Altered Function of the SCN1A Voltage-gated Sodium Channel Leads to ��-Aminobutyric Acid-ergic (GABAergic) Interneuron Abnormalities

Voltage-gated sodium channels are required for the initiation and propagation of action potentials. Mutations in the neuronal voltage-gated sodium channel SCN1A are associated with a growing number of disorders including generalized epilepsy with febrile seizures plus (GEFS+),⁷ severe myoclonic epilepsy of infancy, and familial hemiplegic migraine. To gain insight into the effect of SCN1A mutations on neuronal excitability, we introduced the human GEFS+ mutation SCN1A-R1648H into the orthologous mouse gene. Scn1a^RH/RH mice homozygous for the R1648H mutation exhibit spontaneous generalized seizures and premature death between P16 and P26, whereas Scn1a^RH/+ heterozygous mice exhibit infrequent spontaneous generalized seizures, reduced threshold and accelerated propagation of febrile seizures, and decreased threshold to flurothyl-induced seizures. Inhibitory cortical interneurons from P5-P15 Scn1a^RH/+ and Scn1a^RH/RH mice demonstrated slower recovery from inactivation, greater use-dependent inactivation, and reduced action potential firing compared with wild-type cells. Excitatory cortical pyramidal neurons were mostly unaffected. These results suggest that this SCN1A mutation predominantly impairs sodium channel activity in interneurons, leading to decreased inhibition. Decreased inhibition may be a common mechanism underlying clinically distinct SCN1A-derived disorders.     

Mapping genetic modifiers of survival in a mouse model of Dravet syndrome

Epilepsy is a common neurological disorder affecting approximately 1% of the population. Mutations in voltage‐gated sodium channels are responsible for several monogenic epilepsy syndromes. More than 800 mutations in the voltage‐gated sodium channel SCN1A have been reported in patients with generalized epilepsy with febrile seizures plus and Dravet syndrome. Heterozygous loss‐of‐function mutations in SCN1A result in Dravet syndrome, a severe infant‐onset epileptic encephalopathy characterized by intractable seizures, developmental delays and increased mortality. A common feature of monogenic epilepsies is variable expressivity among individuals with the same mutation, suggesting that genetic modifiers may influence clinical severity. Mice with heterozygous deletion of Scn1a (Scn1a^+/?) model a number of Dravet syndrome features, including spontaneous seizures and premature lethality. Phenotype severity in Scn1a^+/? mice is strongly dependent on strain background. On the 129S6/SvEvTac strain Scn1a^+/? mice exhibit no overt phenotype, whereas on the (C57BL/6J × 129S6/SvEvTac)F1 strain Scn1a^+/? mice exhibit spontaneous seizures and early lethality. To systematically identify loci that influence premature lethality in Scn1a^+/? mice, we performed genome scans on reciprocal backcrosses. Quantitative trait locus mapping revealed modifier loci on mouse chromosomes 5, 7, 8 and 11. RNA‐seq analysis of strain‐dependent gene expression, regulation and coding sequence variation provided a list of potential functional candidate genes at each locus. Identification of modifier genes that influence survival in Scn1a^+/? mice will improve our understanding of the pathophysiology of Dravet syndrome and may suggest novel therapeutic strategies for improved treatment of human patients.     

Mutations in the Scn8a DIIS4 voltage sensor reveal new distinctions among hypomorphic and null Nav1.6 sodium channels

Mutations in the voltage‐gated sodium channel gene SCN8A cause a broad range of human diseases, including epilepsy, intellectual disability, and ataxia. Here we describe three mouse lines on the C57BL/6J background with novel, overlapping mutations in the Scn8a DIIS4 voltage sensor: an in‐frame 9 bp deletion (Δ9), an in‐frame 3 bp insertion (?3) and a 35 bp deletion that results in a frameshift and the generation of a null allele (Δ35). Scn8a ^Δ9/+ and Scn8a ^?3/+ heterozygous mutants display subtle motor deficits, reduced acoustic startle response, and are resistant to induced seizures, suggesting that these mutations reduce activity of the Scn8a channel protein, Na_v1.6. Heterozygous Scn8a ^Δ35/+ mutants show no alterations in motor function or acoustic startle response, but are resistant to induced seizures. Homozygous mutants from each line exhibit premature lethality and severe motor impairments, ranging from uncoordinated gait with tremor (Δ9 and ?3) to loss of hindlimb control (Δ35). Scn8a ^Δ9/Δ9 and Scn8a ^?3/?3 homozygous mutants also exhibit impaired nerve conduction velocity, while normal nerve conduction was observed in Scn8a ^Δ35/Δ35 homozygous mice. Our results suggest that hypomorphic mutations that reduce Na_v1.6 activity will likely result in different clinical phenotypes compared to null alleles. These three mouse lines represent a valuable opportunity to examine the phenotypic impacts of hypomorphic and null Scn8a mutations without the confound of strain‐specific differences.     

Genetic modifiers affecting severity of epilepsy caused by mutation of sodium channelScn2a

Mutations in the voltage-gated sodium channels SCN1A and SCN2A are responsible for several types of human epilepsy. Variable expressivity among family members is a common feature of these inherited epilepsies, suggesting that genetic modifiers may influence the clinical manifestation of epilepsy. The transgenic mouse model Scn2a^Q54 has an epilepsy phenotype as a result of a mutation in Scn2a that slows channel inactivation. The mice display progressive epilepsy that begins with short-duration partial seizures that appear to originate in the hippocampus. The partial seizures become more frequent and of longer duration with age and often induce secondary generalized seizures. Clinical severity of the Scn2a^Q54 phenotype is influenced by genetic background. Congenic C57BL/6J.Q54 mice exhibit decreased incidence of spontaneous seizures, delayed seizure onset, and longer survival in comparison with [C57BL/6J × SJL/J]F₁.Q54 mice. This observation indicates that strain SJL/J carries dominant modifier alleles at one or more loci that determine the severity of the epilepsy phenotype. Genome-wide interval mapping in an N₂ backcross revealed two modifier loci on Chromosomes 11 and 19 that influence the clinical severity of of this sodium channel-induced epilepsy. Modifier genes affecting clinical severity in the Scn2a^Q54 mouse model may contribute to the variable expressivity seen in epilepsy patients with sodium channel mutations.     

Dysfunction of the Scn8a Voltage-gated Sodium Channel Alters Sleep Architecture,Reduces Diurnal Corticosterone Levels,and Enhances Spatial Memory

Voltage-gated sodium channels (VGSCs) are responsible for the initiation and propagation of transient depolarizing currents and play a critical role in the electrical signaling between neurons. A null mutation in the VGSC gene SCN8A, which encodes the transmembrane protein Na_v1.6, was identified previously in a human family. Heterozygous mutation carriers displayed a range of phenotypes, including ataxia, cognitive deficits, and emotional instability. A possible role for SCN8A was also proposed in studies examining the genetic basis of attempted suicide and bipolar disorder. In addition, mice with a Scn8a loss-of-function mutation (Scn8a^med-Tg/+) show altered anxiety and depression-like phenotypes. Because psychiatric abnormalities are often associated with altered sleep and hormonal patterns, we evaluated heterozygous Scn8a^med-jo/+ mutants for alterations in sleep-wake architecture, diurnal corticosterone levels, and behavior. Compared with their wild-type littermates, Scn8a^med-jo/+ mutants experience more non-rapid eye movement (non-REM) sleep, a chronic impairment of REM sleep generation and quantity, and a lowered and flattened diurnal rhythm of corticosterone levels. No robust differences were observed between mutants and wild-type littermates in locomotor activity or in behavioral paradigms that evaluate anxiety or depression-like phenotypes; however, Scn8a^med-jo/+ mutants did show enhanced spatial memory. This study extends the spectrum of phenotypes associated with mutations in Scn8a and suggests a novel role for altered sodium channel function in human sleep disorders.     

Generation and basic characterization of a gene-trap knockout mouse model of Scn2a with a substantial reduction of voltage-gated sodium channel Nav1.2 expression

Large-scale genetic studies revealed SCN2A as one of the most frequently mutated genes in patients with neurodevelopmental disorders. SCN2A encodes for the voltage-gated sodium channel isoform 1.2 (Na_v1.2) expressed in the neurons of the central nervous system. Homozygous knockout (null) of Scn2a in mice is perinatal lethal, whereas heterozygous knockout of Scn2a (Scn2a^+/−) results in mild behavior abnormalities. The Na_v1.2 expression level in Scn2a^+/− mice is reported to be around 50–60% of the wild-type (WT) level, which indicates that a close to 50% reduction of Na_v1.2 expression may not be sufficient to lead to major behavioral phenotypes in mice. To overcome this barrier, we characterized a novel mouse model of severe Scn2a deficiency using a targeted gene-trap knockout (gtKO) strategy. This approach produces viable homozygous mice (Scn2a^gtKO/gtKO) that can survive to adulthood, with about a quarter of Na_v1.2 expression compared to WT mice. Innate behaviors like nesting and mating were profoundly disrupted in Scn2a^gtKO/gtKO mice. Notably, Scn2a^gtKO/gtKO mice have a significantly decreased center duration compared to WT in the open field test, suggesting anxiety-like behaviors in a novel, open space. These mice also have decreased thermal and cold tolerance. Additionally, Scn2a^gtKO/gtKO mice have increased fix-pattern exploration in the novel object exploration test and a slight increase in grooming, indicating a detectable level of repetitive behaviors. They bury little to no marbles and have decreased interaction with novel objects. These Scn2a gene-trap knockout mice thus provide a unique model to study pathophysiology associated with severe Scn2a deficiency.     

Genome-wide linkage of febrile seizures and epilepsy to the FEB4 locus at 5q14.3-q23.1 and no MASS1 mutation

Febrile seizures (FS) represent the most common seizure disorder in childhood and contribution of a genetic predisposition has been clearly proven. In some families FS is associated with a wide variety of afebrile seizures. Generalized epilepsy with febrile seizures plus (GEFS+) is a familial epilepsy syndrome with a spectrum of phenotypes including FS, atypical febrile seizures (FS+) and afebrile generalized and partial seizures. Mutations in the genes SCN1B, SCN1A and GABRG2 were identified in GEFS+ families. GEFS+ is genetically heterogeneous and mutations in these three genes were detected in only a minority of the families. We performed a 10 cM density genome-wide scan in a multigenerational family with febrile seizures and epilepsy and obtained a maximal multipoint LOD score of 3.12 with markers on chromosome 5q14.3-q23.1. Fine mapping and segregation analysis defined a genetic interval of ≈33 cM between D5S2103 and D5S1975. This candidate region overlapped with a previously reported locus for febrile seizures (FEB4) in the Japanese population, in which MASS1 was proposed as disease gene. Mutation analysis of the exons and exon–intron boundaries of MASS1 in our family did not reveal a disease causing mutation. Our linkage data confirm for the first time that a locus on chromosome 5q14-q23 plays a role in idiopathic epilepsies. However, our mutation data is negative and do not support a role for MASS1 suggesting that another gene within or near the FEB4 locus might exist.     

Scn1a missense mutation impairs GABAA receptor-mediated synaptic transmission in the rat hippocampus

Mutations of the Na_v1.1 channel subunit SCN1A have been implicated in the pathogenesis of human febrile seizures (FS). We have recently developed hyperthermia-induced seizure-susceptible (Hiss) rat, a novel rat model of FS, which carries a missense mutation (N1417H) in Scn1a[1]. Here, we conducted electrophysiological studies to clarify the influences of the Scn1a mutation on the hippocampal synaptic transmission, specifically focusing on the GABAergic system. Hippocampal slices were prepared from Hiss or F344 (control) rats and maintained in artificial cerebrospinal fluid saturated with 95% O₂ and 5% CO₂in vitro. Single neuron activity was recorded from CA1 pyramidal neurons and their responses to the test (unconditioned) or paired pulse (PP) stimulation of the Schaffer collateral/commissural fibers were evaluated. Hiss rats were first tested for pentylenetetrazole-induced seizures and confirmed to show high seizure susceptibility to the blockade of GAGA_A receptors. The Scn1a mutation in Hiss rats did not directly affect spike generation (i.e., number of evoked spikes and firing threshold) of the CA1 pyramidal neurons elicited by the Schaffer collateral/commissural stimulation. However, GABA_A receptor-mediated inhibition of pyramidal neurons by the PP stimulation was significantly disrupted in Hiss rats, yielding a significant increase in the number of PP-induced firings at PP intervals of 32-256 ms. The present study shows that the Scn1a missense mutation preferentially impairs GABA_A receptor-mediated synaptic transmission without directly altering the excitability of the pyramidal neurons in the hippocampus, which may be linked to the pathogenesis of FS.     

Variable Nav1.5 Protein Expression from the Wild-Type Allele Correlates with the Penetrance of Cardiac Conduction Disease in the Scn5a +/? Mouse Model

Background

Loss-of-function mutations in SCN5A, the gene encoding Na_v1.5 Na⁺ channel, are associated with inherited cardiac conduction defects and Brugada syndrome, which both exhibit variable phenotypic penetrance of conduction defects. We investigated the mechanisms of this heterogeneity in a mouse model with heterozygous targeted disruption of Scn5a (Scn5a ^+/− mice) and compared our results to those obtained in patients with loss-of-function mutations in SCN5A.

Methodology/Principal Findings

Based on ECG, 10-week-old Scn5a ^+/− mice were divided into 2 subgroups, one displaying severe ventricular conduction defects (QRS interval>18 ms) and one a mild phenotype (QRS≤18 ms; QRS in wild-type littermates: 10–18 ms). Phenotypic difference persisted with aging. At 10 weeks, the Na⁺ channel blocker ajmaline prolonged QRS interval similarly in both groups of Scn5a ^+/− mice. In contrast, in old mice (>53 weeks), ajmaline effect was larger in the severely affected subgroup. These data matched the clinical observations on patients with SCN5A loss-of-function mutations with either severe or mild conduction defects. Ventricular tachycardia developed in 5/10 old severely affected Scn5a ^+/− mice but not in mildly affected ones. Correspondingly, symptomatic SCN5A–mutated Brugada patients had more severe conduction defects than asymptomatic patients. Old severely affected Scn5a ^+/− mice but not mildly affected ones showed extensive cardiac fibrosis. Mildly affected Scn5a ^+/− mice had similar Na_v1.5 mRNA but higher Na_v1.5 protein expression, and moderately larger I_Na current than severely affected Scn5a ^+/− mice. As a consequence, action potential upstroke velocity was more decreased in severely affected Scn5a ^+/− mice than in mildly affected ones.

Conclusions

Scn5a ^+/− mice show similar phenotypic heterogeneity as SCN5A-mutated patients. In Scn5a ^+/− mice, phenotype severity correlates with wild-type Na_v1.5 protein expression.     

Single-channel properties of human NaV1.1 and mechanism of channel dysfunction in SCN1A-associated epilepsy

Mutations in genes encoding neuronal voltage-gated sodium channel subunits have been linked to inherited forms of epilepsy. The majority of mutations (>100) associated with generalized epilepsy with febrile seizures plus (GEFS+) and severe myoclonic epilepsy of infancy (SMEI) occur in SCN1A encoding the NaV1.1 neuronal sodium channel alpha-subunit. Previous studies demonstrated functional heterogeneity among mutant SCN1A channels, revealing a complex relationship between clinical and biophysical phenotypes. To further understand the mechanisms responsible for mutant SCN1A behavior, we performed a comprehensive analysis of the single-channel properties of heterologously expressed recombinant WT-SCN1A channels. Based on these data, we then determined the mechanisms for dysfunction of two GEFS+-associated mutations (R1648H, R1657C) both affecting the S4 segment of domain 4. WT-SCN1A has a slope conductance (17 pS) similar to channels found in native mammalian neurons. The mean open time is approximately 0.3 ms in the -30 to -10 mV range. The R1648H mutant, previously shown to display persistent sodium current in whole-cell recordings, exhibited similar slope conductance but had an increased probability of late reopening and a subfraction of channels with prolonged open times. We did not observe bursting behavior and found no evidence for a gating mode shift to explain the increased persistent current caused by R1648H. Cells expressing R1657C exhibited conductance, open probability, mean open time, and latency to first opening similar to WT channels but reduced whole-cell current density, suggesting decreased number of functional channels at the plasma membrane. In summary, our findings define single-channel properties for WT-SCN1A, detail the functional phenotypes for two human epilepsy-associated sodium channel mutants, and clarify the mechanism for increased persistent sodium current induced by the R1648H allele.     

Modeling human epilepsy by TALEN targeting of mouse sodium channel Scn8a

To evaluate the efficiency of TALEN technology for introducing mutations into the mouse genome we targeted Scn8a, a member of a multigene family with nine closely related paralogs. Our goal was to generate a model of early onset epileptic encephalopathy by introduction of the Scn8a missense mutation p.Asn1768Asp. We used a pair of TALENs that were highly active in transfected cells. The targeting template for homologous recombination contained a 4 kb genomic fragment. Microinjection of TALENs with the targeting construct into the pronucleus of 350 fertilized mouse eggs generated 67 live‐born potential founders, of which 5 were heterozygous for the pathogenic mutation, a yield of 7% correctly targeted mice. Twenty‐four mice carried one or two Scn8a indels, including 12 frameshift mutations and the novel amino acid deletion p.Asn1759del. Nine off‐site mutations in the paralogs sodium channel genes Scn5a and Scn4a were identified. The data demonstrate the feasibility and efficiency of targeting members of multigene families using TALENs. The Scn8a^tm1768DMm mouse model will be useful for investigation of the pathogenesis and therapy of early onset seizure disorders. genesis 52:141–148. © 2013 The Authors genesis Published by Wiley Periodicals, Inc.     

MeCP2 R168X male and female mutant mice exhibit Rett‐like behavioral deficits

Rett syndrome (RTT) is a regressive developmental disorder characterized by motor and breathing abnormalities, anxiety, cognitive dysfunction and seizures. Approximately 95% of RTT cases are caused by more than 200 different mutations in the X‐linked gene encoding methyl‐CpG‐binding protein 2 (MeCP2). While numerous transgenic mice have been created modeling common mutations in MeCP2, the behavioral phenotype of many of these male and, especially, female mutant mice has not been well characterized. Thorough phenotyping of additional RTT mouse models will provide valuable insight into the effects of Mecp2 mutations on behavior and aid in the selection of appropriate models, ages, sexes and outcome measures for preclinical trials. In this study, we characterize the phenotype of male and female mice containing the early truncating MeCP2 R168X nonsense point mutation, one of the most common in RTT individuals, and compare the phenotypes to Mecp2 null mutants. Mecp2^R168X mutants mirror many clinical features of RTT. Mecp2^R168X/y males exhibit impaired motor and cognitive function and reduced anxiety. The behavioral phenotype is less severe and with later onset in Mecp2^R168X/+ females. Seizures were noted in 3.7% of Mecp2^R168X mutant females. The phenotype in Mecp2^R168X/y mutant males is remarkably similar to our previous characterizations of Mecp2 null males, whereas Mecp2^R168X/+ females exhibit a number of phenotypic differences from females heterozygous for a null Mecp2 mutation. This study describes a number of highly robust behavioral paradigms that can be used in preclinical drug trials and underscores the importance of including Mecp2 mutant females in preclinical studies .     

Fine mapping of an epilepsy modifier gene on mouse Chromosome 19

Mutations in voltage-gated sodium channels are associated with several types of human epilepsy. Variable expressivity and penetrance are common features of inherited epilepsy caused by sodium channel mutations, suggesting that genetic modifiers may influence clinical severity. The mouse model Scn2a ^Q54 has an epilepsy phenotype due to a mutation in Scn2a that results in elevated persistent sodium current. Phenotype severity in Scn2a ^Q54 mice is dependent on the genetic background. Congenic C57BL/6J.Q54 mice have delayed onset and low seizure frequency compared to (C57BL/6J × SJL/J)F1.Q54 mice. Previously, we identified two modifier loci that influence the Scn2a ^Q54 epilepsy phenotype: Moe1 (modifier of epilepsy 1) on Chromosome 11 and Moe2 on Chromosome 19. We have constructed interval-specific congenic strains to further refine the position of Moe2 on Chromosome 19 to a 5-Mb region. Sequencing and expression analyses of genes in the critical interval suggested two potential modifier candidates: (1) voltage-gated potassium channel subunit subfamily V, member 2 (Kcnv2), and (2) SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a, member 2 (Smarca2). Based on its biological role in regulating membrane excitability and the association between ion channel variants and seizures, Kcnv2 is a strong functional candidate for Moe2. Modifier genes affecting the epilepsy phenotype of Scn2a ^Q54 mice may contribute to variable expressivity and penetrance in human epilepsy patients with sodium channel mutations.     

Scn3b knockout mice exhibit abnormal ventricular electrophysiological properties

We report for the first time abnormalities in cardiac ventricular electrophysiology in a genetically modified murine model lacking the Scn3b gene (Scn3b^−/−). Scn3b^−/− mice were created by homologous recombination in embryonic stem (ES) cells. RT-PCR analysis confirmed that Scn3b mRNA was expressed in the ventricles of wild-type (WT) hearts but was absent in the Scn3b^−/− hearts. These hearts also showed increased expression levels of Scn1b mRNA in both ventricles and Scn5a mRNA in the right ventricles compared to findings in WT hearts. Scn1b and Scn5a mRNA was expressed at higher levels in the left than in the right ventricles of both Scn3b^−/− and WT hearts. Bipolar electrogram and monophasic action potential recordings from the ventricles of Langendorff-perfused Scn3b^−/− hearts demonstrated significantly shorter ventricular effective refractory periods (VERPs), larger ratios of electrogram duration obtained at the shortest and longest S₁–S₂ intervals, and ventricular tachycardias (VTs) induced by programmed electrical stimulation. Such arrhythmogenesis took the form of either monomorphic or polymorphic VT. Despite shorter action potential durations (APDs) in both the endocardium and epicardium, Scn3b^−/− hearts showed ΔAPD₉₀ values that remained similar to those shown in WT hearts. The whole-cell patch-clamp technique applied to ventricular myocytes isolated from Scn3b^−/− hearts demonstrated reduced peak Na⁺ current densities and inactivation curves that were shifted in the negative direction, relative to those shown in WT myocytes. Together, these findings associate the lack of the Scn3b gene with arrhythmic tendencies in intact perfused hearts and electrophysiological features similar to those in Scn5a^+/− hearts.     

De novo mutations in the sodium-channel gene SCN1A cause severe myoclonic epilepsy of infancy

Severe myoclonic epilepsy of infancy (SMEI) is a rare disorder that occurs in isolated patients. The disease is characterized by generalized tonic, clonic, and tonic-clonic seizures that are initially induced by fever and begin during the first year of life. Later, patients also manifest other seizure types, including absence, myoclonic, and simple and complex partial seizures. Psychomotor development stagnates around the second year of life. Missense mutations in the gene that codes for a neuronal voltage-gated sodium-channel alpha-subunit (SCN1A) were identified in families with generalized epilepsy with febrile seizures plus (GEFS+). GEFS+ is a mild type of epilepsy associated with febrile and afebrile seizures. Because both GEFS+ and SMEI involve fever-associated seizures, we screened seven unrelated patients with SMEI for mutations in SCN1A. We identified a mutation in each patient: four had frameshift mutations, one had a nonsense mutation, one had a splice-donor mutation, and one had a missense mutation. All mutations are de novo mutations and were not observed in 184 control chromosomes.     

Nontruncating SCN1A Mutations Associated with Severe Myoclonic Epilepsy of Infancy Impair Cell Surface Expression

Mutations in SCN1A, encoding the voltage-gated sodium channel Na_V1.1, are the most common cause of severe myoclonic epilepsy of infancy (SMEI) or Dravet syndrome. SMEI is most often associated with premature truncations of Na_V1.1 that cause loss of function, but nontruncating mutations also occur. We hypothesized that some nontruncating mutations might impair trafficking of Na_V1.1 to the plasma membrane. Here we demonstrated that seven nontruncating missense or in-frame deletion mutations (L986F, delF1289, R1648C, F1661S, G1674R, and G1979E) exhibited reduced cell surface expression relative to wild type (WT) Na_V1.1 consistent with impaired trafficking. We tested whether two commonly prescribed antiepileptic drugs (phenytoin, lamotrigine), as well as the cystic fibrosis transmembrane conductance regulator (CFTR) trafficking corrector VRT-325, could rescue cell surface and functional expression of two representative Na_V1.1 mutants (R1648C, G1674R). Treatment of cells with phenytoin increased cell surface expression of WT-Na_V1.1 and both mutant channels, whereas lamotrigine only increased surface expression of R1648C. VRT-325 did not alter surface expression of WT-Na_V1.1 or mutant channels. Although phenytoin increased surface expression of G1674R, channel function was not restored, suggesting that this mutation also causes an intrinsic loss of function. Both phenytoin and lamotrigine increased functional expression of R1648C, but lamotrigine also increased persistent sodium current evoked by this mutation. Our findings indicate that certain nontruncating SCN1A mutations associated with SMEI have impaired cell surface expression and that some alleles may be amenable to pharmacological rescue of this defect. However, rescue of dysfunctional Na_V1.1 channels to the plasma membrane could contribute to exacerbating rather than ameliorating the disease.     

A novel SCN1A mutation associated with generalized epilepsy with febrile seizures plus--and prevalence of variants in patients with epilepsy

We recently described mutations of the neuronal sodium-channel alpha-subunit gene, SCN1A, on chromosome 2q24 in two families with generalized epilepsy with febrile seizures plus (GEFS+) type 2. To assess the contribution that SCN1A makes to other types of epilepsy, 226 patients with either juvenile myoclonic epilepsy, absence epilepsy, or febrile convulsions were screened by conformation-sensitive gel electrophoresis and manual sequencing of variants; the sample included 165 probands from multiplex families and 61 sporadic cases. The novel mutation W1204R was identified in a family with GEFS+. Seven other coding changes were observed; three of these are potential disease-causing mutations. Two common haplotypes, with frequencies of .67 and .33, were defined by five single-nucleotide polymorphisms (SNPs) spanning a 14-kb region of linkage disequilibrium. An SNP located 18 bp upstream of the splice-acceptor site for exon 3 was observed in 7 of the 226 patients but was not present in 185 controls, suggesting possible association with a disease mutation. This work has confirmed the role of SCN1A in GEFS+, by identification of a novel mutation in a previously undescribed family. Although a few candidate disease alleles were identified, the patient survey suggests that SCN1A is not a major contributor to idiopathic generalized epilepsy. The SCN1A haplotypes and SNPs identified here will be useful in future association and linkage studies.     

Arrhythmogenic substrate and its modification by nicorandil in a murine model of long QT type 3 syndrome

The gain-of-function Scn5a+/ΔKPQ mutation in the cardiac Na⁺ channel causes human long QT type 3 syndrome (LQT3) associated with ventricular arrhythmogenesis. The K_ATP channel-opener nicorandil (20 μM) significantly reduced arrhythmic incidence in Langendorff-perfused Scn5a+/Δ hearts during programmed electrical stimulation; wild-types (WTs) showed a total absence of arrhythmogenicity. These observations precisely correlated with alterations in recently established criteria for re-entrant excitation reflected in: (1) shortened left-ventricular epicardial but not endocardial monophasic action potential durations at 90% repolarization (APD₉₀) that (2) restored transmural repolarization gradients, ΔAPD₉₀. Scn5a+/Δ hearts showed longer epicardial but not endocardial APD₉₀s, giving shorter ΔAPD₉₀s than WT hearts. Nicorandil reduced epicardial APD₉₀ in both Scn5a+/Δ and WT hearts thereby increasing ΔAPD₉₀. (3) Reduced epicardial critical intervals for re-excitation; Scn5a+/Δ hearts showed greater differences between APD₉₀ and ventricular effective refractory period than WT hearts that were reduced by nicorandil. (4) Reduced APD₉₀ alternans. Scn5a+/Δ hearts showed greater epicardial and endocardial alternans than WTs, which increased with pacing rate. Nicorandil reduced these in Scn5a+/Δ hearts to levels indistinguishable from untreated WTs. (5) Flattened restitution curves. Scn5a+/Δ hearts showed larger epicardial and endocardial critical diastolic intervals than WT hearts. Nicorandil decreased these in Scn5a+/Δ and WT hearts. The presence or absence of arrhythmogenesis in Scn5a+/Δ and WT hearts thus agreed with previously established criteria for re-entrant excitation, and alterations in these precisely correlated with the corresponding antiarrhythmic effects of nicorandil. Together these findings implicate spatial and temporal re-entrant mechanisms in arrhythmogenesis in LQT3 and their reversal by nicorandil.