Pure haploinsufficiency for Dravet syndrome Na(V)1.1 (SCN1A) sodium channel truncating mutations

Purpose: Dravet syndrome (DS), a devastating epileptic encephalopathy, is mostly caused by mutations of the SCN1A gene, coding for the voltage‐gated Na⁺ channel Na_V1.1 α subunit. About 50% of SCN1A DS mutations truncate Na_V1.1, possibly causing complete loss of its function. However, it has not been investigated yet if Na_V1.1 truncated mutants are dominant negative, if they impair expression or function of wild‐type channels, as it has been shown for truncated mutants of other proteins (e.g., Ca_V channels). We studied the effect of two DS truncated Na_V1.1 mutants, R222* and R1234*, on coexpressed wild‐type Na⁺ channels. Methods: We engineered R222* or R1234* in the human cDNA of Na_V1.1 (hNa_V1.1) and studied their effect on coexpressed wild‐type hNa_V1.1, hNa_V1.2 or hNa_V1.3 cotransfecting tsA‐201 cells, and on hNa_V1.6 transfecting an human embryonic kidney (HEK) cell line stably expressing this channel. We also studied hippocampal neurons dissociated from Na_V1.1 knockout (KO) mice, an animal model of DS expressing a truncated Na_V1.1 channel. Key Findings: We found no modifications of current amplitude coexpressing the truncated mutants with hNa_V1.1, hNa_V1.2, or hNa_V1.3, but a 30% reduction coexpressing them with hNa_V1.6. However, we showed that also coexpression of functional full‐length hNa_V1.1 caused a similar reduction. Therefore, this effect should not be involved in the pathomechanism of DS. Some gating properties of hNa_V1.1, hNa_V1.3, and hNa_V1.6 were modified, but recordings of hippocampal neurons dissociated from Na_V1.1 KO mice did not show any significant modifications of these properties. Therefore, Na_V1.1 truncated mutants are not dominant negative, consistent with haploinsufficiency as the cause of DS. Significance: We have better clarified the pathomechanism of DS, pointed out an important difference between pathogenic truncated Ca_V2.1 mutants and hNa_V1.1 ones, and shown that hNa_V1.6 expression can be reduced in physiologic conditions by coexpression of hNa_V1.1. Moreover, our data may provide useful information for the development of therapeutic approaches.     

SCN1A/NaV1.1 channelopathies: Mechanisms in expression systems,animal models,and human iPSC models

Pathogenic SCN1A/Na_V1.1 mutations cause well‐defined epilepsies, including genetic epilepsy with febrile seizures plus (GEFS+) and the severe epileptic encephalopathy Dravet syndrome. In addition, they cause a severe form of migraine with aura, familial hemiplegic migraine. Moreover, SCN1A/Na_V1.1 variants have been inferred as risk factors in other types of epilepsy. We review here the advancements obtained studying pathologic mechanisms of SCN1A/Na_V1.1 mutations with experimental systems. We present results gained with in vitro expression systems, gene‐targeted animal models, and the induced pluripotent stem cell (iPSC) technology, highlighting advantages, limits, and pitfalls for each of these systems. Overall, the results obtained in the last two decades confirm that the initial pathologic mechanism of epileptogenic SCN1A/Na_V1.1 mutations is loss‐of‐function of Na_V1.1 leading to hypoexcitability of at least some types of γ‐aminobutyric acid (GABA)ergic neurons (including cortical and hippocampal parvalbumin‐positive and somatostatin‐positive ones). Conversely, more limited results point to Na_V1.1 gain‐of‐function for familial hemiplegic migraine (FHM) mutations. Behind these relatively simple pathologic mechanisms, an unexpected complexity has been observed, in part generated by technical issues in experimental studies and in part related to intrinsically complex pathophysiologic responses and remodeling, which yet remain to be fully disentangled.     

SCN1A‐related phenotypes: Epilepsy and beyond

SCN1A, encoding the alpha 1 subunit of the sodium channel, is associated with several epilepsy syndromes and a range of other diseases. SCN1A represents the archetypal channelopathy associated with a wide phenotypic spectrum of epilepsies ranging from genetic epilepsy with febrile seizures plus (GEFS+), to developmental and epileptic encephalopathies (DEEs). SCN1A disorders also result in other diseases such as hemiplegic migraine and autism spectrum disorder (ASD). Dravet syndrome (DS) is the prototypic DEE with an early onset of febrile status epilepticus, hemiclonic or generalized tonic‐clonic seizures, and later onset of additional seizure types. Electroencephalography (EEG) and magnetic resonance imaging (MRI) are normal at onset. Development is normal in the first year of life but plateaus rapidly, with most patients ultimately having intellectual disability. Epilepsy is drug‐resistant and necessitates polytherapy. Most pathogenic variants occur de novo in the affected child, but they are inherited from mosaic affected or unaffected parents in rare cases. The molecular finding of haploinsufficiency is consistent with a loss‐of‐function defect in cells and animal models. Although seizures are the most commonly reported symptom in DS, many additional issues critically affect patients’ cognitive and behavioral functioning. Hemiplegic migraine (HM) is a rare form of migraine with aura, characterized by the emergence of hemiparesis as part of the aura phase. All SCN1A mutations reported in sporadic/familial HM3 are missense mutations. Most of the experimental results show that they cause a gain of function of Na_V1.1 as opposed to the loss of function of the epileptogenic Na_V1.1 mutations. SCN1A and SCN2A pathogenic variants have been identified in genetic studies of cohorts of patients with ASD. In addition, ASD features are often reported in patients with Dravet syndrome and other DEEs.     

A sodium channel mutation linked to epilepsy increases ramp and persistent current of Nav1.3 and induces hyperexcitability in hippocampal neurons

Voltage-gated sodium channelopathies underlie many excitability disorders. Genes SCN1A, SCN2A and SCN9A, which encode pore-forming α-subunits Na_V1.1, Na_V1.2 and Na_V1.7, are clustered on human chromosome 2, and mutations in these genes have been shown to underlie epilepsy, migraine, and somatic pain disorders. SCN3A, the gene which encodes Na_V1.3, is part of this cluster, but until recently was not associated with any mutation. A charge-neutralizing mutation, K345Q, in the Na_V1.3 DI/S5-6 linker has recently been identified in a patient with cryptogenic partial epilepsy. Pathogenicity of the Na_V1.3/K354Q mutation has been inferred from the conservation of this residue in all sodium channels and its absence from control alleles, but functional analysis has been limited to the corresponding substitution in the cardiac muscle sodium channel Na_V1.5. Since identical mutations may produce different effects within different sodium channel isoforms, we assessed the K354Q mutation within its native Na_V1.3 channel and studied the effect of the mutant Na_V1.3/K354Q channels on hippocampal neuron excitability. We show here that the K354Q mutation enhances the persistent and ramp currents of Na_V1.3, reduces current threshold and produces spontaneous firing and paroxysmal depolarizing shift-like complexes in hippocampal neurons. Our data provide a pathophysiological basis for the pathogenicity of the first epilepsy-linked mutation within Na_V1.3 channels and hippocampal neurons.     

A sodium channel gene SCN9A polymorphism that increases nociceptor excitability

Sodium channel Na_V1.7, encoded by the SCN9A gene, is preferentially expressed in nociceptive primary sensory neurons, where it amplifies small depolarizations. In studies on a family with inherited erythromelalgia associated with Na_V1.7 gain‐of‐function mutation A863P, we identified a nonsynonymous single‐nucleotide polymorphism within SCN9A in the affected proband and several unaffected family members; this polymorphism (c. 3448C&T, Single Nucleotide Polymorphisms database rs6746030, which produces the amino acid substitution R1150W in human Na_V1.7 [hNa_V1.7]) is present in 1.1 to 12.7% of control chromosomes, depending on ethnicity. In this study, we examined the effect of the R1150W substitution on function of the hNa_V1.7 channel, and on the firing of dorsal root ganglion (DRG) neurons in which this channel is normally expressed. We show that this polymorphism depolarizes activation (7.9–11mV in different assays). Current‐clamp analysis shows that the 1150W allele depolarizes (6mV) resting membrane potential and increases (～2‐fold) the firing frequency in response to depolarization in DRG neurons in which it is present. Our results suggest that polymorphisms in the Na_V1.7 channel may influence susceptibility to pain. Ann Neurol 2009;66:862–866     

A small molecule activator of Nav1.1 channels increases fast‐spiking interneuron excitability and GABAergic transmission in vitro and has anti‐convulsive effects in vivo

Na_v1.1 (SCN1A) channels primarily located in gamma‐aminobutyric acid (GABA)ergic fast‐spiking interneurons are pivotal for action potential generation and propagation in these neurons. Inappropriate function of fast‐spiking interneurons, leading to disinhibition of pyramidal cells and network desynchronization, correlates with decreased cognitive capability. Further, reduced functionality of Na_v1.1 channels is linked to various diseases in the central nervous system. There is, at present, however no subtype selective pharmacological activators of Na_v1.1 channels available for studying pharmacological modulation of interneuron function. In the current study, we identified a small molecule Na_v1.1 activator, 3‐amino‐5‐(4‐methoxyphenyl)thiophene‐2‐carboxamide, named AA43279, and provided an in vitro to in vivo characterization of the compound. In HEK‐293 cells expressing human Na_v1.1 channels, AA43279 increased the Na_v1.1‐mediated current in a concentration‐dependent manner mainly by impairing the fast inactivation kinetics of the channels. In rat hippocampal brain slices, AA43279 increased the firing activity of parvalbumin‐expressing, fast‐spiking GABAergic interneurons and increased the spontaneous inhibitory post‐synaptic currents (sIPSCs) recorded from pyramidal neurons. When tested in vivo, AA43279 had anti‐convulsive properties in the maximal electroshock seizure threshold test. AA43279 was tested for off‐target effects on 72 different proteins, including Na_v1.2, Na_v1.4, Na_v1.5, Na_v1.6 and Na_v1.7 and exhibited reasonable selectivity. Taken together, AA43279 might constitute a valuable tool compound for revealing biological functions of Na_v1.1 channels.     

Neuropathy‐associated NaV1.7 variant I228M impairs integrity of dorsal root ganglion neuron axons

Small‐fiber neuropathy (SFN) is characterized by injury to small‐diameter peripheral nerve axons and intraepidermal nerve fibers (IENF). Although mechanisms underlying loss of IENF in SFN are poorly understood, available data suggest that it results from axonal degeneration and reduced regenerative capacity. Gain‐of‐function variants in sodium channel Na_V1.7 that increase firing frequency and spontaneous firing of dorsal root ganglion (DRG) neurons have recently been identified in ～30% of patients with idiopathic SFN. In the present study, to determine whether these channel variants can impair axonal integrity, we developed an in vitro assay of DRG neurite length, and examined the effect of 3 SFN‐associated variant Na_V1.7 channels, I228M, M932L/V991L (ML/VL), and I720K, on DRG neurites in vitro. At 3 days after culturing, DRG neurons transfected with I228M channels exhibited ～20% reduced neurite length compared to wild‐type channels; DRG neurons transfected with ML/VL and I720K variants displayed a trend toward reduced neurite length. I228M‐induced reduction in neurite length was ameliorated by the use‐dependent sodium channel blocker carbamazepine and by a blocker of reverse Na‐Ca exchange. These in vitro observations provide evidence supporting a contribution of the I228M variant Na_V1.7 channel to impaired regeneration and/or degeneration of sensory axons in idiopathic SFN, and suggest that enhanced sodium channel activity and reverse Na‐Ca exchange can contribute to a decrease in length of peripheral sensory axons. Ann Neurol 2012     

Comparative study of lacosamide and classical sodium channel blocking antiepileptic drugs on sodium channel slow inactivation

Many antiepileptic drugs (AEDs) exert their therapeutic activity by modifying the inactivation properties of voltage‐gated sodium (Na_v) channels. Lacosamide is unique among AEDs in that it selectively enhances the slow inactivation component. Although numerous studies have investigated the effects of AEDs on Na_v channel inactivation, a direct comparison of results cannot be made because of varying experimental conditions. In this study, the effects of different AEDs on Na_v channel steady‐state slow inactivation were investigated under identical experimental conditions using whole‐cell patch‐clamp in N1E‐115 mouse neuroblastoma cells. All drugs were tested at 100 μM, and results were compared with those from time‐matched control groups. Lacosamide significantly shifted the voltage dependence of Na_v current (I_Na) slow inactivation toward more hyperpolarized potentials (by ?33 ± 7 mV), whereas the maximal fraction of slow inactivated channels and the curve slope did not differ significantly. Neither SPM6953 (lacosamide inactive enantiomer), nor carbamazepine, nor zonisamide affected the voltage dependence of I_Na slow inactivation, the maximal fraction of slow inactivated channels, or the curve slope. Phenytoin significantly increased the maximal fraction of slow inactivated channels (by 28% ± 9%) in a voltage‐independent manner but did not affect the curve slope. Lamotrigine slightly increased the fraction of inactivated currents (by 15% ± 4%) and widened the range of the slow inactivation voltage dependence. Lamotrigine and rufinamide induced weak, but significant, shifts of I_Na slow inactivation toward more depolarized potentials. The effects of lacosamide on Na_v channel slow inactivation corroborate previous observations that lacosamide has a unique mode of action among AEDs that act on Na_v channels. © 2012 Wiley Periodicals, Inc.     

SCN2A channelopathies: Mechanisms and models

Variants in the SCN2A gene, encoding the voltage‐gated sodium channel Na_V1.2, cause a variety of neuropsychiatric syndromes with different severity ranging from self‐limiting epilepsies with early onset to developmental and epileptic encephalopathy with early or late onset and intellectual disability (ID), as well as ID or autism without seizures. Functional analysis of channel defects demonstrated a genotype‐phenotype correlation and suggested effective treatment options for one group of affected patients carrying gain‐of‐function variants. Here, we sum up the functional mechanisms underlying different phenotypes of patients with SCN2A channelopathies and present currently available models that can help in understanding SCN2A‐related disorders.     

Nav1.4 slow‐inactivation: Is it a player in the warm‐up phenomenon of myotonic disorders?

Myotonia is a heritable disorder in which patients are unable to willfully relax their muscles. The physiological basis for myotonia lies in well‐established deficiencies of skeletal muscle chloride and sodium conductances. What is unclear is how normal muscle function can temporarily return with repeated movement, the so‐called “warm‐up” phenomenon. Electrophysiological analyses of the skeletal muscle voltage‐gated sodium channel Na_v1.4 (gene name SCN4A), a key player in myotonia, have revealed several parallels between the Na_v1.4 biophysical signature, specifically slow‐inactivation, and myotonic warm‐up, which suggest that Na_v1.4 is critical not only in producing the myotonic reaction, but also in mediating the warm‐up. Muscle Nerve, 2013     

Co-expression of β Subunits with the Voltage-Gated Sodium Channel Na<Subscript>V</Subscript>1.7: the Importance of Subunit Association and Phosphorylation and Their Effects on Channel Pharmacology and Biophysics

The voltage-gated sodium ion channel Na_V1.7 is crucial in pain signaling. We examined how auxiliary β2 and β3 subunits and the phosphorylation state of the channel influence its biophysical properties and pharmacology. The human Na_V1.7α subunit was co-expressed with either β2 or β3 subunits in HEK-293 cells. The β2 subunits and the Na_V1.7α, however, were barely associated as evidenced by immunoprecipitation. Therefore, the β2 subunits did not change the biophysical properties of the channel. In contrast, β3 subunit was clearly associated with Na_V1.7α. This subunit had a significant degree of glycosylation, and only the fully glycosylated β3 subunit was associated with the Na_V1.7α. Electrophysiological characterisation revealed that the β3 subunit had small but consistent effects: a right-hand shift of the steady-state inactivation and faster recovery from inactivation. Furthermore, the β3 subunit reduced the susceptibility of Na_V1.7α to several sodium channel blockers. In addition, we assessed the functional effect of Na_V1.7α phosphorylation. Inhibition of kinase activity increased channel inactivation, while the blocking phosphatases produced the opposite effect. In conclusion, co-expression of β subunits with Na_V1.7α, to better mimic the native channel properties, may be ineffective in cases when subunits are not associated, as shown in our experiments with β2. The β3 subunit significantly influences the function of Na_V1.7α and, together with the phosphorylation of the channel, regulates its biophysical and pharmacological properties. These are important findings to take into account when considering the role of Na_V1.7 channel in pain signaling.     

Partial epilepsy with antecedent febrile seizures and seizure aggravation by antiepileptic drugs: Associated with loss of function of Nav1.1

Purpose: Generalized epilepsy with febrile seizures plus (GEFS+) and severe myoclonic epilepsy in infancy (SMEI) are associated with sodium channel α‐subunit type‐1 gene (SCN1A) mutations. Febrile seizures and partial seizures occur in both GEFS+ and SMEI; sporadic onset and seizure aggravation by antiepileptic drugs (AEDs) are features of SMEI. We thus searched gene mutations in isolated cases of partial epilepsy with antecedent FS (PEFS+) that showed seizure aggravations by AEDs. Methods: Genomic DNA from four patients was screened for mutations in SCN1A, SCN2A, SCN1B, and GABRG2 using denaturing high‐performance liquid chromatography (dHPLC) and sequencing. Whole‐cell patch clamp analysis was used to characterize biophysical properties of two newly defined mutants of Na_v1.1 in tsA201 cells. Results: Two heterozygous de novo mutations of SCN1A (R946H and F1765L) were detected, which were proven to cause loss of function of Na_v1.1. When the functional defects of mutants reported previously are compared, it is found that all mutants from PEFS+ have features of loss of function, whereas GEFS+ shows mild dysfunction excluding loss of function, coincident with mild clinical manifestations. PEFS+ is similar to SMEI clinically with possible AED‐induced seizure aggravation and biophysiologically with features of loss of function, and different from SMEI by missense mutation without changes in hydrophobicity or polarity of the residues. Conclusions: Isolated milder PEFS+ may associate with SCN1A mutations and loss of function of Na_v1.1, which may be the basis of seizure aggravation by sodium channel–blocking AEDs. This study characterized phenotypes biologically, which may be helpful in understanding the pathophysiologic basis, and further in management of the disease.     

An SCN2A mutation in a family with infantile seizures from Madagascar reveals an increased subthreshold Na+ current

Missense mutations in SCN2A, encoding the brain sodium channel Na_V1.2, have been described in benign familial neonatal‐infantile seizures (BFNIS), a self‐limiting disorder, whereas several SCN2A de novo nonsense mutations have been found in patients with more severe phenotypes including epileptic encephalopathy. We report a family with BFNIS originating from Madagascar. Onset extended from 3 to 9 months of age. Interictal EEGs were normal. In two patients, ictal electroencephalography (EEG) studies showed partial seizure patterns with secondary generalization in one. Seizures remitted before 18 months of age, with or without medication. Intellectual development was normal. A novel missense mutation of SCN2A, c.4766A>G/p.Tyr1589Cys, was found in a highly conserved region of Na_V1.2 (D4/S2‐S3). Functional studies using heterologous expression in tsA201 cells and whole‐cell patch clamping revealed a depolarizing shift of steady‐state inactivation, increased persistent Na⁺ current, a slowing of fast inactivation and an acceleration of its recovery, thus a gain‐of‐function. Using an action potential waveform in a voltage‐clamp experiment we indicated an increased inward Na⁺ current at subthreshold voltages, which can explain a neuronal hyperexcitability. Our results suggest that this mutation induces neuronal hyperexcitability, resulting in infantile epilepsy with favorable outcome.     

Painful neuropathies: the emerging role of sodium channelopathies

Pain is a frequent debilitating feature reported in peripheral neuropathies with involvement of small nerve (Aδ and C) fibers. Voltage‐gated sodium channels are responsible for the generation and conduction of action potentials in the peripheral nociceptive neuronal pathway where Na_V1.7, Na_V1.8, and Na_V1.9 sodium channels (encoded by SCN9A, SCN10A, and SCN11A) are preferentially expressed. The human genetic pain conditions inherited erythromelalgia and paroxysmal extreme pain disorder were the first to be linked to gain‐of‐function SCN9A mutations. Recent studies have expanded this spectrum with gain‐of‐function SCN9A mutations in patients with small fiber neuropathy and in a new syndrome of pain, dysautonomia, and small hands and small feet (acromesomelia). In addition, painful neuropathies have been recently linked to SCN10A mutations. Patch‐clamp studies have shown that the effect of SCN9A mutations is dependent upon the cell‐type background. The functional effects of a mutation in dorsal root ganglion (DRG) neurons and sympathetic neuron cells may differ per mutation, reflecting the pattern of expression of autonomic symptoms in patients with painful neuropathies who carry the mutation in question. Peripheral neuropathies may not always be length‐dependent, as demonstrated in patients with initial facial and scalp pain symptoms with SCN9A mutations showing hyperexcitability in both trigeminal ganglion and DRG neurons. There is some evidence suggesting that gain‐of‐function SCN9A mutations can lead to degeneration of peripheral axons. This review will focus on the emerging role of sodium channelopathies in painful peripheral neuropathies, which could serve as a basis for novel therapeutic strategies.     

Functional properties and differential neuromodulation of Na(v)1.6 channels

The voltage-gated sodium channel Na_v1.6 plays unique roles in the nervous system, but its functional properties and neuromodulation are not as well established as for Na_V1.2 channels. We found no significant differences in voltage-dependent activation or fast inactivation between Na_V1.6 and Na_V1.2 channels expressed in non-excitable cells. In contrast, the voltage dependence of slow inactivation was more positive for Na_v1.6 channels, they conducted substantially larger persistent sodium currents than Na_v1.2 channels, and they were much less sensitive to inhibition by phosphorylation by cAMP-dependent protein kinase and protein kinase C. Resurgent sodium current, a hallmark of Na_v1.6 channels in neurons, was not observed for Na_V1.6 expressed alone or with the auxiliary β₄ subunit. The unique properties of Na_V1.6 channels, together with the resurgent currents that they conduct in neurons, make these channels well-suited to provide the driving force for sustained repetitive firing, a crucial property of neurons.     

Mechanisms Underlying the Selective Therapeutic Efficacy of Carbamazepine for Attenuation of Trigeminal Nerve Injury Pain

Different peripheral nerve injuries cause neuropathic pain through distinct mechanisms. Even the site of injury may impact underlying mechanisms, as indicated by the clinical finding that the antiseizure drug carbamazepine (CBZ) relieves pain because of compression injuries of trigeminal but not somatic nerves. We leveraged this observation in the present study hypothesizing that because CBZ blocks voltage-gated sodium channels (VGSCs), its therapeutic selectivity reflects differences between trigeminal and somatic nerves with respect to injury-induced changes in VGSCs. CBZ diminished ongoing and evoked pain behavior in rats with chronic constriction injury (CCI) to the infraorbital nerve (ION) but had minimal effect in rats with sciatic nerve CCI. This difference in behavior was associated with a selective increase in the potency of CBZ-induced inhibition of compound action potentials in the ION, an effect mirrored in human trigeminal versus somatic nerves. The increase in potency was associated with a selective increase in the efficacy of the Na_V1.1 channel blocker ICA-121431 and Na_V1.1 protein in the ION, but no change in Na_V1.1 mRNA in trigeminal ganglia. Importantly, local ICA-121431 administration reversed ION CCI-induced hypersensitivity. Our results suggest a novel therapeutic target for the treatment of trigeminal neuropathic pain.SIGNIFICANCE STATEMENT This study is based on evidence of differences in pain and its treatment depending on whether the pain is above (trigeminal) or below (somatic) the neck, as well as evidence that voltage-gated sodium channels (VGSCs) may contribute to these differences. The focus of the present study was on channels underlying action potential propagation in peripheral nerves. There were differences between somatic and trigeminal nerves in VGSC subtypes underlying action potential propagation both in the absence and presence of injury. Importantly, because the local block of Na_V1.1 in the trigeminal nerve reverses nerve injury-induced mechanical hypersensitivity, the selective upregulation of Na_V1.1 in trigeminal nerves suggests a novel therapeutic target for the treatment of pain associated with trigeminal nerve injury.     

A Subtle Alternative Splicing Event of the Na<Subscript>V</Subscript>1.8 Voltage-Gated Sodium Channel is Conserved in Human,Rat, and Mouse

The voltage-gated sodium channel subtype Na_V1.8 (SCN10A) is exclusively expressed in dorsal root ganglia (DRG) and plays a critical role in pain perception. We isolated mRNA from human, rat, and mouse DRGs and screened for alternatively spliced isoforms of the SCN10A mRNA using 454 sequencing. In all three species, we found an event of subtle alternative splicing at a NAGNAG tandem acceptor that results in isoforms including or lacking glutamine 1030 (Na_V1.8+Q and Na_V1.8-Q, respectively) within the cytoplasmic loop between domains II and III. The relative amount of Na_V1.8-Q mRNA in adult DRG was measured with 14.1 ± 0.1% in humans and 11.2 ± 0.2% in rats. This is in contrast to an abundance of 64.3 ± 0.3% in mouse DRG. Thus, the NAGNAG tandem acceptor in SCN10A is conserved among rodents and humans but its alternative usage apparently occurs with species-specific abundance. Analysis of human Na_V1.8+Q and -Q isoforms in whole-cell patch-clamp experiments after heterologous expression in the neuroblastoma cell line Neuro-2A revealed no obvious impact of the splicing event on channel function.     

Focal and generalized seizure activity after local hippocampal or cortical ablation of NaV1.1 channels in mice

Early onset seizures are a hallmark of Dravet syndrome. Previous studies in rodent models have shown that the epileptic phenotype is caused by loss-of-function of voltage-gated Na_V1.1 sodium channels, which are chiefly expressed in γ-aminobutyric acid (GABA)ergic neurons. Recently, a possibly critical role has been attributed to the hippocampus in the seizure phenotype, as local hippocampal ablation of Na_V1.1 channels decreased the threshold for hyperthermia-induced seizures. However, the effect of ablation of Na_V1.1 channels restricted to cortical sites has not been tested. Here we studied local field potential (LFP) and behavior in mice following local hippocampal and cortical ablation of Scn1a, a gene encoding the α1 subunit of Na_V1.1 channels, and we compared seizure characteristics with those of heterozygous global knockout Scn1^-/+ mice. We found a high incidence of spontaneous seizures following either local hippocampal or cortical ablation, notably during a transient time window, similar to Scn1a^-/+ mice. Nonconvulsive seizure activity in the injected area was common and preceded generalized seizures. Moreover, mice were susceptible to hyperthermia-induced seizures. In conclusion, local ablation of Na_V1.1 channels in the hippocampus and cortex results in focal seizure activity that can generalize. These data indicate that spontaneous epileptic activity may initiate in multiple brain regions in Dravet syndrome.     

Anticonvulsant pharmacology of voltage-gated Na+ channels in hippocampal neurons of control and chronically epileptic rats

Voltage-gated Na+ channels are a main target of many first-line anticonvulsant drugs and their mechanism of action has been extensively investigated in cell lines and native neurons. Nevertheless, it is unknown whether the efficacy of these drugs might be altered following chronic epileptogenesis. We have, therefore, analysed the effects of phenytoin (100 micro m), lamotrigine (100 micro m) and valproate (600 micro m) on Na+ currents in dissociated rat hippocampal granule neurons in the pilocarpine model of chronic epilepsy. In control animals, all three substances exhibited modest tonic blocking effects on Na+ channels in their resting state. These effects of phenytoin and lamotrigine were reduced (by 77 and 64%) in epileptic compared with control animals. Phenytoin and valproate caused a shift in the voltage dependence of fast inactivation in a hyperpolarizing direction, while all three substances shifted the voltage dependence of activation in a depolarizing direction. The anticonvulsant effects on Na+ channel voltage dependence proved to be similar in control and epileptic animals. The time course of fast recovery from inactivation was potently slowed by lamotrigine and phenytoin in control animals, while valproate had no effect. Interestingly, the effects of phenytoin on fast recovery from inactivation were significantly reduced in chronic epilepsy. Taken together, these results reveal that different anticonvulsant drugs may exert a distinct pattern of effects on native Na+ channels. Furthermore, the reduction of phenytoin and, to a less pronounced extent, lamotrigine effects in chronic epilepsy raises the possibility that reduced pharmacosensitivity of Na+ channels may contribute to the development of drug resistance.