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 α-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 ∼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

Epilepsy-Associated Dysfunction in the Voltage-Gated Neuronal Sodium Channel SCN1A

Mutations in SCN1A, the gene encoding the brain voltage-gated sodium channel subunit (NaV1.1) are associated with at least two forms of epilepsy, generalized epilepsy with febrile seizures plus (GEFS+) and severe myoclonic epilepsy of infancy (SMEI). We examined the functional properties of four GEFS+ alleles and one SMEI allele using whole-cell patch-clamp analysis of heterologously expressed recombinant human SCN1A. One previously reported GEFS+ mutation (I1656M) and an additional novel allele (R1657C), both affecting residues in a voltage-sensing S4 segment, exhibited a similar depolarizing shift in the voltage dependence of activation. Additionally, R1657C showed a 50% reduction in current density and accelerated recovery from slow inactivation. Unlike three other GEFS+ alleles that we recently characterized, neither R1657C nor I1656M gave rise to a persistent, noninactivating current. In contrast, two other GEFS+ mutations (A1685V and V1353L) and L986F, an SMEI-associated allele, exhibited complete loss of function. In conclusion, our data provide evidence for a wide spectrum of sodium channel dysfunction in familial epilepsy and demonstrate that both GEFS+ and SMEI can be associated with nonfunctional SCN1A alleles

The Nav channel bench series: Plasmid preparation

Research involving recombinant voltage-gated sodium (Nav) channels has unique challenges. Multiple factors contribute, but undoubtedly at the top of the list is these channels’ DNA instability. Once introduced into bacterial hosts, Nav channel plasmid DNA will almost invariably emerge mutagenized and unusable, unless special conditions are adopted. This is particularly true for Nav1.1 (gene name SCN1A), Nav1.2 (SCN2A), and Nav1.6 (SCN8A), but less so for Nav1.4 (SCN4A) and Nav1.5 (SCN5A) while other Nav channel isoforms such as Nav1.7 (SCN9A) lie in between. The following recommendations for Nav plasmid DNA amplification and preparation address this problem. Three points are essential: • Bacterial propagation using Stbl2 cells at or below 30 °C. • Bias toward slow-growing, small bacterial colonies. • Comprehensive sequencing of the entire Nav channel coding region

Cardiotoxicity screening: a review of rapid-throughput in vitro approaches

Cardiac toxicity represents one of the leading causes of drug failure along different stages of drug development. Multiple very successful pharmaceuticals had to be pulled from the market or labeled with strict usage warnings due to adverse cardiac effects. In order to protect clinical trial participants and patients, the International Conference on Harmonization published guidelines to recommend that all new drugs to be tested preclinically for hERG (Kv11.1) channel sensitivity before submitting for regulatory reviews. However, extensive studies have demonstrated that measurement of hERG activity has limitations due to the multiple molecular targets of drug compound through which it may mitigate or abolish a potential arrhythmia, and therefore, a model measuring multiple ion channel effects is likely to be more predictive. Several phenotypic rapid-throughput methods have been developed to predict the potential cardiac toxic compounds in the early stages of drug development using embryonic stem cells- or human induced pluripotent stem cell-derived cardiomyocytes. These rapid-throughput methods include microelectrode array-based field potential assay, impedance-based or Ca(2+) dynamics-based cardiomyocytes contractility assays. This review aims to discuss advantages and limitations of these phenotypic assays for cardiac toxicity assessment

Neuroactive steroids for the treatment of status epilepticus

Benzodiazepines are the current first-line standard-of-care treatment for status epilepticus but fail to terminate seizures in about one third of cases. Synaptic GABAA receptors, which mediate phasic inhibition in central circuits, are the molecular target of benzodiazepines. As status epilepticus progresses, these receptors are internalized and become functionally inactivated, conferring benzodiazepine resistance, which is believed to be a major cause of treatment failure. GABAA receptor positive allosteric modulator neuroactive steroids, such as allopregnanolone, also potentiate synaptic GABAA receptors, but in addition they enhance extrasynaptic GABAA receptors that mediate tonic inhibition. Extrasynaptic GABAA receptors are not internalized, and desensitization of these receptors does not occur during continuous seizures in status epilepticus models. Here we review the broad-spectrum antiseizure activity of allopregnanolone in animal seizure models and the evidence for its activity in models of status epilepticus. We also demonstrate that allopregnanolone inhibits ongoing behavioral and electrographic seizures in a model of status epilepticus, even when there is benzodiazepine resistance. Parenteral allopregnanolone may provide an improved treatment for refractory status epilepticus

Novel HCN2 mutation contributes to febrile seizures by shifting the channels kinetics in a temperature-dependent manner.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channel-mediated currents, known as I h, are involved in the control of rhythmic activity in neuronal circuits and in determining neuronal properties including the resting membrane potential. Recent studies have shown that HCN channels play a role in seizure susceptibility and in absence and limbic epilepsy including temporal lobe epilepsy following long febrile seizures (FS). This study focused on the potential contributions of abnormalities in the HCN2 isoform and their role in FS. A novel heterozygous missense mutation in HCN2 exon 1 leading to p.S126L was identified in two unrelated patients with FS. The mutation was inherited from the mother who had suffered from FS in a pedigree. To determine the effect of this substitution we conducted whole-cell patch clamp electrophysiology. We found that mutant channels had elevated sensitivity to temperature. More specifically, they displayed faster kinetics at higher temperature. Kinetic shift by change of temperature sensitivity rather than the shift of voltage dependence led to increased availability of I h in conditions promoting FS. Responses to cyclic AMP did not differ between wildtype and mutant channels. Thus, mutant HCN2 channels cause significant cAMP-independent enhanced availability of I h during high temperatures, which may contribute to hyperthermia-induced neuronal hyperexcitability in some individuals with FS

Novel HCN2 Mutation Contributes to Febrile Seizures by Shifting the Channel's Kinetics in a Temperature-Dependent Manner

<div><p>Hyperpolarization-activated cyclic nucleotide-gated (HCN) channel-mediated currents, known as <i>I</i>_h, are involved in the control of rhythmic activity in neuronal circuits and in determining neuronal properties including the resting membrane potential. Recent studies have shown that HCN channels play a role in seizure susceptibility and in absence and limbic epilepsy including temporal lobe epilepsy following long febrile seizures (FS). This study focused on the potential contributions of abnormalities in the HCN2 isoform and their role in FS. A novel heterozygous missense mutation in <i>HCN2</i> exon 1 leading to p.S126L was identified in two unrelated patients with FS. The mutation was inherited from the mother who had suffered from FS in a pedigree. To determine the effect of this substitution we conducted whole-cell patch clamp electrophysiology. We found that mutant channels had elevated sensitivity to temperature. More specifically, they displayed faster kinetics at higher temperature. Kinetic shift by change of temperature sensitivity rather than the shift of voltage dependence led to increased availability of <i>I</i>_h in conditions promoting FS. Responses to cyclic AMP did not differ between wildtype and mutant channels. Thus, mutant HCN2 channels cause significant cAMP-independent enhanced availability of <i>I</i>_h during high temperatures, which may contribute to hyperthermia-induced neuronal hyperexcitability in some individuals with FS.</p></div