIG4 deficiency. Synthetic ligands of the TRPML1 may become a potential therapy against diseases with FIG4 deficiency.

Use-Dependent Block of Human Cardiac Sodium Channels by GS967.

GS-458967, 6-(4-(Trifluoromethoxy)phenyl)-3-(trifluoromethyl)-[1,2,4]triazolo[4,3-a]pyridine (GS967) is a recently described, novel, sodium channel inhibitor exhibiting potent antiarrhythmic effects in various in vitro and in vivo models. The antiarrhythmic mechanism has been attributed to preferential suppression of late sodium current. However, there has been no reported systematic investigation of the effects of this compound on isolated sodium channels. Here, we examined the effects of GS967 on peak (INaP) and late (INaL) sodium current recorded from cells that heterologously expressed human cardiac voltage-gated sodium channel, the principle cardiac sodium channel. As previously described, we observed that GS967 exerted tonic block of INaL (63%) to a significantly greater extent than INaP (19%). However, GS967 also caused a reduction of INaP in a frequency-dependent manner, consistent with use-dependent block (UDB). GS967 evoked more potent UDB of INaP (IC50 = 0.07 µM) than ranolazine (16 µM) and lidocaine (17 µM). Use-dependent block was best explained by a significant slowing of recovery from fast and slow inactivation with a significant enhancement of slow inactivation in the presence of GS967. Furthermore, GS967 was found to exert these same effects on a prototypical long QT syndrome mutation (delKPQ). An engineered mutation at an interaction site for local anesthetic agents (F1760A) partially attenuated the effect of GS967 on UDB, but had no effect on tonic INaL block. We conclude that GS967 is a preferential inhibitor of INaL, but it also exerts previously unreported strong effects on slow inactivation and recovery from inactivation, resulting in substantial UDB that is not entirely dependent on a known interaction site for local anesthetic agents.

Personalized biochemistry and biophysics.

Whole human genome sequencing of individuals is becoming rapid and inexpensive, enabling new strategies for using personal genome information to help diagnose, treat, and even prevent human disorders for which genetic variations are causative or are known to be risk factors. Many of the exploding number of newly discovered genetic variations alter the structure, function, dynamics, stability, and/or interactions of specific proteins and RNA molecules. Accordingly, there are a host of opportunities for biochemists and biophysicists to participate in (1) developing tools to allow accurate and sometimes medically actionable assessment of the potential pathogenicity of individual variations and (2) establishing the mechanistic linkage between pathogenic variations and their physiological consequences, providing a rational basis for treatment or preventive care. In this review, we provide an overview of these opportunities and their associated challenges in light of the current status of genomic science and personalized medicine, the latter often termed precision medicine.

De novo KCNB1 mutations in epileptic encephalopathy.

OBJECTIVE: Numerous studies have demonstrated increased load of de novo copy number variants or single nucleotide variants in individuals with neurodevelopmental disorders, including epileptic encephalopathies, intellectual disability, and autism. METHODS: We searched for de novo mutations in a family quartet with a sporadic case of epileptic encephalopathy with no known etiology to determine the underlying cause using high-coverage whole exome sequencing (WES) and lower-coverage whole genome sequencing. Mutations in additional patients were identified by WES. The effect of mutations on protein function was assessed in a heterologous expression system. RESULTS: We identified a de novo missense mutation in KCNB1 that encodes the KV 2.1 voltage-gated potassium channel. Functional studies demonstrated a deleterious effect of the mutation on KV 2.1 function leading to a loss of ion selectivity and gain of a depolarizing inward cation conductance. Subsequently, we identified 2 additional patients with epileptic encephalopathy and de novo KCNB1 missense mutations that cause a similar pattern of KV 2.1 dysfunction. INTERPRETATION: Our genetic and functional evidence demonstrate that KCNB1 mutation can result in early onset epileptic encephalopathy. This expands the locus heterogeneity associated with epileptic encephalopathies and suggests that clinical WES may be useful for diagnosis of epileptic encephalopathies of unknown etiology.

Purification and structural study of the voltage-sensor domain of the human KCNQ1 potassium ion channel.

KCNQ1 (also known as KV7.1 or KVLQT1) is a voltage-gated potassium channel modulated by members of the KCNE protein family. Among multiple functions, KCNQ1 plays a critical role in the cardiac action potential. This channel is also subject to inherited mutations that cause certain cardiac arrhythmias and deafness. In this study, we report the overexpression, purification, and preliminary structural characterization of the voltage-sensor domain (VSD) of human KCNQ1 (Q1-VSD). Q1-VSD was expressed in Escherichia coli and purified into lyso-palmitoylphosphatidylglycerol micelles, conditions under which this tetraspan membrane protein yields excellent nuclear magnetic resonance (NMR) spectra. NMR studies reveal that Q1-VSD shares a common overall topology with other channel VSDs, with an S0 helix followed by transmembrane helices S1-S4. The exact sequential locations of the helical spans do, however, show significant variations from those of the homologous segments of previously characterized VSDs. The S4 segment of Q1-VSD was seen to be α-helical (with no 310 component) and underwent rapid backbone amide H-D exchange over most of its length. These results lay the foundation for more advanced structural studies and can be used to generate testable hypotheses for future structure-function experiments.

Novel SCN3A variants associated with focal epilepsy in children.

Voltage-gated sodium (NaV) channels are essential for initiating and propagating action potentials in the brain. More than 800 mutations in genes encoding neuronal NaV channels including SCN1A and SCN2A have been associated with human epilepsy. Only one epilepsy-associated mutation has been identified in SCN3A encoding the NaV1.3 neuronal sodium channel. We performed a genetic screen of pediatric patients with focal epilepsy of unknown cause and identified four novel SCN3A missense variants: R357Q, D766N, E1111K and M1323V. We determined the functional consequences of these variants along with the previously reported K354Q mutation using heterologously expressed human NaV1.3. Functional defects were heterogeneous among the variants. The most severely affected was R357Q, which had a significantly smaller current density and slower activation than the wild-type (WT) channel as well as depolarized voltage dependences of activation and inactivation. Also notable was E1111K, which evoked a significantly greater level of persistent sodium current than WT channels. Interestingly, a common feature shared by all variant channels was increased current activation in response to depolarizing voltage ramps revealing a functional property consistent with conferring neuronal hyper-excitability. Discovery of a common biophysical defect among variants identified in unrelated pediatric epilepsy patients suggests that SCN3A may contribute to neuronal hyperexcitability and epilepsy.

Strain- and age-dependent hippocampal neuron sodium currents correlate with epilepsy severity in Dravet syndrome mice.

Heterozygous loss-of-function SCN1A mutations cause Dravet syndrome, an epileptic encephalopathy of infancy that exhibits variable clinical severity. We utilized a heterozygous Scn1a knockout (Scn1a(+/-)) mouse model of Dravet syndrome to investigate the basis for phenotype variability. These animals exhibit strain-dependent seizure severity and survival. Scn1a(+/-) mice on strain 129S6/SvEvTac (129.Scn1a(+/-)) have no overt phenotype and normal survival compared with Scn1a(+/-) mice bred to C57BL/6J (F1.Scn1a(+/-)) that have severe epilepsy and premature lethality. We tested the hypothesis that strain differences in sodium current (INa) density in hippocampal neurons contribute to these divergent phenotypes. Whole-cell voltage-clamp recording was performed on acutely-dissociated hippocampal neurons from postnatal days 21-24 (P21-24) 129.Scn1a(+/-) or F1.Scn1a(+/-) mice and wild-type littermates. INa density was lower in GABAergic interneurons from F1.Scn1a(+/-) mice compared to wild-type littermates, while on the 129 strain there was no difference in GABAergic interneuron INa density between 129.Scn1a(+/-) mice and wild-type littermate controls. By contrast, INa density was elevated in pyramidal neurons from both 129.Scn1a(+/-) and F1.Scn1a(+/-) mice, and was correlated with more frequent spontaneous action potential firing in these neurons, as well as more sustained firing in F1.Scn1a(+/-) neurons. We also observed age-dependent differences in pyramidal neuron INa density between wild-type and Scn1a(+/-) animals. We conclude that preserved INa density in GABAergic interneurons contributes to the milder phenotype of 129.Scn1a(+/-) mice. Furthermore, elevated INa density in excitatory pyramidal neurons at P21-24 correlates with age-dependent onset of lethality in F1.Scn1a(+/-) mice. Our findings illustrate differences in hippocampal neurons that may underlie strain- and age-dependent phenotype severity in a Dravet syndrome mouse model, and emphasize a contribution of pyramidal neuron excitability.

Antiepileptic activity of preferential inhibitors of persistent sodium current.

OBJECTIVE: Evidence from basic neurophysiology and molecular genetics has implicated persistent sodium current conducted by voltage-gated sodium (NaV ) channels as a contributor to the pathogenesis of epilepsy. Many antiepileptic drugs target NaV channels and modulate neuronal excitability, mainly by a use-dependent block of transient sodium current, although suppression of persistent current may also contribute to the efficacy of these drugs. We hypothesized that a drug or compound capable of preferential inhibition of persistent sodium current would have antiepileptic activity. METHODS: We examined the antiepileptic activity of two selective persistent sodium current blockers ranolazine, a U.S. Food and Drug Administration (FDA)-approved drug for treatment of angina pectoris, and GS967, a novel compound with more potent effects on persistent current, in the epileptic Scn2a(Q54) mouse model. We also examined the effect of GS967 in the maximal electroshock model and evaluated effects of the compound on neuronal excitability, propensity for hilar neuron loss, development of mossy fiber sprouting, and survival of Scn2a(Q54) mice. RESULTS: We found that ranolazine was capable of reducing seizure frequency by approximately 50% in Scn2a(Q54) mice. The more potent persistent current blocker GS967 reduced seizure frequency by >90% in Scn2a(Q54) mice and protected against induced seizures in the maximal electroshock model. GS967 greatly attenuated abnormal spontaneous action potential firing in pyramidal neurons acutely isolated from Scn2a(Q54) mice. In addition to seizure suppression in vivo, GS967 treatment greatly improved the survival of Scn2a(Q54) mice, prevented hilar neuron loss, and suppressed the development of hippocampal mossy fiber sprouting. SIGNIFICANCE: Our findings indicate that the selective persistent sodium current blocker GS967 has potent antiepileptic activity and that this compound could inform development of new agents.

Voltage-gated potassium channel KCNV2 (Kv8.2) contributes to epilepsy susceptibility.

Mutations in voltage-gated ion channels are responsible for several types of epilepsy. Genetic epilepsies often exhibit variable severity in individuals with the same mutation, which may be due to variation in genetic modifiers. The Scn2a(Q54) transgenic mouse model has a sodium channel mutation and exhibits epilepsy with strain-dependent severity. We previously mapped modifier loci that influence Scn2a(Q54) phenotype severity and identified Kcnv2, encoding the voltage-gated potassium channel subunit Kv8.2, as a candidate modifier. In this study, we demonstrate a threefold increase in hippocampal Kcnv2 expression associated with more severe epilepsy. In vivo exacerbation of the phenotype by Kcnv2 transgenes supports its identification as an epilepsy modifier. The contribution of KCNV2 to human epilepsy susceptibility is supported by identification of two nonsynonymous variants in epilepsy patients that alter function of Kv2.1/Kv8.2 heterotetrameric potassium channels. Our results demonstrate that altered potassium subunit function influences epilepsy susceptibility and implicate Kcnv2 as an epilepsy gene.

Engineered cocultures of iPSC-derived atrial cardiomyocytes and atrial fibroblasts for modeling atrial fibrillation.

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia treatable with antiarrhythmic drugs; however, patient responses remain highly variable. Human induced pluripotent stem cell-derived atrial cardiomyocytes (iPSC-aCMs) are useful for discovering precision therapeutics, but current platforms yield phenotypically immature cells and are not easily scalable for high-throughput screening. Here, primary adult atrial, but not ventricular, fibroblasts induced greater functional iPSC-aCM maturation, partly through connexin-40 and ephrin-B1 signaling. We developed a protein patterning process within multiwell plates to engineer patterned iPSC-aCM and atrial fibroblast coculture (PC) that significantly enhanced iPSC-aCM structural, electrical, contractile, and metabolic maturation for 6+ weeks compared to conventional mono-/coculture. PC displayed greater sensitivity for detecting drug efficacy than monoculture and enabled the modeling and pharmacological or gene editing treatment of an AF-like electrophysiological phenotype due to a mutated sodium channel. Overall, PC is useful for elucidating cell signaling in the atria, drug screening, and modeling AF.