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     

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.     

Voltage-gated sodium channel expression in rat spiral ganglion neurons

The spiral ganglion neurons (SGN) provide the afferent innervation of the hair cells in the organ of Corti and relay auditory information from the inner ear to the brain. Voltage-gated sodium channels (Na_V) initiate and propagate action potentials that encode this sensory information but little is known regarding the subtypes expressed in these cells. We have used RT-PCR and immunohistochemistry to study the compliment and anatomical distribution of Na_V channels in rodent SGN. Na_V1.1, Na_V1.6 and Na_V1.7 were all detected at the mRNA level. Fluorescence or streptavidin–horseradish peroxidase immunohistochemistry extended these findings, demonstrating predominant localisation of Na_V1.6 and Na_V1.7 on SGN cell bodies and Na_V1.1 on axonal processes. Dual labelling with peripherin demonstrated higher Na_V1.6 and Na_V1.7 expression on Type I rather than Type II neurons. These results provide evidence for selective expression and variations in the distribution of VGSC in the rodent SGN, which may guide further studies into afferent function in the auditory pathway and therapeutic approaches for diseases such as hearing loss and tinnitus.     

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 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.     

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.     

Impaired θ-γ Coupling Indicates Inhibitory Dysfunction and Seizure Risk in a Dravet Syndrome Mouse Model

Dravet syndrome (DS) is an epileptic encephalopathy that still lacks biomarkers for epileptogenesis and its treatment. Dysfunction of Na_V1.1 sodium channels, which are chiefly expressed in inhibitory interneurons, explains the epileptic phenotype. Understanding the network effects of these cellular deficits may help predict epileptogenesis. Here, we studied θ-γ coupling as a potential marker for altered inhibitory functioning and epileptogenesis in a DS mouse model. We found that cortical θ-γ coupling was reduced in both male and female juvenile DS mice and persisted only if spontaneous seizures occurred. θ-γ Coupling was partly restored by cannabidiol (CBD). Locally disrupting Na_V1.1 expression in the hippocampus or cortex yielded early attenuation of θ-γ coupling, which in the hippocampus associated with fast ripples, and which was replicated in a computational model when voltage-gated sodium currents were impaired in basket cells (BCs). Our results indicate attenuated θ-γ coupling as a promising early indicator of inhibitory dysfunction and seizure risk in DS.     

Sodium channel subtypes are differentially localized to pre‐ and post‐synaptic sites in rat hippocampus

Voltage‐gated Na⁺ channels (Na_v) modulate neuronal excitability, but the roles of the various Na_v subtypes in specific neuronal functions such as synaptic transmission are unclear. We investigated expression of the three major brain Na_v subtypes (Na_v1.1, Na_v1.2, Na_v1.6) in area CA1 and dentate gyrus of rat hippocampus. Using light and electron microscopy, we found labeling for all three Na_v subtypes on dendrites, dendritic spines, and axon terminals, but the proportion of pre‐ and post‐synaptic labeling for each subtype varied within and between subregions of CA1 and dentate gyrus. In the central hilus (CH) of the dentate gyrus, Na_v1.1 immunoreactivity was selectively expressed in presynaptic profiles, while Na_v1.2 and Na_v1.6 were expressed both pre‐ and post‐synaptically. In contrast, in the stratum radiatum (SR) of CA1, Na_v1.1, Na_v1.2, and Na_v1.6 were selectively expressed in postsynaptic profiles. We next compared differences in Na_v subtype expression between CH and SR axon terminals and between CH and SR dendrites and spines. Na_v1.1 and Na_v1.2 immunoreactivity was preferentially localized to CH axon terminals compared to SR, and in SR dendrites and spines compared to CH. No differences in Na_v1.6 immunoreactivity were found between axon terminals of CH and SR or between dendrites and spines of CH and SR. All Na_v subtypes in both CH and SR were preferentially associated with asymmetric synapses rather than symmetric synapses. These findings indicate selective presynaptic and postsynaptic Na_v expression in glutamatergic synapses of CH and SR supporting neurotransmitter release and synaptic plasticity.     

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.     

Impaired NaV1.2 function and reduced cell surface expression in benign familial neonatal-infantile seizures

Purpose: Mutations in SCN2A, the gene encoding the brain voltage‐gated sodium channel α‐subunit Na _V 1.2, are associated with inherited epilepsies including benign familial neonatal‐infantile seizures (BFNIS). Functional characterization of three BFNIS mutations was performed to identify defects in channel function that underlie this disease. Methods: We examined three BFNIS mutations (R1319Q, L1330F, and L1563V) using whole‐cell patch‐clamp recording of heterologously expressed human Na _V 1.2. Membrane biotinylation was employed to examine the cell surface protein expression of the four Na _V 1.2 alleles. Results: R1319Q displayed mixed effects on activation and fast inactivation gating, consistent with a net loss of channel function. L1563V exhibited impaired fast inactivation predicting a net gain of channel function. The L1330F mutation significantly decreased overall channel availability during repetitive stimulation. Patch‐clamp analysis also revealed that cells expressing BFNIS mutants exhibited lower levels of sodium current compared to wild type (WT) Na _V 1.2. Biochemical experiments demonstrated that all three BFNIS mutations exhibited a significant reduction in cell surface expression compared to WT. Discussion: Our findings indicate that BFNIS is associated with a range of biophysical defects accompanied by reduced levels of channel protein at the plasma membrane.     

Voltage-gated potassium channel (K(v) 1) autoantibodies in patients with chagasic gut dysmotility and distribution of K(v) 1 channels in human enteric neuromusculature (autoantibodies in GI dysmotility)

Background Autoantibodies directed against specific neuronal antigens are found in a significant number of patients with gastrointestinal neuromuscular diseases (GINMDs) secondary to neoplasia. This study examined the presence of antineuronal antibodies in idiopathic GINMD and GINMD secondary to South American Trypanosomiasis. The GI distribution of voltage‐gated potassium channels (VGKCs) was also investigated. Methods Seventy‐three patients were included in the study with diagnoses of primary achalasia, enteric dysmotility, chronic intestinal pseudo‐obstruction, esophageal or colonic dysmotility secondary to Chagas’ disease. Sera were screened for specific antibodies to glutamic acid decarboxylase, voltage‐gated calcium channels (VGCCs; P/Q subtype), nicotinic acetylcholine receptors (nAChRs; α3 subtype), and voltage‐gated potassium channels (VGKCs, K_V1 subtype) using validated immunoprecipitation assays. The distribution of six VGKC subunits (K_V1.1–1.6), including those known to be antigenic targets of anti‐VGKC antibodies was immunohistochemically investigated in all main human GI tract regions. Key Results Three patients (14%) with chagasic GI dysmotility were found to have positive anti‐VGKC antibody titers. No antibodies were detected in patients with idiopathic GINMD. The VGKCs were found in enteric neurons at every level of the gut in unique yet overlapping distributions. The VGKC expression in GI smooth muscle was found to be limited to the esophagus. Conclusions & Inferences A small proportion of patients with GI dysfunction secondary to Chagas’ disease have antibodies against VGKCs. The presence of these channels in the human enteric nervous system may have pathological relevance to the growing number of GINMDs with which anti‐VGKC antibodies have been associated.     

Induction of sodium channel Nax (SCN7A) expression in rat and human hippocampus in temporal lobe epilepsy

Purpose: In a recent large‐scale gene‐expression study in a rat model of temporal lobe epilepsy (TLE) a persistent up‐regulation in the expression of the SCN7A gene was revealed. The SCN7A gene encodes an atypical sodium channel (Na_x), which is involved in osmoregulation via a sensing mechanism for the extracellular sodium concentration. Herein we investigated the expression and cellular distribution of SCN7A mRNA and protein in normal and epileptic rat and human hippocampus. Methods: SCN7A/Na_x expression analysis was performed by polymerase chain reaction (PCR), immunocytochemistry, and western blot analysis. Results: Increased expression of SCN7A/Na_x mRNA/protein was observed during epileptogenesis and in the chronic epileptic phase in the post–status epilepticus (SE) model of TLE. The up‐regulation was confirmed in human hippocampal tissue resected from pharmacoresistant patients with hippocampal sclerosis (HS). In both epileptic rat and human hippocampus, increased Na_x expression was observed in neurons and reactive astrocytes compared to control tissue. Conclusions: The increased and persistent expression of SCN7A/Na_x in the epileptic rat and human hippocampus supports the possible involvement of this channel in the complex reorganization occurring within the hippocampus during the epileptogenic process in TLE. Further studies are needed for a complete understanding of the functional role of SCN7A in epilepsy.     

Untangling the Dravet Syndrome Seizure Network: The Changing Face of a Rare Genetic Epilepsy

Dravet syndrome (also known as Severe Myoclonic Epilepsy of Infancy) is a rare genetic epilepsy syndrome commonly associated with loss-of-function mutations in SCN1A, the gene encoding the α subunit of the voltage-gated sodium channel Na_V1.1, resulting in haploinsufficiency. Like other voltage-gated sodium channels, Na_V1.1 function contributes to the rising phase of the neuronal action potential; thus, the observation that loss-of-function mutations in this channel gene are associated with seizures has created a paradox for the field. Major work has been done to untangle this paradox during the past decade, resulting in the development of two distinct hypotheses to explain seizures in Dravet syndrome. Here, we review the history of these two hypotheses and speculate as to what the history of Dravet syndrome research might tell us about its future.Dravet syndrome (DS, also known as Severe Myoclonic Epilepsy of Infancy or SMEI) is a genetic form of pediatric epilepsy that affects between 1 of 20,000 and 1 of 40,000 live births (1). Patients with DS typically experience their first seizure at 5 to 8 months of age. This is typically a clonic, generalized, or unilateral seizure triggered by fever. Within weeks to months, patients experience additional febrile seizures and then begin to exhibit temperature-independent seizures, including tonic, myoclonic, atypical absence, focal, generalized clonic, or generalized tonic–clonic seizures. Starting in the second year of life, many DS patients begin to show signs of other neurologic symptoms, including cognitive deficits (reviewed in [2]) and motor dysfunction. Cognitive dysfunction stabilizes (but in general does not improve) in the second half of the first decade of life, and seizures tend to decline into adolescence and adulthood (for a complete review of the clinical presentation of DS, see [1]).The first mutations linked to DS were identified in 2001 in SCN1A, the gene encoding the α subunit of the voltage-gated sodium channel Na_V1.1 (3). To date, 70 to 80 percent of patients with DS have identified SCN1A mutations (4). Other genes have been implicated in DS by testing patients who do not have identifiable SCN1A mutations. These include GABARG2 (5), SCN1B (6), and SCN2A (7). Several genes have been linked to seizure syndromes that are phenotypically very similar to DS, notably PCDH19 (8) (a gene associated with Epilepsy in Females with Mental Retardation [EFMR]), and SCN8A (9). To further complicate the situation, it is becoming clear that single gene mutations do not tell the whole story and that modifier genes play important roles in disease severity (10).Studies of mutant SCN1A cDNAs from DS patients have shown that these mutations typically produce nonfunctional Na_v1.1 channels (11, 12). Approximately half of SCN1A patient mutations are truncations that are predicted to result in no protein from one allele. These studies, in addition to work suggesting that most DS SCN1A mutations arise de novo (3, 13–15) have led to a generally accepted idea that DS mainly arises from de novo SCN1A haploinsufficiency. However, there is a paradox that confronts this idea: How is it that reduced expression of a gene encoding an essential depolarizing current predisposes cortical networks to excitability and synchrony? This review aims to summarize the research community''s attempts to untangle this paradox, and ultimately raises questions regarding what might be the most informative tools and questions to move the field forward.     

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.     

Histamine resets the circadian clock in the suprachiasmatic nucleus through the H1R‐CaV1.3‐RyR pathway in the mouse

Histamine, a neurotransmitter/neuromodulator implicated in the control of arousal state, exerts a potent phase‐shifting effect on the circadian clock in the rodent suprachiasmatic nucleus (SCN). In this study, the mechanisms by which histamine resets the circadian clock in the mouse SCN were investigated. As a first step, Ca²⁺‐imaging techniques were used to demonstrate that histamine increases intracellular Ca²⁺ concentration ([Ca²⁺]_i) in acutely dissociated SCN neurons and that this increase is blocked by the H1 histamine receptor (H1R) antagonist pyrilamine, the removal of extracellular Ca²⁺ and the L‐type Ca²⁺ channel blocker nimodipine. The histamine‐induced Ca²⁺ transient is reduced, but not blocked, by application of the ryanodine receptor (RyR) blocker dantrolene. Immunohistochemical techniques indicated that Ca_V1.3 L‐type Ca²⁺ channels are expressed mainly in the somata of SCN cells along with the H1R, whereas Ca_V1.2 channels are located primarily in the processes. Finally, extracellular single‐unit recordings demonstrated that the histamine‐elicited phase delay of the circadian neural activity rhythm recorded from SCN slices is blocked by pyrilamine, nimodipine and the knockout of Ca_V1.3 channel. Again, application of dantrolene reduced but did not block the histamine‐induced phase delays. Collectively, these results indicate that, to reset the circadian clock, histamine increases [Ca²⁺]_i in SCN neurons by activating Ca_V1.3 channels through H1R, and secondarily by causing Ca²⁺‐induced Ca²⁺ release from RyR‐mediated internal stores.     

Deletion of the L‐type calcium channel CaV1.3 but not CaV1.2 results in a diminished sAHP in mouse CA1 pyramidal neurons

Trains of action potentials in CA1 pyramidal neurons are followed by a prolonged calcium‐dependent postburst afterhyperpolarization (AHP) that serves to limit further firing to a sustained depolarizing input. A reduction in the AHP accompanies acquisition of several types of learning and increases in the AHP are correlated with age‐related cognitive impairment. The AHP develops primarily as the result of activation of outward calcium‐activated potassium currents; however, the precise source of calcium for activation of the AHP remains unclear. There is substantial experimental evidence suggesting that calcium influx via voltage‐gated L‐type calcium channels (L‐VGCCs) contributes to the generation of the AHP. Two L‐VGCC subtypes are predominately expressed in the hippocampus, Ca_V1.2 and Ca_V1.3; however, it is not known which L‐VGCC subtype is involved in generation of the AHP. This ambiguity is due in large part to the fact that at present there are no subunit‐specific agonists or antagonists. Therefore, using mice in which the gene encoding Ca_V1.2 or Ca_V1.3 was deleted, we sought to determine the impact of alterations in levels of these two L‐VCGG subtypes on neuronal excitability. No differences in any AHP measure were seen between neurons from Ca_V1.2 knockout mice and controls. However, the total area of the AHP was significantly smaller in neurons from Ca_V1.3 knockout mice as compared with neurons from wild‐type controls. A significant reduction in the amplitude of the AHP was also seen at the 1 s time point in neurons from Ca_V1.3 knockout mice as compared with those from controls. Reductions in both the area and 1 s amplitude suggest the involvement of calcium influx via Ca_V1.3 in the slow AHP (sAHP). Thus, the results of our study demonstrate that deletion of Ca_V1.3, but not Ca_V1.2, significantly impacts the generation of the sAHP. © 2009 Wiley‐Liss, Inc.     

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     

SCN1A testing for epilepsy: Application in clinical practice

This report is a practical reference guide for genetic testing of SCN1A, the gene encoding the α1 subunit of neuronal voltage‐gated sodium channels (protein name: Na_v1.1). Mutations in this gene are frequently found in Dravet syndrome (DS), and are sometimes found in genetic epilepsy with febrile seizures plus (GEFS+), migrating partial seizures of infancy (MPSI), other infantile epileptic encephalopathies, and rarely in infantile spasms. Recommendations for testing: (1) Testing is particularly useful for people with suspected DS and sometimes in other early onset infantile epileptic encephalopathies such as MPSI because genetic confirmation of the clinical diagnosis may allow optimization of antiepileptic therapy with the potential to improve seizure control and developmental outcome. In addition, a molecular diagnosis may prevent the need for unnecessary investigations, as well as inform genetic counseling. (2) SCN1A testing should be considered in people with possible DS where the typical initial presentation is of a developmentally normal infant presenting with recurrent, febrile or afebrile prolonged, hemiclonic seizures or generalized status epilepticus. After age 2, the clinical diagnosis of DS becomes more obvious, with the classical evolution of other seizure types and developmental slowing. (3) In contrast to DS, the clinical utility of SCN1A testing for GEFS+ remains questionable. (4) The test is not recommended for children with phenotypes that are not clearly associated with SCN1A mutations such as those characterized by abnormal development or neurologic deficits apparent at birth or structural abnormalities of the brain. Interpreting test results: (1) Mutational testing of SCN1A involves both conventional DNA sequencing of the coding regions and analyses to detect genomic rearrangements within the relevant chromosomal region: 2q24. Interpretation of the test results must always be done in the context of the electroclinical syndrome and often requires the assistance of a medical geneticist, since many genomic variations are possible and it is essential to differentiate benign polymorphisms from pathogenic mutations. (2) Missense variants may have no apparent effect on the phenotype (benign polymorphisms) or may represent mutations underlying DS, MPSI, GEFS+, and related syndromes and can provide a challenge in interpretation. (3) Conventional methods do not detect variations in introns or promoter or regulatory regions; therefore, a negative test does not exclude a pathogenic role of SCN1A in a specific phenotype. (4) It is important to note that a negative test does not rule out the clinical diagnosis of DS or other conditions because genes other than SCN1A may be involved. Obtaining written informed consent and genetic counseling should be considered prior to molecular testing, depending on the clinical situation and local regulations.