Comparative structural analysis of human Nav1.1 and Nav1.5 reveals mutational hotspots for sodium channelopathies.

Among the nine subtypes of human voltage-gated sodium (Nav) channels, the brain and cardiac isoforms, Nav1.1 and Nav1.5, each carry more than 400 missense mutations respectively associated with epilepsy and cardiac disorders. High-resolution structures are required for structure-function relationship dissection of the disease variants. We report the cryo-EM structures of the full-length human Nav1.1-ß4 complex at 3.3 Å resolution here and the Nav1.5-E1784K variant in the accompanying paper. Up to 341 and 261 disease-related missense mutations in Nav1.1 and Nav1.5, respectively, are resolved. Comparative structural analysis reveals several clusters of disease mutations that are common to both Nav1.1 and Nav1.5. Among these, the majority of mutations on the extracellular loops above the pore domain and the supporting segments for the selectivity filter may impair structural integrity, while those on the pore domain and the voltage-sensing domains mostly interfere with electromechanical coupling and fast inactivation. Our systematic structural delineation of these mutations provides important insight into their pathogenic mechanism, which will facilitate the development of precise therapeutic interventions against various sodium channelopathies.

Selective spider toxins reveal a role for the Nav1.1 channel in mechanical pain.

Voltage-gated sodium (Nav) channels initiate action potentials in most neurons, including primary afferent nerve fibres of the pain pathway. Local anaesthetics block pain through non-specific actions at all Nav channels, but the discovery of selective modulators would facilitate the analysis of individual subtypes of these channels and their contributions to chemical, mechanical, or thermal pain. Here we identify and characterize spider (Heteroscodra maculata) toxins that selectively activate the Nav1.1 subtype, the role of which in nociception and pain has not been elucidated. We use these probes to show that Nav1.1-expressing fibres are modality-specific nociceptors: their activation elicits robust pain behaviours without neurogenic inflammation and produces profound hypersensitivity to mechanical, but not thermal, stimuli. In the gut, high-threshold mechanosensitive fibres also express Nav1.1 and show enhanced toxin sensitivity in a mouse model of irritable bowel syndrome. Together, these findings establish an unexpected role for Nav1.1 channels in regulating the excitability of sensory nerve fibres that mediate mechanical pain.

Inhibitory effects of cannabidiol on voltage-dependent sodium currents.

Cannabis sativa contains many related compounds known as phytocannabinoids. The main psychoactive and nonpsychoactive compounds are Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD), respectively. Much of the evidence for clinical efficacy of CBD-mediated antiepileptic effects has been from case reports or smaller surveys. The mechanisms for CBD's anticonvulsant effects are unclear and likely involve noncannabinoid receptor pathways. CBD is reported to modulate several ion channels, including sodium channels (Nav). Evaluating the therapeutic mechanisms and safety of CBD demands a richer understanding of its interactions with central nervous system targets. Here, we used voltage-clamp electrophysiology of HEK-293 cells and iPSC neurons to characterize the effects of CBD on Nav channels. Our results show that CBD inhibits hNav1.1-1.7 currents, with an IC50 of 1.9-3.8 µm, suggesting that this inhibition could occur at therapeutically relevant concentrations. A steep Hill slope of ∼3 suggested multiple interactions of CBD with Nav channels. CBD exhibited resting-state blockade, became more potent at depolarized potentials, and also slowed recovery from inactivation, supporting the idea that CBD binding preferentially stabilizes inactivated Nav channel states. We also found that CBD inhibits other voltage-dependent currents from diverse channels, including bacterial homomeric Nav channel (NaChBac) and voltage-gated potassium channel subunit Kv2.1. Lastly, the CBD block of Nav was temperature-dependent, with potency increasing at lower temperatures. We conclude that CBD's mode of action likely involves 1) compound partitioning in lipid membranes, which alters membrane fluidity affecting gating, and 2) undetermined direct interactions with sodium and potassium channels, whose combined effects are loss of channel excitability.

Late sodium current blocker GS967 inhibits persistent currents induced by familial hemiplegic migraine type 3 mutations of the SCN1A gene.

BACKGROUND: Familial hemiplegic migraine (FHM) is a group of genetic migraine, associated with hemiparesis and aura. Three causative different genes have been identified, all of which are involved in membrane ion transport. Among these, SCN1A encodes the voltage-gated Na+ channel Nav1.1, and FHM caused by mutations of SCN1A is named FHM3. For 7 of the 12 known FHM3-causing SCNA1 mutations functional consequences have been investigated, and even if gain of function effect seems to be a predominant phenotype, for several mutations conflicting results have been obtained and the available data do not reveal a univocal FHM3 pathomechanism. METHODS: To obtain a more complete picture, here, we characterized by patch clamp approach the remaining 5 mutations (Q1489H, I1498M, F1499 L, M1500 V, F1661 L) in heterologous expression systems. RESULTS: With the exception of I1498M, all mutants exhibited the same current density as WT and exhibited a shift of the steady state inactivation to more positive voltages, an accelerated recovery from inactivation, and an increase of the persistent current, revealing that most FHM3 mutations induce a gain of function. We also determined the effect of GS967, a late Na+ current blocker, on the above mentioned mutants as well as on previously characterized ones (L1649Q, L1670 W, F1774S). GS967 inhibited persistent currents of all SCNA1 FMH3-related mutants and dramatically slowed the recovery from fast inactivation of WT and mutants, consistent with the hypothesis that GS967 specifically binds to and thereby stabilizes the fast inactivated state. Simulation of neuronal firing showed that enhanced persistent currents cause an increase of ionic fluxes during action potential repolarization and consequent accumulation of K+ and/or exhaustion of neuronal energy resources. In silico application of GS967 largely reduced net ionic currents in neurons without impairing excitability. CONCLUSION: In conclusion, late Na+ current blockers appear a promising specific pharmacological treatment of FHM3.

Design, synthesis, biological evaluation, homology modeling and docking studies of (E)-3-(benzo[d][1,3]dioxol-5-ylmethylene) pyrrolidin-2-one derivatives as potent anticonvulsant agents.

A series of (E)-3-(benzo[d][1,3]dioxol-5-ylmethylene)pyrrolidin-2-one derivatives were designed, synthesized, and evaluated for their anticonvulsant activities. In the preliminary screening, compounds 5, 6a-6f and 6h-6i showed promising anticonvulsant activities in MES model, while 6f and 6g represented protection against seizures at doses of 100 mg/kg and 0.5 h in scPTZ model. The most active compound 6d had a high-degree protection against the MES-induced seizures with ED50 value of 4.3 mg/kg and TD50 value of 160.9 mg/kg after intraperitoneal (i.p.) injection in mice, which provided 6d in a high protective index (TD50/ED50) of 37.4 comparable to the reference drugs. Beyond that, 6d has been selected and evaluated in vitro experiment to estimate the activation impact. Apparently, 6d clearly inhibits the Nav1.1 channel. Our preliminary results provide new insights for the development of small-molecule activators targeting specifically Nav1.1 channels to design potential drugs for treating epilepsy. The computational parameters, such as homology modeling, docking study, and ADME prediction, were made to exploit the results.

The Effect of Ca2+, Lobe-Specificity, and CaMKII on CaM Binding to NaV1.1.

Calmodulin (CaM) is well known as an activator of calcium/calmodulin-dependent protein kinase II (CaMKII). Voltage-gated sodium channels (VGSCs) are basic signaling molecules in excitable cells and are crucial molecular targets for nervous system agents. However, the way in which Ca2+/CaM/CaMKII cascade modulates NaV1.1 IQ (isoleucine and glutamine) domain of VGSCs remains obscure. In this study, the binding of CaM, its mutants at calcium binding sites (CaM12, CaM34, and CaM1234), and truncated proteins (N-lobe and C-lobe) to NaV1.1 IQ domain were detected by pull-down assay. Our data showed that the binding of Ca2+/CaM to the NaV1.1 IQ was concentration-dependent. ApoCaM (Ca2+-free form of calmodulin) bound to NaV1.1 IQ domain preferentially more than Ca2+/CaM. Additionally, the C-lobe of CaM was the predominant domain involved in apoCaM binding to NaV1.1 IQ domain. By contrast, the N-lobe of CaM was predominant in the binding of Ca2+/CaM to NaV1.1 IQ domain. Moreover, CaMKII-mediated phosphorylation increased the binding of Ca2+/CaM to NaV1.1 IQ domain due to one or several phosphorylation sites in T1909, S1918, and T1934 of NaV1.1 IQ domain. This study provides novel mechanisms for the modulation of NaV1.1 by the Ca2+/CaM/CaMKII axis. For the first time, we uncover the effect of Ca2+, lobe-specificity and CaMKII on CaM binding to NaV1.1.

The Receptor Site and Mechanism of Action of Sodium Channel Blocker Insecticides.

Sodium channels are excellent targets of both natural and synthetic insecticides with high insect selectivity. Indoxacarb, its active metabolite DCJW, and metaflumizone (MFZ) belong to a relatively new class of sodium channel blocker insecticides (SCBIs) with a mode of action distinct from all other sodium channel-targeting insecticides, including pyrethroids. Electroneutral SCBIs preferably bind to and trap sodium channels in the inactivated state, a mechanism similar to that of cationic local anesthetics. Previous studies identified several SCBI-sensing residues that face the inner pore of sodium channels. However, the receptor site of SCBIs, their atomic mechanisms, and the cause of selective toxicity of MFZ remain elusive. Here, we have built a homology model of the open-state cockroach sodium channel BgNav1-1a. Our computations predicted that SCBIs bind in the inner pore, interact with a sodium ion at the focus of P1 helices, and extend their aromatic moiety into the III/IV domain interface (fenestration). Using model-driven mutagenesis and electrophysiology, we identified five new SCBI-sensing residues, including insect-specific residues. Our study proposes the first three-dimensional models of channel-bound SCBIs, sheds light on the molecular basis of MFZ selective toxicity, and suggests that a sodium ion located in the inner pore contributes to the receptor site for electroneutral SCBIs.

Evidence for Dual Binding Sites for 1,1,1-Trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) in Insect Sodium Channels.

1,1,1-Trichloro-2,2-bis(p-chlorophenyl)ethane (DDT), the first organochlorine insecticide, and pyrethroid insecticides are sodium channel agonists. Although the use of DDT is banned in most of the world due to its detrimental impact on the ecosystem, indoor residual spraying of DDT is still recommended for malaria control in Africa. Development of resistance to DDT and pyrethroids is a serious global obstacle for managing disease vectors. Mapping DDT binding sites is necessary for understanding mechanisms of resistance and modulation of sodium channels by structurally different ligands. The pioneering model of the housefly sodium channel visualized the first receptor for pyrethroids, PyR1, in the II/III domain interface and suggested that DDT binds within PyR1. Previously, we proposed the second pyrethroid receptor, PyR2, at the I/II domain interface. However, whether DDT binds to both pyrethroid receptor sites remains unknown. Here, using computational docking of DDT into the Kv1.2-based mosquito sodium channel model, we predict that two DDT molecules can bind simultaneously within PyR1 and PyR2. The bulky trichloromethyl group of each DDT molecule fits snugly between four helices in the bent domain interface, whereas two p-chlorophenyl rings extend into two wings of the interface. Model-driven mutagenesis and electrophysiological analysis confirmed these propositions and revealed 10 previously unknown DDT-sensing residues within PyR1 and PyR2. Our study proposes a dual DDT-receptor model and provides a structural background for rational development of new insecticides.

Prokaryotic NavMs channel as a structural and functional model for eukaryotic sodium channel antagonism.

Voltage-gated sodium channels are important targets for the development of pharmaceutical drugs, because mutations in different human sodium channel isoforms have causal relationships with a range of neurological and cardiovascular diseases. In this study, functional electrophysiological studies show that the prokaryotic sodium channel from Magnetococcus marinus (NavMs) binds and is inhibited by eukaryotic sodium channel blockers in a manner similar to the human Nav1.1 channel, despite millions of years of divergent evolution between the two types of channels. Crystal complexes of the NavMs pore with several brominated blocker compounds depict a common antagonist binding site in the cavity, adjacent to lipid-facing fenestrations proposed to be the portals for drug entry. In silico docking studies indicate the full extent of the blocker binding site, and electrophysiology studies of NavMs channels with mutations at adjacent residues validate the location. These results suggest that the NavMs channel can be a valuable tool for screening and rational design of human drugs.

Benchmarking the stability of human detergent-solubilised voltage-gated sodium channels for structural studies using eel as a reference.

With the ultimate goal of detailed structural analysis of mammalian and particularly human voltage-gated sodium channels (VGSCs), we have investigated the relative stability of human and rat VGSCs and compared them with electric eel VGSC. We found that NaV1.3 from rat was the most stable after detergent solubilisation. The order of stability was rNaV1.3>hNaV1.2>hNaV1.1>hNaV1.6>hNaV1.3>hNaV1.4. However, a comparison with the VGSC from Electrophorus electricus, which is most similar to NaV1.4, shows that the eel VGSC is considerably more stable in detergent than the human VGSCs examined. We conclude that current methods of structural analysis, such as single particle electron cryomicroscopy (cryoEM), may be most usefully targeted to eel VGSC or rNaV1.3, but that structural analysis on the full spectrum of VGSCs, by methods that require greater stability such as crystallisation and X-ray crystallography, will require further stabilisation of the channel.

Nonfunctional NaV1.1 familial hemiplegic migraine mutant transformed into gain of function by partial rescue of folding defects.

Familial hemiplegic migraine (FHM) is a rare subtype of migraine with aura. Mutations causing FHM type 3 have been identified in SCN1A, the gene encoding the Nav1.1 Na(+) channel, which is also a major target of epileptogenic mutations and is particularly important for the excitability of GABAergic neurons. However, functional studies of NaV1.1 FHM mutations have generated controversial results. In particular, it has been shown that the NaV1.1-L1649Q mutant is nonfunctional when expressed in a human cell line because of impaired plasma membrane expression, similarly to NaV1.1 mutants that cause severe epilepsy, but we have observed gain-of-function effects for other NaV1.1 FHM mutants. Here we show that NaV1.1-L1649Q is nonfunctional because of folding defects that are rescuable by incubation at lower temperatures or coexpression of interacting proteins, and that a partial rescue is sufficient for inducing an overall gain of function because of the modifications in gating properties. Strikingly, when expressed in neurons, the mutant was partially rescued and was a constitutive gain of function. A computational model showed that 35% rescue can be sufficient for inducing gain of function. Interestingly, previously described folding-defective epileptogenic NaV1.1 mutants show loss of function also when rescued. Our results are consistent with gain of function as the functional effect of NaV1.1 FHM mutations and hyperexcitability of GABAergic neurons as the pathomechanism of FHM type 3.

Rotational Symmetry of Two Pyrethroid Receptor Sites in the Mosquito Sodium Channel.

Voltage-gated sodium channels are the primary target of pyrethroid insecticides. Although it is well known that specific mutations in insect sodium channels confer knockdown resistance (kdr) to pyrethroids, the atomic mechanisms of pyrethroid-sodium channel interactions are not clearly understood. Previously, computer modeling and mutational analysis predicted two pyrethroid receptors, pyrethroid receptor site 1 (PyR1) (initial) and pyrethroid receptor site 2 (PyR2), located in the domain interfaces II/III and I/II, respectively. The models differ in ligand orientation and the number of transmembrane helices involved. In this study, we elaborated a revised PyR1 model of the mosquito sodium channel. Computational docking in the Kv1.2-based open channel model yielded a complex in which a pyrethroid (deltamethrin) binds between the linker helix IIL45 and transmembrane helices IIS5, IIS6, and IIIS6 with its dibromoethenyl and diphenylether moieties oriented in the intra- and extracellular directions, respectively. The PyR2 and revised PyR1 models explained recently discovered kdr mutations and predicted new deltamethrin-channel contacts. Further model-driven mutagenesis identified seven new pyrethroid-sensing residues, three in the revised PyR1 and four in PyR2. Our data support the following conclusions: 1) each pyrethroid receptor is formed by a linker-helix L45 and three transmembrane helices (S5 and two S6s); 2) IIS6 contains four residues that contribute to PyR1 and another four to PyR2; 3) seven pairs of pyrethroid-sensing residues are located in symmetric positions within PyR1 and PyR2; and 4) pyrethroids bind to PyR1 and PyR2 in similar orientations, penetrating deeply into the respective domain interfaces. Our study elaborates the dual pyrethroid-receptor sites concept and provides a structural background for rational development of new insecticides.

Rescuable folding defective NaV1.1 (SCN1A) mutants in epilepsy: properties, occurrence, and novel rescuing strategy with peptides targeted to the endoplasmic reticulum.

Mutations of the voltage gated Na(+) channel Na(V)1.1 (SCN1A) are important causes of different genetic epilepsies and can also cause familial hemiplegic migraine (FHM-III). In previous studies, some rescuable epileptogenic folding defective mutants located in domain IV of Na(V)1.1 have been identified, showing partial loss of function also with maximal rescue. Variable rescue may be one of the causes of phenotypic variability, and rescue might be exploited for therapeutic approaches. Recently, we have identified a folding defective FHM-III Na(V)1.1 mutant that showed overall gain of function when rescued, consistent with a differential pathomechanism. Here, we have evaluated functional properties and cell surface expression of six Na(V)1.1 epileptogenic missense mutations in different rescuing conditions, including a novel one that we have developed expressing a selective sodium channel toxin (CsEI) targeted to the endoplasmic reticulum (ER). All the mutants showed loss of function and reduced cell surface expression, consistently with possibility of rescue. Four of them were rescuable by incubation at low temperature and interactions with different co-expressed proteins or a pharmacological chaperone (phenytoin). Notably, CsEI was able to rescue four mutants. Thus, Na(V)1.1 folding defective mutants can be relatively common and mutations inducing rescuable folding defects are spread in all Na(V)1.1 domains. Importantly, epileptogenic mutants showed overall loss of function even upon rescue, differently than FHM-III ones. The effectiveness of CsEI demonstrates that interactions in the ER are sufficient for inducing rescue, and provides a proof of concept for developing possible therapeutic approaches that may overcome some limitations of pharmacological chaperones.

Ranolazine reduces neuronal excitability by interacting with inactivated states of brain sodium channels.

Ranolazine is an approved drug for chronic stable angina that acts by suppressing a noninactivating current conducted by the cardiac sodium channel [persistent sodium ion current (INa)]. Ranolazine has also been shown to inhibit the increased persistent INa carried by NaV1.1 channels encoding epilepsy- and migraine-associated mutations. Here, we investigate the antiepileptic properties of ranolazine exhibited through the reduction of hippocampal neuronal excitability. At therapeutically relevant concentrations, ranolazine reduced action potential firing frequency of hippocampal neurons in response to repetitive depolarizing current injections. Similarly, using a single current injection paradigm, ranolazine required a long depolarization (4 seconds) to produce significant inhibition of excitability, which was similar to that observed for the anticonvulsants phenytoin (slowly binds to the fast-inactivated state) and lacosamide (binds to the slow-inactivated state). Ranolazine enhanced the development of fast and slow inactivation assessed with conditioning prepulses of 100, 1000, or 10,000 milliseconds. Recovery of channels from inactivated states was also slowed in the presence of ranolazine. Interestingly, the use-dependent inhibition of hippocampal neurons was dependent on the duration of the voltage step, suggesting ranolazine does not selectively affect the open state and may also interact with inactivated states. NEURON (Yale University, New Haven, CT) computational simulations predict equal inhibition of action potential generation for binding to either fast-inactivated or slow-inactivated states. Binding of ranolazine to either preopen or open states did not affect the excitability of the simulation. Ranolazine was able to significantly reduce the epileptiform activity of the neuronal cultures, suggesting possible antiepileptic activity.

High-resolution molecular genomic autopsy reveals complex sudden unexpected death in epilepsy risk profile.

Advanced variant detection in genes underlying risk of sudden unexpected death in epilepsy (SUDEP) can uncover extensive epistatic complexity and improve diagnostic accuracy of epilepsy-related mortality. However, the sensitivity and clinical utility of diagnostic panels based solely on established cardiac arrhythmia genes in the molecular autopsy of SUDEP is unknown. We applied the established clinical diagnostic panels, followed by sequencing and a high density copy number variant (CNV) detection array of an additional 253 related ion channel subunit genes to analyze the overall genomic variation in a SUDEP of the 3-year-old proband with severe myoclonic epilepsy of infancy (SMEI). We uncovered complex combinations of single nucleotide polymorphisms and CNVs in genes expressed in both neurocardiac and respiratory control pathways, including SCN1A, KCNA1, RYR3, and HTR2C. Our findings demonstrate the importance of comprehensive high-resolution variant analysis in the assessment of personally relevant SUDEP risk. In this case, the combination of de novo single nucleotide polymorphisms (SNPs) and CNVs in the SCN1A and KCNA1 genes, respectively, is suspected to be the principal risk factor for both epilepsy and premature death. However, consideration of the overall biologically relevant variant complexity with its extensive functional epistatic interactions reveals potential personal risk more accurately.

Identification of the cysteine residue responsible for disulfide linkage of Na+ channel α and ß2 subunits.

Voltage-gated Na(+) channels in the brain are composed of a single pore-forming α subunit, one non-covalently linked ß subunit (ß1 or ß3), and one disulfide-linked ß subunit (ß2 or ß4). The final step in Na(+) channel biosynthesis in central neurons is concomitant α-ß2 disulfide linkage and insertion into the plasma membrane. Consistent with this, Scn2b (encoding ß2) null mice have reduced Na(+) channel cell surface expression in neurons, and action potential conduction is compromised. Here we generated a series of mutant ß2 cDNA constructs to investigate the cysteine residue(s) responsible for α-ß2 subunit covalent linkage. We demonstrate that a single cysteine-to-alanine substitution at extracellular residue Cys-26, located within the immunoglobulin (Ig) domain, abolishes the covalent linkage between α and ß2 subunits. Loss of α-ß2 covalent complex formation disrupts the targeting of ß2 to nodes of Ranvier in a myelinating co-culture system and to the axon initial segment in primary hippocampal neurons, suggesting that linkage with α is required for normal ß2 subcellular localization in vivo. WT ß2 subunits are resistant to live cell Triton X-100 detergent extraction from the hippocampal axon initial segment, whereas mutant ß2 subunits, which cannot form disulfide bonds with α, are removed by detergent. Taken together, our results demonstrate that α-ß2 covalent association via a single, extracellular disulfide bond is required for ß2 targeting to specialized neuronal subcellular domains and for ß2 association with the neuronal cytoskeleton within those domains.

The SCN1A gene variants and epileptic encephalopathies.

The voltage-gated sodium channels are fundamental units that evoke the action potential in excitable cells such as neurons. These channels are integral membrane proteins typically consisting of one α-subunit, which forms the larger central pore of the channel, and two smaller auxiliary ß-subunits, which modulate the channel functions. Genetic alterations in the SCN1A gene coding for the α-subunit of the neuronal voltage-gated sodium ion channel, type 1 (NaV 1.1), is associated with a spectrum of seizure-related disorders in human, ranging from a relatively milder form of febrile seizures to a more severe epileptic condition known as the Dravet syndrome. Among the epilepsy genes, the SCN1A gene perhaps known to have the largest number of disease-associated alleles. Here we present a meta-analysis on the SCN1A gene variants and provide comprehensive information on epilepsy-associated gene variants, their frequency, the predicted effect on the protein, the ethnicity of the affected along with the inheritance pattern and the associated epileptic phenotype. We also summarize our current understanding on the pathophysiology of the SCN1A gene defects, disease mechanism, genetic modifiers and their clinical and diagnostic relevance.

Discovery of a selective, state-independent inhibitor of NaV1.7 by modification of guanidinium toxins.

The voltage-gated sodium channel isoform NaV1.7 is highly expressed in dorsal root ganglion neurons and is obligatory for nociceptive signal transmission. Genetic gain-of-function and loss-of-function NaV1.7 mutations have been identified in select individuals, and are associated with episodic extreme pain disorders and insensitivity to pain, respectively. These findings implicate NaV1.7 as a key pharmacotherapeutic target for the treatment of pain. While several small molecules targeting NaV1.7 have been advanced to clinical development, no NaV1.7-selective compound has shown convincing efficacy in clinical pain applications. Here we describe the discovery and characterization of ST-2262, a NaV1.7 inhibitor that blocks the extracellular vestibule of the channel with an IC50 of 72 nM and greater than 200-fold selectivity over off-target sodium channel isoforms, NaV1.1-1.6 and NaV1.8. In contrast to other NaV1.7 inhibitors that preferentially inhibit the inactivated state of the channel, ST-2262 is equipotent in a protocol that favors the resting state of the channel, a protocol that favors the inactivated state, and a high frequency protocol. In a non-human primate study, animals treated with ST-2262 exhibited reduced sensitivity to noxious heat. These findings establish the extracellular vestibule of the sodium channel as a viable receptor site for the design of selective ligands targeting NaV1.7.

NaV1.1 and NaV1.6 selective compounds reduce the behavior phenotype and epileptiform activity in a novel zebrafish model for Dravet Syndrome.

Dravet syndrome is caused by dominant loss-of-function mutations in SCN1A which cause reduced activity of Nav1.1 leading to lack of neuronal inhibition. On the other hand, gain-of-function mutations in SCN8A can lead to a severe epileptic encephalopathy subtype by over activating NaV1.6 channels. These observations suggest that Nav1.1 and Nav1.6 represent two opposing sides of the neuronal balance between inhibition and activation. Here, we hypothesize that Dravet syndrome may be treated by either enhancing Nav1.1 or reducing Nav1.6 activity. To test this hypothesis we generated and characterized a novel DS zebrafish model and tested new compounds that selectively activate or inhibit the human NaV1.1 or NaV1.6 channel respectively. We used CRISPR/Cas9 to generate two separate Scn1Lab knockout lines as an alternative to previous zebrafish models generated by random mutagenesis or morpholino oligomers. Using an optimized locomotor assay, spontaneous burst movements were detected that were unique to Scn1Lab knockouts and disappear when introducing human SCN1A mRNA. Besides the behavioral phenotype, Scn1Lab knockouts show sudden, electrical discharges in the brain that indicate epileptic seizures in zebrafish. Scn1Lab knockouts showed increased sensitivity to the GABA antagonist pentylenetetrazole and a reduction in whole organism GABA levels. Drug screenings further validated a Dravet syndrome phenotype. We tested the NaV1.1 activator AA43279 and two novel NaV1.6 inhibitors MV1369 and MV1312 in the Scn1Lab knockouts. Both type of compounds significantly reduced the number of spontaneous burst movements and seizure activity. Our results show that selective inhibition of NaV1.6 could be just as efficient as selective activation of NaV1.1 and these approaches could prove to be novel potential treatment strategies for Dravet syndrome and other (genetic) epilepsies. Compounds tested in zebrafish however, should always be further validated in other model systems for efficacy in mammals and to screen for potential side effects.

De novo SCN1A, SCN8A, and CLCN2 mutations in childhood absence epilepsy.

This study aimed to identify monogenic mutations from Chinese patients with childhood absence epilepsy (CAE) and summarize their characteristics. A total of 100 patients with CAE were recruited in Peking University First Hospital from 2005 to 2016 and underwent telephone and outpatient follow-up review. We used targeted disease-specific gene capture sequencing (involving 300 genes) to identify pathogenic variations for these patients. We identified three de novo epilepsy-related gene mutations, including missense mutations of SCN1A (c. 5399 T > A; p. Val1800Asp), SCN8A (c. 2371 G > T; p. Val791Phe), and CLCN2 (c. 481 G > A; p. Gly161Ser), from three patients, separately. All recruited patients presented typical CAE features and good prognosis. To date, CAE has been considered a complex disease caused by multiple susceptibility genes. In this study, we observed that 3% of typical CAE patients had a de novo mutation of a known monogenic epilepsy-related gene. Our study suggests that a significant proportion of typical CAE cases may be monogenic forms of epilepsy. For genetic generalized epilepsies, such as CAE, further studies are needed to clarify the contributions of de novo or inherited rare monogenic coding, noncoding and copy number variants.