Voltage-gated Sodium Channel Alpha Subunit Research Articles

High Threshold, Proximal Initiation, and Slow Conduction Velocity of Action Potentials in Dentate Granule Neuron Mossy Fibers

Dentate granule neurons give rise to some of the smallest unmyelinated fibers in the mammalian CNS, the hippocampal mossy fibers. These neurons are also key regulators of physiological and pathophysiological information flow through the hippocampus. We took a comparative approach to studying mossy fiber action potential initiation and propagation in hippocampal slices from juvenile rats. Dentate granule neurons exhibited axonal action potential initiation significantly more proximal than CA3 pyramidal neurons. This conclusion was suggested by phase plot analysis of somatic action potentials and by local tetrodotoxin application to the axon and somatodendritic compartments. This conclusion was also verified by immunostaining for voltage-gated sodium channel alpha subunits and by direct dual soma/axonal recordings. Dentate neurons exhibited a significantly higher action potential threshold and slower axonal conduction velocity than CA3 neurons. We conclude that while the electrotonically proximal axon location of action potential initiation allows granule neurons to sensitively detect and integrate synaptic inputs, the neurons are sluggish to initiate and propagate an action potential.

Severe neonatal non‐dystrophic myotonia secondary to a novel mutation of the voltage‐gated sodium channel (SCN4A) gene

We report on a patient with a severe, rare neonatal form of non-dystrophic myotonia. The patient presented with facial dysmorphism, muscle hypertrophy, severe constipation, psychomotor delay, and frequent cold-induced episodes of myotonia and muscle weakness leading to severe hypoxia and loss of consciousness. Muscle biopsy was non-specific and electromyography revealed intense generalized myotonia. The myotonic episodes improved after introducing oral mexiletine and maintaining room temperature at 28 degrees C. The patient died at 20 months of age following a bronchopulmonary infection. A previously undescribed de novo heterozygous c.3891C > A change, which predicts p.N1297K in the SCN4A gene. Mutations within the voltage-gated sodium channel alpha-subunit gene (SCN4A) have been described in association with several phenotypes including paramyotonia congenita, hyperkalemic or hypokalemic periodic paralysis, and potassium-aggravated myotonias. The cold-sensitive episodes of stiffness followed by weakness suggested the diagnosis of channelopathy in our patient. However, her neonatal onset, the triggering of severe episodes by exposure to modest decreases in temperature, involvement of respiratory muscles with prolonged apnea, early-onset muscle hypertrophy, psychomotor retardation, and fatal outcome are evocative of a distinct clinical subtype. Our observation expands the phenotypic spectrum of sodium channelopathies.

NMR Analysis of Interaction of LqhαIT Scorpion Toxin with a Peptide Corresponding to the D4/S3−S4 Loop of Insect Para Voltage-Gated Sodium Channel

Voltage-gated sodium channels (Navs) are large transmembrane proteins that initiate action potential in electrically excitable cells. This central role in the nervous system has made them a primary target for a large number of neurotoxins. Scorpion alpha-neurotoxins bind to Navs with high affinity and slow their inactivation, causing a prolonged action potential. Despite the similarity in their mode of action and three-dimensional structure, alpha-toxins exhibit great variations in selectivity toward insect and mammalian Navs, suggesting differences in the binding surfaces of the toxins and the channels. The scorpion alpha-toxin binding site, termed neurotoxin receptor site 3, has been shown to involve the extracellular S3-S4 loop in domain 4 of the alpha-subunit of voltage-gated sodium channels (D4/S3-S4). In this study, the binding site for peptides corresponding to the D4/S3-S4 loop of the para insect Nav was mapped on the highly insecticidal alpha-neurotoxin, LqhalphaIT, from the scorpion Leiurus quinquestriatus hebraeus, by following changes in the toxin amide 1H and 15N chemical shifts upon binding. This analysis suggests that the five-residue turn (residues LqK8-LqC12) of LqhalphaIT and those residues in its vicinity interact with the D4/S3-S4 loop of Nav. Residues LqR18, LqW38, and LqA39 could also form a patch contributing to the interaction with D4/S3-S4. Moreover, a new bioactive residue, LqV13, was identified as being important for Nav binding and specifically for the interaction with the D4/S3-S4 loop. The contribution of LqV13 to NaV binding was further verified by mutagenesis. Future studies involving other extracellular regions of Navs are required for further characterization of the structure of the LqhalphaIT-Navs binding site.

Rasmussen encephalitis associated with SCN1A mutation

Mutations in the SCN 1 A gene, encoding the neuronal voltage-gated sodium channel alpha1 subunit, cause SMEI, GEFS+, and related epileptic syndromes. We herein report the R1575C-SCN 1 A mutation identified in a patient with Rasmussen encephalitis. R1575C were constructed in a recombinant human SCN 1 A and then heterologously expressed in HEK293 cells along with the human beta1 and beta2 sodium channel accessory subunits. Whole-cell patch-clamp recording was used to define biophysical properties. The R1575C channels exhibited increased channel availability and an increased persistent sodium current in comparison to the wild-type. These defects of electrophysiological properties can result in neuronal hyperexitability. The seizure susceptibility allele may influence the pathogenesis of Rasmussen encephalitis in this case.

Paramyotonie congénitale d’Eulenburg

Paramyotonia congenita is an autosomal dominant sodium channelopathy, caused by mutations in gene coding for muscle voltage-gated sodium channel alpha subunit. We report the case of a 38-year-old man who described since childhood muscle stiffness with attacks ok weakness induced by two provocative stimuli: cold exposure and exercise. It primarily concerned eyelids and hands, occasionally limbs. Family history suggested an autosomal dominant mode of transmission. Clinical examination revealed myotonia at the thenar eminence percussion. Generalized myotonic discharges were observed on electromyography. Molecular diagnosis reported an Arg1448Cys mutation in exon 24 in gene coding for muscle voltage-gated sodium channel alpha subunit (SCN4A) in chromosome 17. Paramyotonia congenita is not evolutive. Treatment is essentially preventive. Some medications could be proposed: membrane stabilizing agents like antiarrhythmic drugs (mexiletine, tocainide), or the carbonic anhydrase inhibitor (acetazolamide). Precautions may be taken during general anaesthesia because of diaphragm myotonia risk.

Brevetoxin-induced phosphorylation of Pyk2 and Src in murine neocortical neurons involves distinct signaling pathways

Brevetoxins (PbTx-1 to PbTx-10) are potent lipid soluble polyether neurotoxins produced by the marine dinoflagellate Karenia brevis. Brevetoxins bind to site 5 of the alpha-subunit of voltage-gated sodium channels (VGSCs) and augment Na(+) influx. In neocortical neurons brevetoxins elevate intracellular Ca(2+) and augment NMDA receptor signaling. In this study, we explored the effects of PbTx-2 on Pyk2 and Src activation in neocortical neurons. We found that both Pyk2 and Src were activated following PbTx-2 exposure. PbTx-2-induced Pyk2 Tyr402 phosphorylation was dependent on elevation of Ca(2+) influx through NMDA receptors. Moreover, Pyk2 Tyr402 phosphorylation was also found to require PKC activation inasmuch as RO-31-8425 and GF 109203x both attenuated the response. In contrast, PbTx-2-induced Src Tyr416 phosphorylation involved a Gq-coupled receptor inasmuch as U73122, a specific PLC inhibitor, abolished the response. This Gq-coupled receptor appears to be mGluR 5. The PKCdelta inhibitor rottlerin abolished PbTx-2-induced Src activation demonstrating that this isoform of PKC is involved in the activation of Src by PbTx-2. Considered together these data suggest that although activation of neuronal Pyk2 and Src result from PbTx-2 stimulation of VGSC, engagement of these two non-receptor tyrosine kinases involves distinct signaling pathways.

β-Subunits of voltage-gated sodium channels in human prostate cancer: quantitative in vitro and in vivo analyses of mRNA expression

We previously identified high levels of Na(v)1.7 voltage-gated sodium channel alpha-subunit (VGSCalpha) mRNA and protein in human prostate cancer cells and tissues. Here, we investigated auxillary beta-subunit (VGSCbetas) expression. In vitro, the combined expression of all four VGSCbetas was significantly (approximately 4.5-fold) higher in strongly compared to weakly metastatic cells. This was mainly due to increased beta1-expression, which was under androgenic control. In vivo, beta1-beta4 mRNAs were detectable and their expression in CaP vs non-CaP tissues generally reflected the in vitro levels in relation to metastatic potential. The possible role(s) of VGSCbetas (VGSCalpha-associated and VGSCalpha-independent) in prostate cancer are discussed.

Detection of Pyrethroid Resistance Gene in Head Lice in Schoolchildren from Bobigny, France

The head louse, Pediculus humanus capitis (De Geer), is an hematophagous ectoparasite that affects mainly children. Resistance to insecticides belonging to pyrethroids and other pediculicides, such as malathion, is responsible for frequently reported treatment failures. Recent studies showed that a M815I-T929I-L932F kdr-like mutation in the voltage-gated sodium channel alpha-subunit gene was associated with permethrin resistance in head lice from several countries worldwide. We searched for the presence ofpyrethroid resistance gene in head lice populations obtained in schoolchildren in an urban area of France. All the 15 primary schools of Bobigny, a city located 3 km north of Paris, were selected to participate. Of 3,493 children enrolled, 3,345 (95.8%) children were screened for head lice by using fine-toothed antilouse combs. Live head lice were detected in 112 (3.3%) of children screened. A subsample of 90 lice was processed for DNA study. The amplification of a 332-bp portion of the kdr-like gene spanning the codon 929 was performed, and polymerase chain reaction products were submitted to the restriction enzyme SspI. Twenty of these lice (22.2%) were homozygous susceptible, 33 (36.7%) were homozygous resistant, and 37 (41.1%) were heterozygotes. Globally, the frequency of the T929I mutation was 0.57. The prevalence of pediculosis in schoolchildren of Bobigny seemed relatively low in comparison with findings of other European studies. The presence of the T929I mutation associated with permethrin resistance probably reflected the frequent local use of this insecticide. Further studies are now required to evaluate the prevalence of the kdr-like mutant allele in head lice in French schools.

Expression of Nav1.1 in rat retinal AII amacrine cells

In retinal ganglion cells (RGCs), the expression of various types of voltage-gated sodium channel (Nav) alpha-subunits (Nav1.1, Nav1.2, Nav1.3, and Nav1.6) has been reported. Like RGCs, certain subsets of retinal amacrine cells, including AII amacrine cells, generate tetrodotoxin (TTX)-sensitive action potentials in response to light; however, the Nav subtypes expressed in these cells have not been identified. We examined the Nav subtypes expressed in rat retinal amacrine cells by in situ hybridization (ISH) using RNA probes specific for TTX-sensitive Na(v)s (Nav1.1, Nav1.2, Nav1.3, Nav1.6, and Nav1.7). Our results confirmed that Nav1.1, Nav1.2, Nav1.3, and Nav1.6 are localized in the ganglion cell layer (GCL). Interestingly, Nav1.1 was expressed not only in the GCL, but also in the inner nuclear layer (INL). The cell bodies of the Nav1.1-positive cells in the INL were located at the INL/inner plexiform layer (IPL) border. The cell bodies of AII amacrine cells are located close to the INL/IPL border, and these cells can be labeled with antibodies against parvalbumin (PV). Therefore, we combined ISH with immunohistochemistry and discovered that most of the PV-immunoreactive cells located at the INL/IPL border express Nav1.1. Our results show that AII amacrine cells express Nav1.1.

A comparative study of voltage‐gated sodium channels in the Insecta: implications for pyrethroid resistance in Anopheline and other Neopteran species

We report the complete cDNA sequence of the Anopheles gambiae voltage-gated sodium channel (VGSC) alpha-subunit isolated from mature adult mosquitoes. The genomic DNA contains 35 deduced exons with a predicted translation of <or= 2139 amino acid cDNAs. The transcription of the gene is, however, complex, alternate splicing being evident for at least five optional exons (or exon segments) and two sets of mutually exclusive exons. Overall gene organization was also compared with that of other VGSCs within the Insecta. Several insecticides used in mosquito control (including DDT and synthetic pyrethroids) target the VGSC. Isolation of the sodium channel cDNA for An. gambiae: (1) allows prediction of likely single nucleotide polymorphisms that may arise at residue L1014 to cause resistance to insecticides; (2) defines An. gambiae exon usage in key areas of the VGSC protein that are known (from previous studies in a range of different pest species) to have roles in altering insecticide susceptibility and in generating resistance; and (3) is a critical first step towards development of refined malarial control strategies and of new diagnostics for resistance monitoring.

Microdeletions involving theSCN1A gene may be common inSCN1A-mutation-negative SMEI patients

Severe myoclonic epilepsy of infancy (SMEI) or Dravet syndrome is a rare epilepsy syndrome. In 30 to 70% of SMEI patients, truncating and missense mutations in the neuronal voltage-gated sodium-channel alpha-subunit gene (SCN1A) have been identified. The majority of patients have truncating mutations that are predicted to be loss-of-function alleles. Because mutation detection studies use PCR-based sequencing or conformation sensitive gel electrophoresis (CSGE), microdeletions, which are also predicted to be loss-of-function alleles, can easily escape detection. We selected 11 SMEI patients with or without additional features who had no SCN1A mutation detectable with sequencing analysis. In addition, none of the patients was heterozygous for any of the SNPs in SCN1A, indicating that they were either homozygous for all SNPs or hemizygous due to a microdeletion of the gene. We subsequently analyzed these patients for the presence of microdeletions in SCN1A using a quantitative PCR method named multiplex amplicon quantification (MAQ), and observed three patients missing one copy of the SCN1A gene. All three microdeletions were confirmed by fluorescence in situ hybridization (FISH). These findings demonstrate that a substantial percentage of SCN1A-mutation-negative SMEI patients with or without additional features carry a chromosomal microdeletion comprising the SCN1A gene and that haploinsufficiency of the SCN1A gene is a cause of SMEI.

Sodium channel dysfunction in intractable childhood epilepsy with generalized tonic–clonic seizures

Mutations in SCN1A, the gene encoding the brain voltage-gated sodium channel alpha(1) subunit (Na(V)1.1), are associated with genetic forms of epilepsy, including generalized epilepsy with febrile seizures plus (GEFS+ type 2), severe myoclonic epilepsy of infancy (SMEI) and related conditions. Several missense SCN1A mutations have been identified in probands affected by the syndrome of intractable childhood epilepsy with generalized tonic-clonic seizures (ICEGTC), which bears similarity to SMEI. To test whether ICEGTC arises from molecular mechanisms similar to those involved in SMEI, we characterized eight ICEGTC missense mutations by whole-cell patch clamp recording of recombinant human SCN1A heterologously expressed in cultured mammalian cells. Two mutations (G979R and T1709I) were non-functional. The remaining alleles (T808S, V983A, N1011I, V1611F, P1632S and F1808L) exhibited measurable sodium current, but had heterogeneous biophysical phenotypes. Mutant channels exhibited lower (V983A, N1011I and F1808L), greater (T808S) or similar (V1611F and P1632S) peak sodium current densities compared with wild-type (WT) SCN1A. Three mutations (V1611F, P1632S and F1808L) displayed hyperpolarized conductance-voltage relationships, while V983A exhibited a strong depolarizing shift in the voltage dependence of activation. All mutants except T808S had hyperpolarized shifts in the voltage dependence of steady-state channel availability. Three mutants (V1611F, P1632S and F1808L) exhibited persistent sodium current ranging from approximately 1-3% of peak current amplitude that was significantly greater than WT-SCN1A. Several mutants had impaired slow inactivation, with V983A showing the most prominent effect. Finally, all of the functional alleles exhibited reduced use-dependent channel inhibition. In summary, SCN1A mutations associated with ICEGTC result in a wide spectrum of biophysical defects, including mild-to-moderate gating impairments, shifted voltage dependence and reduced use dependence. The constellation of biophysical abnormalities for some mutants is distinct from those previously observed for GEFS+ and SMEI, suggesting possible, but complex, genotype-phenotype correlations.

High Risk for Bradyarrhythmic Complications in Patients With Brugada Syndrome Caused by SCN5AGene Mutations

We carried out a complete screening of the SCN5A gene in 38 Japanese patients with Brugada syndrome to investigate the genotype-phenotype relationship. The gene SCN5A encodes the pore-forming alpha-subunit of voltage-gated cardiac sodium (Na) channel, which plays an important role in heart excitation/contraction. Mutations of SCN5A have been identified in 15% of patients with Brugada syndrome. In 38 unrelated patients with clinically diagnosed Brugada syndrome, we screened for SCN5A gene mutations using denaturing high-performance liquid chromatography and direct sequencing, and conducted a functional assay for identified mutations using whole-cell patch-clamp in heterologous expression system. Four heterozygous mutations were identified (T187I, D356N, K1578fs/52, and R1623X) in 4 of the 38 patients. All of them had bradyarrhythmic complications: three with sick sinus syndrome (SSS) and the other (D356N) with paroxysmal complete atrioventricular block. SCN5A-linked Brugada patients were associated with a higher incidence of bradyarrhythmia (4 of 4) than non-SCN5A-linked Brugada patients (2 of 34). Families with T187I and K1578fs/52 had widespread penetrance of SSS. Notably, the patient with K1578fs/52, who had been diagnosed as having familial SSS without any clinical signs of Brugada syndrome, showed a Brugada-type ST-segment elevation after intravenous administration of pilsicainide and programmed electrical stimulation-induced ventricular tachycardia. All of the mutations encoded non-functional Na channels, and thus were suggested to cause impulse propagation defect underlying bradyarrhythmias. Our findings suggest that loss-of-function SCN5A mutations resulting in Brugada syndrome are distinguished by profound bradyarrhythmias.

Autosomal Dominant Erythermalgia Associated With a Novel Mutation in the Voltage-Gated Sodium Channel α Subunit Nav1.7

Autosomal dominant primary erythermalgia is a rare disorder characterized by recurrent attacks of red, warm, and painful hands and/or feet. To describe the phenotypes and molecular data of a 10-member family with 5 symptomatic living patients with erythermalgia. The clinical phenotype of this family was featured by episodic or continuous symmetrical red swelling, irritating warmth, and burning pain of feet and lower legs provoked or aggravated by warmth and exercise, and relief was always obtained by application of cold, such as putting feet in (ice-) cold water. The symptoms in this family were only partially controlled by analgesics and sedatives. All affected family members were heterozygous for a novel mutation (S241T) of the voltage-gated sodium channel alpha subunit Nav1.7. Primary erythermalgia may be a neuropathic disorder of the small peripheral sensory and sympathetic neurons, and may be caused by hyperexcitability of Nav1.7.

A potential novel marker for human prostate cancer: voltage-gated sodium channel expression in vivo

Functional expression of voltage-gated sodium channel alpha-subunits (VGSCalphas), specifically Nav1.7, is associated with strong metastatic potential in prostate cancer (CaP) in vitro. Furthermore, VGSC activity in vitro directly potentiates processes integral to metastasis. To investigate VGSCalpha expression in CaP in vivo, immunohistochemistry and real-time PCR were performed on human prostate biopsies (n>20). VGSCalpha immunostaining was evident in prostatic tissues and markedly stronger in CaP vs non-CaP patients. Importantly, RT-PCRs identified Nav1.7 as the VGSCalpha most strikingly upregulated (approximately 20-fold) in CaP, and the resultant receiver-operating characteristics curve demonstrated high diagnostic efficacy for the disease. It is concluded that VGSCalpha expression increases significantly in CaP in vivo and that Nav1.7 is a potential functional diagnostic marker.

Expression and distribution of TTX‐sensitive sodium channel alpha subunits in the enteric nervous system

The expression and distribution of TTX-sensitive voltage-gated sodium channel (VGSC) alpha subunits in the enteric nervous system (ENS) has not been described. Using RT-PCR, expression of Na(v)1.2, Na(v)1.3, Na(v)1.6, and Na(v)1.7 mRNA was detected in small and large intestinal preparations from guinea pigs. Expression of Na(v)1.1 mRNA as well as Na(v)1.1-like immunoreactivity (-li) were not observed in any intestinal region investigated. Na(v)1.2-li was primarily observed within the soma of the majority of myenteric and submucosal neurons, although faint immunoreactivity was occasionally observed in ganglionic and internodal fibers. Na(v)1.3-li was observed in dendrites, soma, and axons in a small group of myenteric neurons, as well as in numerous myenteric internodal fibers; immunoreactivity was rarely observed in the submucosal plexus. Na(v)1.6-li was primarily observed in the initial axonal segment of colonic myenteric neurons. Na(v)1.7-li was observed in dorsal root ganglia neurons but not in the myenteric plexus of the small and large intestine. In the ileum, 37% of Na(v)1.2-li cell bodies colocalized with calbindin-li while colocalization with calretinin-li was rare. In contrast, 22% of Na(v)1.3-li cell bodies colocalized with calretinin-li but colocalization with calbindin-li was not observed. In the colon, both Na(v)1.2-li and Na(v)1.3-li cell bodies frequently colocalized with either calretinin-li or calbindin-li. Na(v)1.2-li cell bodies also colocalized with the majority of NeuN-li cells in the small and large intestine. These data suggest that Na(v)1.1 may not be highly expressed in the ENS, but that Na(v)1.2, Na(v)1.3, and Na(v)1.6, and possibly Na(v)1.7, have broadly important and distinct functions in the ENS.

Molecular diversity of voltage-gated sodium channel alpha subunits expressed in neuronal and non-neuronal excitable cells

In order to investigate the role of molecular diversity of voltage-activated sodium channel α-subunits in excitability of neuronal and non-neuronal cells, we carried out patch-clamp recordings and single-cell RT-PCR on two different types of mammalian excitable cells i.e. hippocampal neurons and non-neuronal utricular epithelial hair cells. In each cell type, multiple different combinations of sodium channel α-subunits exist from cell to cell despite similar sodium current properties. The mRNA isoforms, Nav1.2 and Nav1.6, are the most frequently detected by single cell analysis in the two cell types while Nav1.3 and Nav1.7 are also moderately expressed in embryonic hippocampal neurons and in neonatal utricular hair cells respectively. By investigating the particular alternate splice isoforms of Nav1.6 occurring at the exon 18 of the mouse orthologue SCN8A, we revealed that this subunit co-exist in the two cell types under different alternative spliced isoforms. The expression of non-functional isoforms of Nav1.6 in utricular epithelial hair cells excludes the involvement of this subunit in supporting their excitability. Thus, from a functional point of view, the present results suggest that, at the single cell level, both neuronal and non-neuronal excitable cells expressed different and complex patterns of sodium channel gene transcripts but this diversity alone cannot explain the sodium current properties of these cell types.

Tyrosine-phosphorylated and Nonphosphorylated Sodium Channel β1 Subunits Are Differentially Localized in Cardiac Myocytes

Voltage-gated sodium channel alpha and beta subunits expressed in mammalian heart are differentially localized to t-tubules and intercalated disks. Sodium channel beta subunits are multifunctional molecules that participate in channel modulation and cell adhesion. Reversible, receptor-mediated changes in beta1 tyrosine phosphorylation modulate its ability to recruit and associate with ankyrin. The purpose of the present study was to test our hypothesis that tyrosine-phosphorylated beta1 (pYbeta1) and nonphosphorylated beta1 subunits may be differentially localized in heart and thus interact with different cytoskeletal and signaling proteins. We developed an antibody that specifically recognizes pYbeta1 and investigated the differential subcellular localization of beta1 and pYbeta1 in mouse ventricular myocytes. We found that pYbeta1 colocalized with connexin-43, N-cadherin, and Nav1.5 at intercalated disks but was not detected at the t-tubules. Anti-pYbeta1 immunoprecipitates N-cadherin from heart membranes and from cells transfected with beta1 and N-cadherin in the absence of other sodium channel subunits. pYbeta1 does not associate with ankyrinB in heart membranes. N-cadherin and connexin-43 associate with Nav1.5 in heart membranes as assessed by co-immunoprecipitation assays. We propose that sodium channel complexes at intercalated disks of ventricular myocytes are composed of Nav1.5 and pYbeta1 and that these complexes are in close association with both N-cadherin and connexin-43. beta1 phosphorylation appears to regulate its localization to differential subcellular domains.

Functional characterization of the pentapeptide QYNAD on rNav1.2 channels and its NMR structure.

The endogenous pentapeptide QYNAD (Gln-Tyr-Asn-Ala-Asp) is present in human cerebrospinal fluid (CSF), and its concentration is increased in demyelinating diseases. QYNAD was synthesized and its action on the rNav1.2 voltage-gated sodium channel alpha-subunit was studied using whole-cell recordings in a heterologous expression system. The effects were seen only upon equilibration of the peptide in the external bath solution for at least 10 min before the commencement of whole-cell experiments. The steady-state activation curve showed a rightward shift of 10 mV, while the steady-state inactivation curve showed a leftward shift of 5 mV. Frequency-dependent inhibition of the sodium current amplitude was observed at 2-10 Hz, in the presence of external QYNAD, but was not seen when applied internally. Fits of the whole-cell sodium current traces by Hodgkin-Huxley equations revealed subtle changes in the voltage-dependent rate constants governing the transition of the activation and the inactivation gates. Two dimensional NMR spectroscopy revealed the absence of medium and long-range Nuclear Overhauser effects (NOEs), which indicates that the peptide does not adopt any canonical secondary structure in solution. In summary, our studies show that although the pentapeptide QYNAD does not have a defined structure in solution, it has defined actions on the rNav1.2 voltage-gated sodium channel isoform.

Mutations of sodium channel alpha subunit type 1 (SCN1A) in intractable childhood epilepsies with frequent generalized tonic-clonic seizures.

A group of infant onset epilepsies manifest very frequent generalized tonic-clonic seizures (GTC) intractable to medical therapy, which may or may not be accompanied by minor seizures such as myoclonic seizures, absences and partial seizures. They include severe myoclonic epilepsy in infancy (SMEI) and intractable childhood epilepsy with GTC (ICEGTC). They are commonly associated with fever-sensitivity, family history of seizure disorders and developmental decline after seizure onset. Mutations of the neuronal voltage-gated sodium channel alpha subunit type 1 gene (SCN1A) were recently reported in SMEI patients. To clarify the genotypic differences in this group of epilepsies, we searched for SCN1A abnormalities in 25 patients with SMEI and 10 with ICEGTC, together with the family members of 15 patients. Frameshift mutations in SCN1A were observed in four patients, nonsense mutations in five patients, missense mutations in 21 patients, other mutations in two patients and no mutation in five patients. SMEI patients showed nonsense mutations, frameshifts, or missense mutations, while ICEGTC patients showed only missense mutations. Study of both parents of 11 patients revealed that the mutations in these patients were de novo. However, two mothers had the same missense mutations as their ICEGTC children, and they had generalized epilepsy with febrile seizures plus. Here we suggest that SMEI and ICEGTC represent a continuum with minor phenotypic and genotypic differences.