Pathogenic SCN2A variants cause early-stage dysfunction in patient-derived neurons.

Pathogenic heterozygous variants in SCN2A, which encodes the neuronal sodium channel NaV1.2, cause different types of epilepsy or intellectual disability (ID)/autism without seizures. Previous studies using mouse models or heterologous systems suggest that NaV1.2 channel gain-of-function typically causes epilepsy, whereas loss-of-function leads to ID/autism. How altered channel biophysics translate into patient neurons remains unknown. Here, we investigated iPSC-derived early-stage cortical neurons from ID patients harboring diverse pathogenic SCN2A variants [p.(Leu611Valfs*35); p.(Arg937Cys); p.(Trp1716*)] and compared them with neurons from an epileptic encephalopathy (EE) patient [p.(Glu1803Gly)] and controls. ID neurons consistently expressed lower NaV1.2 protein levels. In neurons with the frameshift variant, NaV1.2 mRNA and protein levels were reduced by ~ 50%, suggesting nonsense-mediated decay and haploinsufficiency. In other ID neurons, only protein levels were reduced implying NaV1.2 instability. Electrophysiological analysis revealed decreased sodium current density and impaired action potential (AP) firing in ID neurons, consistent with reduced NaV1.2 levels. In contrast, epilepsy neurons displayed no change in NaV1.2 levels or sodium current density, but impaired sodium channel inactivation. Single-cell transcriptomics identified dysregulation of distinct molecular pathways including inhibition of oxidative phosphorylation in neurons with SCN2A haploinsufficiency and activation of calcium signaling and neurotransmission in epilepsy neurons. Together, our patient iPSC-derived neurons reveal characteristic sodium channel dysfunction consistent with biophysical changes previously observed in heterologous systems. Additionally, our model links the channel dysfunction in ID to reduced NaV1.2 levels and uncovers impaired AP firing in early-stage neurons. The altered molecular pathways may reflect a homeostatic response to NaV1.2 dysfunction and can guide further investigations.

Microglial over-pruning of synapses during development in autism-associated SCN2A-deficient mice and human cerebral organoids.

Autism spectrum disorder (ASD) is a major neurodevelopmental disorder affecting 1 in 36 children in the United States. While neurons have been the focus of understanding ASD, an altered neuro-immune response in the brain may be closely associated with ASD, and a neuro-immune interaction could play a role in the disease progression. As the resident immune cells of the brain, microglia regulate brain development and homeostasis via core functions including phagocytosis of synapses. While ASD has been traditionally considered a polygenic disorder, recent large-scale human genetic studies have identified SCN2A deficiency as a leading monogenic cause of ASD and intellectual disability. We generated a Scn2a-deficient mouse model, which displays major behavioral and neuronal phenotypes. However, the role of microglia in this disease model is unknown. Here, we reported that Scn2a-deficient mice have impaired learning and memory, accompanied by reduced synaptic transmission and lower spine density in neurons of the hippocampus. Microglia in Scn2a-deficient mice are partially activated, exerting excessive phagocytic pruning of post-synapses related to the complement C3 cascades during selective developmental stages. The ablation of microglia using PLX3397 partially restores synaptic transmission and spine density. To extend our findings from rodents to human cells, we established a microglia-incorporated human cerebral organoid model carrying an SCN2A protein-truncating mutation identified in children with ASD. We found that human microglia display increased elimination of post-synapse in cerebral organoids carrying the SCN2A mutation. Our study establishes a key role of microglia in multi-species autism-associated models of SCN2A deficiency from mouse to human cells.

Cellular and behavioral effects of altered NaV1.2 sodium channel ion permeability in Scn2aK1422E mice.

Genetic variants in SCN2A, encoding the NaV1.2 voltage-gated sodium channel, are associated with a range of neurodevelopmental disorders with overlapping phenotypes. Some variants fit into a framework wherein gain-of-function missense variants that increase neuronal excitability lead to developmental and epileptic encephalopathy, while loss-of-function variants that reduce neuronal excitability lead to intellectual disability and/or autism spectrum disorder (ASD) with or without co-morbid seizures. One unique case less easily classified using this framework is the de novo missense variant SCN2A-p.K1422E, associated with infant-onset developmental delay, infantile spasms and features of ASD. Prior structure-function studies demonstrated that K1422E substitution alters ion selectivity of NaV1.2, conferring Ca2+ permeability, lowering overall conductance and conferring resistance to tetrodotoxin (TTX). Based on heterologous expression of K1422E, we developed a compartmental neuron model incorporating variant channels that predicted reductions in peak action potential (AP) speed. We generated Scn2aK1422E mice and characterized effects on neurons and neurological/neurobehavioral phenotypes. Cultured cortical neurons from heterozygous Scn2aK1422E/+ mice exhibited lower current density with a TTX-resistant component and reversal potential consistent with mixed ion permeation. Recordings from Scn2aK1442E/+ cortical slices demonstrated impaired AP initiation and larger Ca2+ transients at the axon initial segment during the rising phase of the AP, suggesting complex effects on channel function. Scn2aK1422E/+ mice exhibited rare spontaneous seizures, interictal electroencephalogram abnormalities, altered induced seizure thresholds, reduced anxiety-like behavior and alterations in olfactory-guided social behavior. Overall, Scn2aK1422E/+ mice present with phenotypes similar yet distinct from other Scn2a models, consistent with complex effects of K1422E on NaV1.2 channel function.

Crosstalk among WEE1 Kinase, AKT, and GSK3 in Nav1.2 Channelosome Regulation.

The signaling complex around voltage-gated sodium (Nav) channels includes accessory proteins and kinases crucial for regulating neuronal firing. Previous studies showed that one such kinase, WEE1-critical to the cell cycle-selectively modulates Nav1.2 channel activity through the accessory protein fibroblast growth factor 14 (FGF14). Here, we tested whether WEE1 exhibits crosstalk with the AKT/GSK3 kinase pathway for coordinated regulation of FGF14/Nav1.2 channel complex assembly and function. Using the in-cell split luciferase complementation assay (LCA), we found that the WEE1 inhibitor II and GSK3 inhibitor XIII reduce the FGF14/Nav1.2 complex formation, while the AKT inhibitor triciribine increases it. However, combining WEE1 inhibitor II with either one of the other two inhibitors abolished its effect on the FGF14/Nav1.2 complex formation. Whole-cell voltage-clamp recordings of sodium currents (INa) in HEK293 cells co-expressing Nav1.2 channels and FGF14-GFP showed that WEE1 inhibitor II significantly suppresses peak INa density, both alone and in the presence of triciribine or GSK3 inhibitor XIII, despite the latter inhibitor's opposite effects on INa. Additionally, WEE1 inhibitor II slowed the tau of fast inactivation and caused depolarizing shifts in the voltage dependence of activation and inactivation. These phenotypes either prevailed or were additive when combined with triciribine but were outcompeted when both WEE1 inhibitor II and GSK3 inhibitor XIII were present. Concerted regulation by WEE1 inhibitor II, triciribine, and GSK3 inhibitor XIII was also observed in long-term inactivation and use dependency of Nav1.2 currents. Overall, these findings suggest a complex role for WEE1 kinase-in concert with the AKT/GSK3 pathway-in regulating the Nav1.2 channelosome.

Ca2+-saturated calmodulin binds tightly to the N-terminal domain of A-type fibroblast growth factor homologous factors.

Voltage-gated sodium channels (Navs) are tightly regulated by multiple conserved auxiliary proteins, including the four fibroblast growth factor homologous factors (FGFs), which bind the Nav EF-hand like domain (EFL), and calmodulin (CaM), a multifunctional messenger protein that binds the NaV IQ motif. The EFL domain and IQ motif are contiguous regions of NaV cytosolic C-terminal domains (CTD), placing CaM and FGF in close proximity. However, whether the FGFs and CaM act independently, directly associate, or operate through allosteric interactions to regulate channel function is unknown. Titrations monitored by steady-state fluorescence spectroscopy, structural studies with solution NMR, and computational modeling demonstrated for the first time that both domains of (Ca2+)4-CaM (but not apo CaM) directly bind two sites in the N-terminal domain (NTD) of A-type FGF splice variants (FGF11A, FGF12A, FGF13A, and FGF14A) with high affinity. The weaker of the (Ca2+)4-CaM-binding sites was known via electrophysiology to have a role in long-term inactivation of the channel but not known to bind CaM. FGF12A binding to a complex of CaM associated with a fragment of the NaV1.2 CTD increased the Ca2+-binding affinity of both CaM domains, consistent with (Ca2+)4-CaM interacting preferentially with its higher-affinity site in the FGF12A NTD. Thus, A-type FGFs can compete with NaV IQ motifs for (Ca2+)4-CaM. During spikes in the cytosolic Ca2+ concentration that accompany an action potential, CaM may translocate from the NaV IQ motif to the FGF NTD, or the A-type FGF NTD may recruit a second molecule of CaM to the channel.

Cryptic prokaryotic promoters explain instability of recombinant neuronal sodium channels in bacteria.

Mutations in genes encoding the human-brain-expressed voltage-gated sodium (NaV) channels NaV1.1, NaV1.2, and NaV1.6 are associated with a variety of human diseases including epilepsy, autism spectrum disorder, familial migraine, and other neurodevelopmental disorders. A major obstacle hindering investigations of the functional consequences of brain NaV channel mutations is an unexplained instability of the corresponding recombinant complementary DNA (cDNA) when propagated in commonly used bacterial strains manifested by high spontaneous rates of mutation. Here, using a combination of in silico analysis, random and site-directed mutagenesis, we investigated the cause for instability of human NaV1.1 cDNA. We identified nucleotide sequences within the NaV1.1 coding region that resemble prokaryotic promoter-like elements, which are presumed to drive transcription of translationally toxic mRNAs in bacteria as the cause of the instability. We further demonstrated that mutations disrupting these elements mitigate the instability. Extending these observations, we generated full-length human NaV1.1, NaV1.2, and NaV1.6 plasmids using one or two introns that interrupt the latent reading frames along with a minimum number of silent nucleotide changes that achieved stable propagation in bacteria. Expression of the stabilized sequences in cultured mammalian cells resulted in functional NaV channels with properties that matched their parental constructs. Our findings explain a widely observed instability of recombinant neuronal human NaV channels, and we describe re-engineered plasmids that attenuate this problem.

SCN2A-related epilepsy of infancy with migrating focal seizures: report of a variant with apparent gain- and loss-of-function effects.

SCN2A encodes a voltage-gated sodium channel (NaV1.2) expressed throughout the central nervous system in predominantly excitatory neurons. Pathogenic variants in SCN2A are associated with epilepsy and neurodevelopmental disorders. Genotype-phenotype correlations have been described, with loss-of-function variants typically being associated with neurodevelopmental delay and later-onset seizures, whereas gain-of-function variants more often result in early infantile-onset epilepsy. However, the true electrophysiological effects of most disease-causing SCN2A variants have yet to be characterized. We report an infant who presented with migrating focal seizures in the neonatal period. She was found to have a mosaic c.2635G>A, p.Gly879Arg variant in SCN2A. Voltage-clamp studies of the variant expressed on adult and neonatal NaV1.2 isoforms demonstrated a mixed gain and loss of function, with predominantly a loss-of-function effect with reduced cell surface expression and current density. Additional small electrophysiological alterations included a decrease in the voltage dependence of activation and an increase in the voltage dependence of inactivation. This finding of a predominantly loss-of-function effect was unexpected, as the infant's early epilepsy onset would have suggested a predominantly gain-of-function effect. This case illustrates that our understanding of genotype-phenotype correlations is still limited and highlights the complexity of the underlying electrophysiological effects of SCN2A variants.NEW & NOTEWORTHY Voltage-gated sodium channels play an important role in the central nervous system, mutations in which have been reported to be responsible for epilepsy. We report here an infant presenting with epilepsy of infancy with migrating focal seizures (EIMFS) in the neonatal period with a mosaic c.2635G>A, resulting in a p.Gly879Arg missense mutation on the SCN2A gene encoding NaV1.2 sodium channels. Biophysical characterization of this variant revealed a mixture of gain- and loss-of-function effects.

Deficiency of autism-related Scn2a gene in mice disrupts sleep patterns and circadian rhythms.

Autism spectrum disorder (ASD) affects ~2% of the population in the US, and monogenic forms of ASD often result in the most severe manifestation of the disorder. Recently, SCN2A has emerged as a leading gene associated with ASD, of which abnormal sleep pattern is a common comorbidity. SCN2A encodes the voltage-gated sodium channel NaV1.2. Predominantly expressed in the brain, NaV1.2 mediates the action potential firing of neurons. Clinical studies found that a large portion of children with SCN2A deficiency have sleep disorders, which severely impact the quality of life of affected individuals and their caregivers. The underlying mechanism of sleep disturbances related to NaV1.2 deficiency, however, is not known. Using a gene-trap Scn2a-deficient mouse model (Scn2atrap), we found that Scn2a deficiency results in increased wakefulness and reduced non-rapid-eye-movement (NREM) sleep. Brain region-specific Scn2a deficiency in the suprachiasmatic nucleus (SCN) containing region, which is involved in circadian rhythms, partially recapitulates the sleep disturbance phenotypes. At the cellular level, we found that Scn2a deficiency disrupted the firing pattern of spontaneously firing neurons in the SCN region. At the molecular level, RNA-sequencing analysis revealed differentially expressed genes in the circadian entrainment pathway including core clock genes Per1 and Per2. Performing a transcriptome-based compound discovery, we identified dexanabinol (HU-211), a putative glutamate receptor modulator, that can partially reverse the sleep disturbance in mice. Overall, our study reveals possible molecular and cellular mechanisms underlying Scn2a deficiency-related sleep disturbances, which may inform the development of potential pharmacogenetic interventions for the affected individuals.

Comprehensive engineering of the tarantula venom peptide huwentoxin-IV to inhibit the human voltage-gated sodium channel hNav1.7.

Pain is a significant public health burden in the United States, and current treatment approaches rely heavily on opioids, which often have limited efficacy and can lead to addiction. In humans, functional loss of the voltage-gated sodium channel Nav1.7 leads to pain insensitivity without deficits in the central nervous system. Accordingly, discovery of a selective Nav1.7 antagonist should provide an analgesic without abuse liability and an improved side-effect profile. Huwentoxin-IV, a component of tarantula venom, potently blocks sodium channels and is an attractive scaffold for engineering a Nav1.7-selective molecule. To define the functional impact of alterations in huwentoxin-IV sequence, we produced a library of 373 point mutants and tested them for Nav1.7 and Nav1.2 activity. We then combined favorable individual changes to produce combinatorial mutants that showed further improvements in Nav1.7 potency (E1N, E4D, Y33W, Q34S-Nav1.7 pIC50 = 8.1 ± 0.08) and increased selectivity over other Nav isoforms (E1N, R26K, Q34S, G36I, Nav1.7 pIC50 = 7.2 ± 0.1, Nav1.2 pIC50 = 6.1 ± 0.18, Nav1.3 pIC50 = 6.4 ± 1.0), Nav1.4 is inactive at 3 µm, and Nav1.5 is inactive at 10 µm We also substituted noncoded amino acids at select positions in huwentoxin-IV. Based on these results, we identify key determinants of huwentoxin's Nav1.7 inhibition and propose a model for huwentoxin-IV's interaction with Nav1.7. These findings uncover fundamental features of huwentoxin involved in Nav1.7 blockade, provide a foundation for additional optimization of this molecule, and offer a basis for the development of a safe and effective analgesic.

Properties of Calmodulin Binding to NaV1.2 IQ Motif and Its Autism-Associated Mutation R1902C.

Voltage-gated sodium channels (VGSCs) are fundamental to the initiation and propagation of action potentials in excitable cells. Ca2+/calmodulin (CaM) binds to VGSC type II (NaV1.2) isoleucine and glutamine (IQ) motif. An autism-associated mutation in NaV1.2 IQ motif, Arg1902Cys (R1902C), has been reported to affect the combination between CaM and the IQ motif compared to that of the wild type IQ motif. However, the detailed properties for the Ca2+-regulated binding of CaM to NaV1.2 IQ (1901Lys-1927Lys, IQwt) and mutant IQ motif (IQR1902C) remains unclear. Here, the binding ability of CaM and CaM's constituent proteins including N- and C lobe to the IQ motif of NaV1.2 and its mutant was investigated by protein pull-down experiments. We discovered that the combination between CaM and the IQ motif was U-shaped with the highest at [Ca2+] ≈ free and the lowest at 100 nM [Ca2+]. In the IQR1902C mutant, Ca2+-dependence of CaM binding was nearly lost. Consequently, the binding of CaM to IQR1902C at 100 and 500 nM [Ca2+] was increased compared to that of IQwt. Both N- and C lobe of CaM could bind with NaV1.2 IQ motif and IQR1902C mutant, with the major effect of C lobe. Furthermore, CaMKII had no impact on the binding between CaM and NaV1.2 IQ motif. This research offers novel insight to the regulation of NaV1.2 IQwt and IQR1902C motif, an autism-associated mutation, by CaM.

Axonal sodium channel NaV1.2 drives granule cell dendritic GABA release and rapid odor discrimination.

Dendrodendritic synaptic interactions between olfactory bulb mitral and granule cells represent a key neuronal mechanism of odor discrimination. Dendritic release of gamma-aminobutyric acid (GABA) from granule cells contributes to stimulus-dependent, rapid, and accurate odor discrimination, yet the physiological mechanisms governing this release and its behavioral relevance are unknown. Here, we show that granule cells express the voltage-gated sodium channel α-subunit NaV1.2 in clusters distributed throughout the cell surface including dendritic spines. Deletion of NaV1.2 in granule cells abolished spiking and GABA release as well as inhibition of synaptically connected mitral cells (MCs). As a consequence, mice required more time to discriminate highly similar odorant mixtures, while odor discrimination learning remained unaffected. In conclusion, we show that expression of NaV1.2 in granule cells is crucial for physiological dendritic GABA release and rapid discrimination of similar odorants with high accuracy. Hence, our data indicate that neurotransmitter-releasing dendritic spines function just like axon terminals.

Role of sodium channel subtype in action potential generation by neocortical pyramidal neurons.

Neocortical pyramidal neurons express several distinct subtypes of voltage-gated Na+ channels. In mature cells, Nav1.6 is the dominant channel subtype in the axon initial segment (AIS) as well as in the nodes of Ranvier. Action potentials (APs) are initiated in the AIS, and it has been proposed that the high excitability of this region is related to the unique characteristics of the Nav1.6 channel. Knockout or loss-of-function mutation of the Scn8a gene is generally lethal early in life because of the importance of this subtype in noncortical regions of the nervous system. Using the Cre/loxP system, we selectively deleted Nav1.6 in excitatory neurons of the forebrain and characterized the excitability of Nav1.6-deficient layer 5 pyramidal neurons by patch-clamp and Na+ and Ca2+ imaging recordings. We now report that, in the absence of Nav1.6 expression, the AIS is occupied by Nav1.2 channels. However, APs are generated in the AIS, and differences in AP propagation to soma and dendrites are minimal. Moreover, the channels that are expressed in the AIS still show a clear hyperpolarizing shift in voltage dependence of activation, compared with somatic channels. The only major difference between Nav1.6-null and wild-type neurons was a strong reduction in persistent sodium current. We propose that the molecular environment of the AIS confers properties on whatever Na channel subtype is present and that some other benefit must be conferred by the selective axonal presence of the Nav1.6 channel.

Elevated ocular pressure reduces voltage-gated sodium channel NaV1.2 protein expression in retinal ganglion cell axons.

Glaucoma is an age-related neurodegenerative disease that is commonly associated with sensitivity to intraocular pressure. The disease selectively targets retinal ganglion cells (RGCs) and constituent axons. RGC axons are rich in voltage-gated sodium channels, which are essential for action potential initiation and regeneration. Here, we identified voltage-dependent sodium channel, NaV1.2, in the retina, examined how this channel contributes to RGC light responses, and monitored NaV1.2 mRNA and protein expression in the retina during progression of modeled glaucoma. We found NaV1.2 is predominately localized in ganglion cell intraretinal axons with dispersed expression in the outer and inner plexiform layers. We showed Phrixotoxin-3, a potent NaV1.2 channel blocker, significantly decreased RGC electrical activity in a dose-dependent manner with an IC50 of 40 nM. Finally, we found four weeks of raised intraocular pressure (30% above baseline) significantly increased NaV1.2 mRNA expression but reduced NaV1.2 protein level in the retina up to 57% (p < 0.001). Following prolonged intraocular pressure elevation, NaV1.2 protein expression particularly diminished at distal sections of ganglion cell intraretinal axons (p ≤ 0.01). Our results suggest NaV1.2 might be a therapeutic target during disease progression to maintain RGC excitability, preserving presynaptic connections through action potential backpropagation.

Biological concepts in human sodium channel epilepsies and their relevance in clinical practice.

OBJECTIVE: Voltage-gated sodium channels (SCNs) share similar amino acid sequence, structure, and function. Genetic variants in the four human brain-expressed SCN genes SCN1A/2A/3A/8A have been associated with heterogeneous epilepsy phenotypes and neurodevelopmental disorders. To better understand the biology of seizure susceptibility in SCN-related epilepsies, our aim was to determine similarities and differences between sodium channel disorders, allowing us to develop a broader perspective on precision treatment than on an individual gene level alone. METHODS: We analyzed genotype-phenotype correlations in large SCN-patient cohorts and applied variant constraint analysis to identify severe sodium channel disease. We examined temporal patterns of human SCN expression and correlated functional data from in vitro studies with clinical phenotypes across different sodium channel disorders. RESULTS: Comparing 865 epilepsy patients (504 SCN1A, 140 SCN2A, 171 SCN8A, four SCN3A, 46 copy number variation [CNV] cases) and analysis of 114 functional studies allowed us to identify common patterns of presentation. All four epilepsy-associated SCN genes demonstrated significant constraint in both protein truncating and missense variation when compared to other SCN genes. We observed that age at seizure onset is related to SCN gene expression over time. Individuals with gain-of-function SCN2A/3A/8A missense variants or CNV duplications share similar characteristics, most frequently present with early onset epilepsy (<3 months), and demonstrate good response to sodium channel blockers (SCBs). Direct comparison of corresponding SCN variants across different SCN subtypes illustrates that the functional effects of variants in corresponding channel locations are similar; however, their clinical manifestation differs, depending on their role in different types of neurons in which they are expressed. SIGNIFICANCE: Variant function and location within one channel can serve as a surrogate for variant effects across related sodium channels. Taking a broader view on precision treatment suggests that in those patients with a suspected underlying genetic epilepsy presenting with neonatal or early onset seizures (<3 months), SCBs should be considered.

CaMKII modulates sodium current in neurons from epileptic Scn2a mutant mice.

Monogenic epilepsies with wide-ranging clinical severity have been associated with mutations in voltage-gated sodium channel genes. In the Scn2aQ54 mouse model of epilepsy, a focal epilepsy phenotype is caused by transgenic expression of an engineered NaV1.2 mutation displaying enhanced persistent sodium current. Seizure frequency and other phenotypic features in Scn2aQ54 mice depend on genetic background. We investigated the neurophysiological and molecular correlates of strain-dependent epilepsy severity in this model. Scn2aQ54 mice on the C57BL/6J background (B6.Q54) exhibit a mild disorder, whereas animals intercrossed with SJL/J mice (F1.Q54) have a severe phenotype. Whole-cell recording revealed that hippocampal pyramidal neurons from B6.Q54 and F1.Q54 animals exhibit spontaneous action potentials, but F1.Q54 neurons exhibited higher firing frequency and greater evoked activity compared with B6.Q54 neurons. These findings correlated with larger persistent sodium current and depolarized inactivation in neurons from F1.Q54 animals. Because calcium/calmodulin protein kinase II (CaMKII) is known to modify persistent current and channel inactivation in the heart, we investigated CaMKII as a plausible modulator of neuronal sodium channels. CaMKII activity in hippocampal protein lysates exhibited a strain-dependence in Scn2aQ54 mice with higher activity in F1.Q54 animals. Heterologously expressed NaV1.2 channels exposed to activated CaMKII had enhanced persistent current and depolarized channel inactivation resembling the properties of F1.Q54 neuronal sodium channels. By contrast, inhibition of CaMKII attenuated persistent current, evoked a hyperpolarized channel inactivation, and suppressed neuronal excitability. We conclude that CaMKII-mediated modulation of neuronal sodium current impacts neuronal excitability in Scn2aQ54 mice and may represent a therapeutic target for the treatment of epilepsy.

New Sea Anemone Toxin RTX-VI Selectively Modulates Voltage-Gated Sodium Channels.

A new neurotoxin RTX-VI that modulates the voltage-gated sodium channels (NaV) was isolated from the ethanolic extract of the sea anemone Heteractis crispa. Its amino acid sequence was determined using the combination of Edman degradation and tandem mass spectrometry. RTX-VI turned out to be an unusual natural analogue of the previously described sea anemone toxin RTX-III. The RTX-VI molecule consists of two disulfide-linked peptide chains and is devoid of Arg13, which is important for the selectivity and affinity of such peptides for the NaV channels. Electrophysiological screening of RTV-VI on NaV channel subtypes showed its selective interaction with the central nervous system (NaV1.2, NaV1.6) and insect (BgNaV1, VdNaV1) sodium channels.

[Effect of calmodulin and its mutants on binding to NaV1.2 IQ].

OBJECTIVE: To investigate the effect of calmodulin (CaM) and its mutants on binding to voltage-gated Na channel isoleucine-glutamine domain (NaV1.2 IQ). METHODS: The cDNA of NaV1.2 IQ was constructed by PCR technique, CaM mutants CaM12, CaM34 and CaM1234 were constructed with QuickchangeTM site-directed mutagenesis kit (QIAGEN). The binding of NaV1.2 IQ to CaM and CaM mutants under calcium and calcium free conditions were detected by pull-down assay. RESULTS: NaV1.2 IQ and CaM were bound to each other at different calcium concentrations, while GST alone did not bind to CaM. The binding affinity of CaM and NaV1.2 IQ at [Ca2+]-free was greater than that at 100 nmol/L [Ca2+] (P < 0.05). In the absence of calcium, the binding amount of CaM wild-type to NaV1.2 IQ was greater than that of its mutant, and the binding affinity of CaM1234 to NaV1.2 IQ was the weakest among the three mutants (P < 0.05). CONCLUSIONS: The binding ability of CaM and CaM mutants to NaV1.2 IQ is Ca2+-dependent. This study has revealed a new mechanism of NaV1.2 regulated by CaM, which would be useful for the study of ion channel related diseases.

Scn2a deletion improves survival and brain-heart dynamics in the Kcna1-null mouse model of sudden unexpected death in epilepsy (SUDEP).

People with epilepsy have greatly increased probability of premature mortality due to sudden unexpected death in epilepsy (SUDEP). Identifying which patients are most at risk of SUDEP is hindered by a complex genetic etiology, incomplete understanding of the underlying pathophysiology and lack of prognostic biomarkers. Here we evaluated heterozygous Scn2a gene deletion (Scn2a+/-) as a protective genetic modifier in the Kcna1 knockout mouse (Kcna1-/-) model of SUDEP, while searching for biomarkers of SUDEP risk embedded in electroencephalography (EEG) and electrocardiography (ECG) recordings. The human epilepsy gene Kcna1 encodes voltage-gated Kv1.1 potassium channels that act to dampen neuronal excitability whereas Scn2a encodes voltage-gated Nav1.2 sodium channels important for action potential initiation and conduction. SUDEP-prone Kcna1-/- mice with partial genetic ablation of Nav1.2 channels (i.e. Scn2a+/-; Kcna1-/-) exhibited a two-fold increase in survival. Classical analysis of EEG and ECG recordings separately showed significantly decreased seizure durations in Scn2a+/-; Kcna1-/- mice compared with Kcna1-/- mice, without substantial modification of cardiac abnormalities. Novel analysis of the EEG and ECG together revealed a significant reduction in EEG-ECG association in Kcna1-/- mice compared with wild types, which was partially restored in Scn2a+/-; Kcna1-/- mice. The degree of EEG-ECG association was also proportional to the survival rate of mice across genotypes. These results show that Scn2a gene deletion acts as protective genetic modifier of SUDEP and suggest measures of brain-heart association as potential indices of SUDEP susceptibility.

PRRT2 controls neuronal excitability by negatively modulating Na+ channel 1.2/1.6 activity.

See Lerche (doi:10.1093/brain/awy073) for a scientific commentary on this article.Proline-rich transmembrane protein 2 (PRRT2) is the causative gene for a heterogeneous group of familial paroxysmal neurological disorders that include seizures with onset in the first year of life (benign familial infantile seizures), paroxysmal kinesigenic dyskinesia or a combination of both. Most of the PRRT2 mutations are loss-of-function leading to haploinsufficiency and 80% of the patients carry the same frameshift mutation (c.649dupC; p.Arg217Profs*8), which leads to a premature stop codon. To model the disease and dissect the physiological role of PRRT2, we studied the phenotype of neurons differentiated from induced pluripotent stem cells from previously described heterozygous and homozygous siblings carrying the c.649dupC mutation. Single-cell patch-clamp experiments on induced pluripotent stem cell-derived neurons from homozygous patients showed increased Na+ currents that were fully rescued by expression of wild-type PRRT2. Closely similar electrophysiological features were observed in primary neurons obtained from the recently characterized PRRT2 knockout mouse. This phenotype was associated with an increased length of the axon initial segment and with markedly augmented spontaneous and evoked firing and bursting activities evaluated, at the network level, by multi-electrode array electrophysiology. Using HEK-293 cells stably expressing Nav channel subtypes, we demonstrated that the expression of PRRT2 decreases the membrane exposure and Na+ current of Nav1.2/Nav1.6, but not Nav1.1, channels. Moreover, PRRT2 directly interacted with Nav1.2/Nav1.6 channels and induced a negative shift in the voltage-dependence of inactivation and a slow-down in the recovery from inactivation. In addition, by co-immunoprecipitation assays, we showed that the PRRT2-Nav interaction also occurs in brain tissue. The study demonstrates that the lack of PRRT2 leads to a hyperactivity of voltage-dependent Na+ channels in homozygous PRRT2 knockout human and mouse neurons and that, in addition to the reported synaptic functions, PRRT2 is an important negative modulator of Nav1.2 and Nav1.6 channels. Given the predominant paroxysmal character of PRRT2-linked diseases, the disturbance in cellular excitability by lack of negative modulation of Na+ channels appears as the key pathogenetic mechanism.

Mutant cycle analysis with modified saxitoxins reveals specific interactions critical to attaining high-affinity inhibition of hNaV1.7.

Improper function of voltage-gated sodium channels (NaVs), obligatory membrane proteins for bioelectrical signaling, has been linked to a number of human pathologies. Small-molecule agents that target NaVs hold considerable promise for treatment of chronic disease. Absent a comprehensive understanding of channel structure, the challenge of designing selective agents to modulate the activity of NaV subtypes is formidable. We have endeavored to gain insight into the 3D architecture of the outer vestibule of NaV through a systematic structure-activity relationship (SAR) study involving the bis-guanidinium toxin saxitoxin (STX), modified saxitoxins, and protein mutagenesis. Mutant cycle analysis has led to the identification of an acetylated variant of STX with unprecedented, low-nanomolar affinity for human NaV1.7 (hNaV1.7), a channel subtype that has been implicated in pain perception. A revised toxin-receptor binding model is presented, which is consistent with the large body of SAR data that we have obtained. This new model is expected to facilitate subsequent efforts to design isoform-selective NaV inhibitors.