Impaired cerebellar plasticity hypersensitizes sensory reflexes in SCN2A-associated ASD.

Children diagnosed with autism spectrum disorder (ASD) commonly present with sensory hypersensitivity or abnormally strong reactions to sensory stimuli. Such hypersensitivity can be overwhelming, causing high levels of distress that contribute markedly to the negative aspects of the disorder. Here, we identify a mechanism that underlies hypersensitivity in a sensorimotor reflex found to be altered in humans and in mice with loss of function in the ASD risk-factor gene SCN2A. The cerebellum-dependent vestibulo-ocular reflex (VOR), which helps maintain one's gaze during movement, was hypersensitized due to deficits in cerebellar synaptic plasticity. Heterozygous loss of SCN2A-encoded NaV1.2 sodium channels in granule cells impaired high-frequency transmission to Purkinje cells and long-term potentiation, a form of synaptic plasticity important for modulating VOR gain. VOR plasticity could be rescued in mice via a CRISPR-activator approach that increases Scn2a expression, demonstrating that evaluation of a simple reflex can be used to assess and quantify successful therapeutic intervention.

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.

A Push-Pull Mechanism Between PRRT2 and ß4-subunit Differentially Regulates Membrane Exposure and Biophysical Properties of NaV1.2 Sodium Channels.

Proline-rich transmembrane protein 2 (PRRT2) is a neuron-specific protein implicated in the control of neurotransmitter release and neural network stability. Accordingly, PRRT2 loss-of-function mutations associate with pleiotropic paroxysmal neurological disorders, including paroxysmal kinesigenic dyskinesia, episodic ataxia, benign familial infantile seizures, and hemiplegic migraine. PRRT2 is a negative modulator of the membrane exposure and biophysical properties of Na+ channels NaV1.2/NaV1.6 predominantly expressed in brain glutamatergic neurons. NaV channels form complexes with ß-subunits that facilitate the membrane targeting and the activation of the α-subunits. The opposite effects of PRRT2 and ß-subunits on NaV channels raises the question of whether PRRT2 and ß-subunits interact or compete for common binding sites on the α-subunit, generating Na+ channel complexes with distinct functional properties. Using a heterologous expression system, we have observed that ß-subunits and PRRT2 do not interact with each other and act as independent non-competitive modulators of NaV1.2 channel trafficking and biophysical properties. PRRT2 antagonizes the ß4-induced increase in expression and functional activation of the transient and persistent NaV1.2 currents, without affecting resurgent current. The data indicate that ß4-subunit and PRRT2 form a push-pull system that finely tunes the membrane expression and function of NaV channels and the intrinsic neuronal excitability.

Modulating effects of FGF12 variants on NaV1.2 and NaV1.6 being associated with developmental and epileptic encephalopathy and Autism spectrum disorder: A case series.

OBJECTIVE: Fibroblast Growth Factor 12 (FGF12) may represent an important modulator of neuronal network activity and has been associated with developmental and epileptic encephalopathy (DEE). We sought to identify the underlying pathomechanism of FGF12-related disorders. METHODS: Patients with pathogenic variants in FGF12 were identified through published case reports, GeneMatcher and whole exome sequencing of own case collections. The functional consequences of two missense and two copy number variants (CNVs) were studied by co-expression of wildtype and mutant FGF12 in neuronal-like cells (ND7/23) with the sodium channels NaV1.2 or NaV1.6, including their beta-1 and beta-2 sodium channel subunits (SCN1B and SCN2B). RESULTS: Four variants in FGF12 were identified for functional analysis: one novel FGF12 variant in a patient with autism spectrum disorder and three variants from previously published patients affected by DEE. We demonstrate the differential regulating effects of wildtype and mutant FGF12 on NaV1.2 and NaV1.6 channels. Here, FGF12 variants lead to a complex kinetic influence on NaV1.2 and NaV1.6, including loss- as well as gain-of function changes in fast and slow inactivation. INTERPRETATION: We could demonstrate the detailed regulating effect of FGF12 on NaV1.2 and NaV1.6 and confirmed the complex effect of FGF12 on neuronal network activity. Our findings expand the phenotypic spectrum related to FGF12 variants and elucidate the underlying pathomechanism. Specific variants in FGF12-associated disorders may be amenable to precision treatment with sodium channel blockers. FUNDING: DFG, BMBF, Hartwell Foundation, National Institute for Neurological Disorders and Stroke, IDDRC, ENGIN, NIH, ITMAT, ILAE, RES and GRIN.

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.

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.

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.

Pharmacological Inhibition of Wee1 Kinase Selectively Modulates the Voltage-Gated Na+ Channel 1.2 Macromolecular Complex.

Voltage-gated Na+ (Nav) channels are a primary molecular determinant of the action potential (AP). Despite the canonical role of the pore-forming α subunit in conferring this function, protein-protein interactions (PPI) between the Nav channel α subunit and its auxiliary proteins are necessary to reconstitute the full physiological activity of the channel and to fine-tune neuronal excitability. In the brain, the Nav channel isoforms 1.2 (Nav1.2) and 1.6 (Nav1.6) are enriched, and their activities are differentially regulated by the Nav channel auxiliary protein fibroblast growth factor 14 (FGF14). Despite the known regulation of neuronal Nav channel activity by FGF14, less is known about cellular signaling molecules that might modulate these regulatory effects of FGF14. To that end, and building upon our previous investigations suggesting that neuronal Nav channel activity is regulated by a kinase network involving GSK3, AKT, and Wee1, we interrogate in our current investigation how pharmacological inhibition of Wee1 kinase, a serine/threonine and tyrosine kinase that is a crucial component of the G2-M cell cycle checkpoint, affects the Nav1.2 and Nav1.6 channel macromolecular complexes. Our results show that the highly selective inhibitor of Wee1 kinase, called Wee1 inhibitor II, modulates FGF14:Nav1.2 complex assembly, but does not significantly affect FGF14:Nav1.6 complex assembly. These results are functionally recapitulated, as Wee1 inhibitor II entirely alters FGF14-mediated regulation of the Nav1.2 channel, but displays no effects on the Nav1.6 channel. At the molecular level, these effects of Wee1 inhibitor II on FGF14:Nav1.2 complex assembly and FGF14-mediated regulation of Nav1.2-mediated Na+ currents are shown to be dependent upon the presence of Y158 of FGF14, a residue known to be a prominent site for phosphorylation-mediated regulation of the protein. Overall, our data suggest that pharmacological inhibition of Wee1 confers selective modulatory effects on Nav1.2 channel activity, which has important implications for unraveling cellular signaling pathways that fine-tune neuronal excitability.

Severe deficiency of the voltage-gated sodium channel NaV1.2 elevates neuronal excitability in adult mice.

Scn2a encodes the voltage-gated sodium channel NaV1.2, a main mediator of neuronal action potential firing. The current paradigm suggests that NaV1.2 gain-of-function variants enhance neuronal excitability, resulting in epilepsy, whereas NaV1.2 deficiency impairs neuronal excitability, contributing to autism. However, this paradigm does not explain why ∼20%-30% of individuals with NaV1.2 deficiency still develop seizures. Here, we report the counterintuitive finding that severe NaV1.2 deficiency results in increased neuronal excitability. Using a NaV1.2-deficient mouse model, we show enhanced intrinsic excitability of principal neurons in the prefrontal cortex and striatum, brain regions known to be involved in Scn2a-related seizures. This increased excitability is autonomous and reversible by genetic restoration of Scn2a expression in adult mice. RNA sequencing reveals downregulation of multiple potassium channels, including KV1.1. Correspondingly, KV channel openers alleviate the hyperexcitability of NaV1.2-deficient neurons. This unexpected neuronal hyperexcitability may serve as a cellular basis underlying NaV1.2 deficiency-related seizures.

Paradoxical hyperexcitability from NaV1.2 sodium channel loss in neocortical pyramidal cells.

Loss-of-function variants in the gene SCN2A, which encodes the sodium channel NaV1.2, are strongly associated with autism spectrum disorder and intellectual disability. An estimated 20%-30% of children with these variants also suffer from epilepsy, with altered neuronal activity originating in neocortex, a region where NaV1.2 channels are expressed predominantly in excitatory pyramidal cells. This is paradoxical, as sodium channel loss in excitatory cells would be expected to dampen neocortical activity rather than promote seizure. Here, we examined pyramidal neurons lacking NaV1.2 channels and found that they were intrinsically hyperexcitable, firing high-frequency bursts of action potentials (APs) despite decrements in AP size and speed. Compartmental modeling and dynamic-clamp recordings revealed that NaV1.2 loss prevented potassium channels from properly repolarizing neurons between APs, increasing overall excitability by allowing neurons to reach threshold for subsequent APs more rapidly. This cell-intrinsic mechanism may, therefore, account for why SCN2A loss-of-function can paradoxically promote seizure.

Precise spatiotemporal control of voltage-gated sodium channels by photocaged saxitoxin.

Here we report the pharmacologic blockade of voltage-gated sodium ion channels (NaVs) by a synthetic saxitoxin derivative affixed to a photocleavable protecting group. We demonstrate that a functionalized saxitoxin (STX-eac) enables exquisite spatiotemporal control of NaVs to interrupt action potentials in dissociated neurons and nerve fiber bundles. The photo-uncaged inhibitor (STX-ea) is a nanomolar potent, reversible binder of NaVs. We use STX-eac to reveal differential susceptibility of myelinated and unmyelinated axons in the corpus callosum to NaV-dependent alterations in action potential propagation, with unmyelinated axons preferentially showing reduced action potential fidelity under conditions of partial NaV block. These results validate STX-eac as a high precision tool for robust photocontrol of neuronal excitability and action potential generation.

NaV1.2 EFL domain allosterically enhances Ca2+ binding to sites I and II of WT and pathogenic calmodulin mutants bound to the channel CTD.

Neuronal voltage-gated sodium channel NaV1.2 C-terminal domain (CTD) binds calmodulin (CaM) constitutively at its IQ motif. A solution structure (6BUT) and other NMR evidence showed that the CaM N domain (CaMN) is structurally independent of the C-domain (CaMC) whether CaM is bound to the NaV1.2IQp (1,901-1,927) or NaV1.2CTD (1,777-1,937) with or without calcium. However, in the CaM + NaV1.2CTD complex, the Ca2+ affinity of CaMN was more favorable than in free CaM, while Ca2+ affinity for CaMC was weaker than in the CaM + NaV1.2IQp complex. The CTD EF-like (EFL) domain allosterically widened the energetic gap between CaM domains. Cardiomyopathy-associated CaM mutants (N53I(N54I), D95V(D96V), A102V(A103V), E104A(E105A), D129G(D130G), and F141L(F142L)) all bound the NaV1.2 IQ motif favorably under resting (apo) conditions and bound calcium normally at CaMN sites. However, only N53I and A102V bound calcium at CaMC sites at [Ca2+] < 100 µM. Thus, they are expected to respond like wild-type CaM to Ca2+ spikes in excitable cells.

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.

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.

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.

Dominant SCN2A mutation with variable phenotype in two generations.

BACKGROUND: SCN2A mutations are some of the commonest causes of neurodevelopmental disorders including epilepsy, movement disorders, autism spectrum disorder, intellectual disability and rarely episodic ataxia. CASE REPORT: We present a patient with a dominantly inherited SCN2A mutation presenting as episodic ataxia in a boy and episodic hemiplegia in his father. We have briefly reviewed the literature of SCN2A mutations presenting with episodic ataxia. CONCLUSION: Our report has expanded the phenotype for SCN2A mutations.

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.

Parabens inhibit hNaV 1.2 channels.

Propylparaben, a commonly used antimicrobial preservative, has been reported as an anticonvulsant agent targeting neuronal Na+ channels (NaV). However, the specific features of the NaV channel inhibition by this agent have so far not been extensively studied. Moreover, it is still unclear if it shares this pharmacological activity with other parabens. Here, we fully characterized the mechanism of action of the inhibitory effect that propylparaben and benzylparaben induce on human NaV 1.2 channel isoform (hNaV1.2). We established a first approach to know the parabens structural determinants for this channel inhibition. The parabens effects on hNaV1.2 channel mediated currents were recorded using the patch-clamp whole-cell configuration on hNaV1.2 stably transfected HEK293 cells. Propylparaben induced a typical state-dependent inhibition on hNaV1.2 channel carried current, characterized by a left-shift in the steady-state inactivation curve, a prolongation in the time needed for recovery from fast inactivation and a frequency-dependent blocking behavior. The state-dependent inhibition is increased for butylparaben and benzylparaben and diminished for methylparaben, ethylparaben and p-hydroxybenzoic acid (the major metabolite of parabens hydrolysis). Particularly, butylparaben and benzylparaben shift the steady-state inactivation curve 2- and 3-times more than propylparaben, respectively. Parabens are blockers of hNaV1.2 channels, sharing the mechanism of action of most of sodium channel blocking antiseizure drugs. The potency of this inhibition increases with the size of the lipophilic alcoholic residue of the ester group. These results provide a basis for rational drug design directed to generate new potential anticonvulsant agents.

Electrophysiological features: The next precise step for SCN2A developmental epileptic encephalopathy.

BACKGROUND: To investigate the relationships among phenotypes, genotypes, and funotypes of SCN2A-related developmental epileptic encephalopathy (DEE). METHODS: We enrolled five DEE patients with five de novo variants of the SCN2A. Functional analysis and pharmacological features of Nav1.2 channel protein expressed in HEK293T cells were characterized by whole-cell patch-clamp recording. RESULTS: The phenotypes of c.4712T>C(p. I1571T), c.2995G>A(p.E999K), and c.4015A>G(p. N1339D) variants showed similar characteristics, including early seizure onset with severe to profound intellectual disability. Electrophysiological recordings revealed a hyperpolarizing shift in the voltage dependence of the activation curve and smaller recovery time constants of fast-inactivation than in wild type, indicating a prominent gain of function (GOF). Moreover, pharmacological electrophysiology showed that phenytoin inhibited over a 70% peak current and was more effective than oxcarbazepine and carbamazepine. In contrast, c.4972C>T (p.P1658S) and c.5317G>A (p.A1773T) led to loss of function (LOF) changes, showing reduced current density and enhanced fast inactivation. Both showed seizure onset after 3 months of age with moderate development delay. Interestingly, we discovered that choreoathetosis was a specific phenotype feature. CONCLUSION: These findings provided the insights into the phenotype-genotype-funotype relationships of SCN2A-related DEE. The preliminary evaluation using the distinct hints of GOF and LOF helped plan the treatment, and the next precise step should be electrophysiological study.