Mutant Nav1 Research Articles

Molecular mechanism for the interaction of MOG1 with the intracellular Loop II of cardiac sodium channel Nav1.5 and its role in Brugada syndrome

Abstract Background SCN5A encodes the cardiac sodium channel a-subunit Nav1.5 with an essential role in generation and conduction of cardiac action potential. Mutations in SCN5A cause Brugada syndrome (BrS) and long QT syndrome (LQTS). The function of Nav1.5, including its cell membrane trafficking and clustering in caveolae, is regulated by MOG1. We recently showed that gene therapy with AAV9-MOG1 reversed disease phenotypes of BrS, dilated cardiomyopathy, cardiac conduction disease and sick sinus syndrome associated with SCN5A mutations. MOG1 is a small chaperon that interacts with multiple sites of Nav1.5, including intracellular Loop I between transmembrane domain I and II and Loop II between domain II and III. We previously characterized the interaction between MOG1 and the intracellular Loop I of Nav1.5, and defined amino acids D24, E36, D44, E53, and E101A of MOG1 as critical residues for its interaction with Nav1.5 Loop I. However, the mechanistic understanding of the interaction between MOG1 and intracellular Loop II of Nav1.5 is needed. Purpose The aim of this study is to perform systematic mutational and electrophysiological studies to identify the key structural elements involved in the interaction between MOG1 and Nav1.5 Loop II and determine the significance of this interaction to the pathogenesis of BrS. Methods Patch-clamping was used to record sodium currents. Glutathione S-transferase (GST) pull-down were used to characterize protein-protein interactions. Results Nav1.5 Loop II contains 765 amino acids, and the domain between amino acids 1157 and 1200 was shown to be the interaction domain for MOG1. Six serial deletions were created, including LoopII△940-982, LoopII△983-1025, LoopII△1026-1068, LoopII△1069-1112, LoopII△1113-1156, and LoopII△1157-1200. GST-pull-down showed that only LoopII△1157-1200 failed to interact with MOG1. MOG1 enhanced sodium current densities all Nav1.5 deletion mutants except for LoopII△1157-1200. Further deletions by every 10-11 amino acids defined the MOG1-interacting domain to 1190-1200. Point mutation analysis defined the MOG1-Nav1.5 Loop II interaction domain to an 8-amino-acid domain of R1193-Y1200 of Nav1.5 Loop II. Mutations R1193W, L1194M and Y1199S associated with BrS reduced the interaction between MOG1 and Nav1.5. MOG1 enhanced sodium current densities from wild type Nav1.5, but not from mutant Nav1.5 with mutations R1193W, L1194M and Y1199S. Conclusions This study identifies the critical elements of Nav1.5 Loop II for interaction with MOG1, and reveals the molecular mechanism by which Nav1.5 mutations R1193W, L1194M and Y1199S cause BrS. Mutations R1193W, L1194M and Y1199S weaken the interaction between Nav1.5 and MOG1, and reduce MOG1-enhanced cardiac sodium current densities.

Complex biophysical changes and reduced neuronal firing in an SCN8A variant associated with developmental delay and epilepsy

Mutations in the SCN8A gene, encoding the voltage-gated sodium channel NaV1.6, are associated with a range of neurodevelopmental syndromes. The p.(Gly1625Arg) (G1625R) mutation was identified in a patient diagnosed with developmental epileptic encephalopathy (DEE). While most of the characterized DEE-associated SCN8A mutations were shown to cause a gain-of-channel function, we show that the G1625R variant, positioned within the S4 segment of domain IV, results in complex effects. Voltage-clamp analyses of NaV1.6G1625R demonstrated a mixture of gain- and loss-of-function properties, including reduced current amplitudes, increased time constant of fast voltage-dependent inactivation, a depolarizing shift in the voltage dependence of activation and inactivation, and increased channel availability with high-frequency repeated depolarization. Current-clamp analyses in transfected cultured neurons revealed that these biophysical properties caused a marked reduction in the number of action potentials when firing was driven by the transfected mutant NaV1.6. Accordingly, computational modeling of mature cortical neurons demonstrated a mild decrease in neuronal firing when mimicking the patients' heterozygous SCN8A expression. Structural modeling of NaV1.6G1625R suggested the formation of a cation-π interaction between R1625 and F1588 within domain IV. Double-mutant cycle analysis revealed that this interaction affects the voltage dependence of inactivation in NaV1.6G1625R. Together, our studies demonstrate that the G1625R variant leads to a complex combination of gain and loss of function biophysical changes that result in an overall mild reduction in neuronal firing, related to the perturbed interaction network within the voltage sensor domain, necessitating personalized multi-tiered analysis for SCN8A mutations for optimal treatment selection.

Anxiety and dysautonomia symptoms in patients with a NaV1.7 mutation and the potential benefits of low-dose short-acting guanfacine.

Guanfacine is an α2A-adrenergic receptor agonist, FDA-approved to treat attention-deficit hyperactivity disorder and high blood pressure, typically as an extended-release formulation up to 7mg/day. In our dysautonomia clinic, we observed that off-label use of short-acting guanfacine at 1mg/day facilitated symptom relief in two families with multiple members presenting withsevere generalized anxiety. We also noted anecdotal improvements in associated dysautonomia symptoms such as hyperhidrosis, cognitive impairment, and palpitations. We postulated that a genetic deficit existed in these patients that might augment guanfacine susceptibility. We used whole-exome sequencing to identify mutations in patients with shared generalized anxiety and dysautonomia symptoms. Guanfacine-induced changes in the function of voltage-gated Na+ channels were investigated using voltage-clamp electrophysiology. Whole-exome sequencing uncovered the p.I739V mutation in SCN9A in the proband of two nonrelated families. Moreover, guanfacine inhibited ionic currents evoked by wild-type and mutant NaV1.7 encoded by SCN9A, as well as other NaV channel subtypes to a varying degree. Our study provides further evidence for a possible pathophysiological role of NaV1.7 in anxiety and dysautonomia. Combined with off-target effects on NaV channel function, daily administration of 1mg short-acting guanfacine may be sufficient to normalize NaV channel mutation-induced changes in sympathetic activity, perhaps aided by partial inhibition of NaV1.7 or other channel subtypes. In a broader context, expanding genetic and functional data about ion channel aberrations may enable the prospect of stratifying patients in which mutation-induced increased sympathetic tone normalization by guanfacine can support treatment strategies for anxiety and dysautonomia symptoms.

GPD1L-A306del modifies sodium current in a family carrying the dysfunctional SCN5A-G1661R mutation associated with Brugada syndrome

Loss-of-function variants of SCN5A, encoding the sodium channel alpha subunit Nav1.5 are associated with high phenotypic variability and multiple cardiac presentations, while underlying mechanisms are incompletely understood. Here we investigated a family with individuals affected by Brugada Syndrome (BrS) of different severity and aimed to unravel the underlying genetic and electrophysiological basis.Next-generation sequencing was used to identify the genetic variants carried by family members. The index patient, who was severely affected by arrhythmogenic BrS, carried previously uncharacterized variants of Nav1.5 (SCN5A-G1661R) and glycerol-3-phosphate dehydrogenase-1-like protein (GPD1L-A306del) in a double heterozygous conformation. Family members exclusively carrying SCN5A-G1661R showed asymptomatic Brugada ECG patterns, while another patient solely carrying GPD1L-A306del lacked any clinical phenotype.To assess functional mechanisms, Nav1.5 channels were transiently expressed in HEK-293 cells in the presence and absence of GPD1L. Whole-cell patch-clamp recordings revealed loss of sodium currents after homozygous expression of SCN5A-G1661R, and reduction of current amplitude to ~ 50% in cells transfected with equal amounts of wildtype and mutant Nav1.5. Co-expression of wildtype Nav1.5 and GPD1L showed a trend towards increased sodium current amplitudes and a hyperpolarizing shift in steady-state activation and -inactivation compared to sole SCN5A expression. Application of the GPD1L-A306del variant shifted steady-state activation to more hyperpolarized and inactivation to more depolarized potentials.In conclusion, SCN5A-G1661R produces dysfunctional channels and associates with BrS. SCN5A mediated currents are modulated by co-expression of GDP1L and this interaction is altered by mutations in both proteins. Thus, additive genetic burden may aggravate disease severity, explaining higher arrhythmogenicity in double mutation carriers.

The dominant-negative effect of Loss of function Nav 1.5 variants is not a critical determinant of phenotype severity

Abstract Introduction Loss-of-function (LOF) variants of SCN5A cause various arrhythmic diseases. Recent studies have demonstrated that some LOF-SCN5A variants can exert a dominant-negative (DN) effect through channel dimerization, resulting in higher penetrance of disease phenotypes. However, the association between the DN effect and phenotype severity remains unclear. In this study, we identified a proband with compound heterozygous SCN5A variants, p.G833R and p.T1396P, in a family with bradyarrhythmia. We analyzed the electrophysiological function of these variants and their association with channel-dimerization to elucidate their effects on phenotype severity. Purpose The purpose of this study is to investigate the effect of sodium channel dimerization on the phenotype severity in a family with bradyarrhythmia Methods We performed functional analysis of the mutant Nav1.5s induced in HEK cells utilizing a whole-cell patch-clamp method. The DN effect was also assessed by co-expressing WT and mutants in each heterozygous combinations, with or without difopein, a peptide working as a channel-coupling canceler. Cell surface expression of mutated channels were measured by flowcytometry. Results We identified SCN5A p.T1396P and p.G833R variants segregating in a family suffering from severe bradyarrhythmia. The proband carried both variants in a compound heterozygous manner, while her mother carried only p.T1396P. The clinical phenotype was more severe and had early onset in the proband; the compound heterozygous case. In the patch-clamp study, HEK 293 cells solely expressing T1396P did not show any measurable sodium current, while those with G833R had comparative current with that of WT channel. Cells expressing WT/T1396P showed significant reduction and delay of inactivation decay, which was normalized by co-expression with difopein. The results implied that loss-of-function variant T1396P manifested DN-effect via channel dimerization. Cells co-expressing T1396P and G833R did not demonstrate a DN-effect, suggesting that Nav1.5-G833R did not form a dimer. Considering more severe phenotype of compound heterozygous member in the family, the DN-cancelation by monomerized Nav1.5-G833R was not associated with a mild phenotype. Conclusion Our result supports the current Nav1.5-dimerization concept and consequent DN-effect by LOF-SCN5A variants. However, the phenotype severity was not simply explainable by the presence of a dominant negative effect. Further multiplanar analyses including assays for channel clustering, localization, and gating modification would be necessary.

Hyperkalemic periodic paralysis associated with a novel missense variant located in the inner pore of Nav1.4

BackgroundHyperkalemic periodic paralysis (HyperPP) is an autosomal dominantly inherited disease characterized by episodic paralytic attacks with hyperkalemia, and is caused by mutations of the SCN4A gene encoding the skeletal muscle type voltage-gated sodium channel Nav1.4. The pathological mechanism of HyperPP was suggested to be associated with gain-of-function changes for Nav1.4 gating, some of which are defects of slow inactivation. Case presentation & MethodsWe identified a HyperPP family consisting of the proband and his mother, who showed a novel heterozygous SCN4A variant, p.V792G, in an inner pore lesion of segment 6 in Domain II of Nav1.4. Clinical and neurophysiological evaluations were conducted for the proband and his mother. We explored the pathogenesis of the variant by whole-cell patch clamp technique using HEK293T cells expressing the mutant Nav1.4 channel. ResultsFunctional analysis of Nav1.4 with the V792G mutation revealed a hyperpolarized shift of voltage-dependent activation and fast inactivation. Moreover, steady-state slow inactivation in V792G was impaired with larger residual currents in comparison with wild-type Nav1.4. ConclusionV792G in SCN4A is a pathogenic variant associated with the HyperPP phenotype and the inner pore lesion of Nav1.4 plays a crucial role in slow inactivation.

Targeting the Nav1.6:GSK3β Protein:Protein Interaction Complex to Mitigate Hippocampal Hyperexcitability in Early‐Stage Alzheimer’s Disease

AbstractBackgroundNav1.6 is the most densely expressed sodium channel isoform in the adult brain and is a primary determinant of action potential initiation and propagation due to its subcellular localization at the axon initial segment (AIS). Importantly, it has been shown that the firing frequency and Na+ currents of Nav 1.6 channels in hippocampal neurons are specifically increased by Aβ exposure in a concentration‐dependent manner. Nav1.6 is regulated through its interactions with key auxiliary proteins, notably Glycogen Synthase Kinase 3‐β (GSK3β), which has increased expression in the hippocampus of AD patients as well as peripheral circulating lymphocytes in both MCI and AD patients. We have shown that GSK3β regulates Nav1.6 channel activity through phosphorylation of and direct binding to the Nav1.6’s intracellular C‐terminal domain (CTD). Furthermore, genetic silencing of GSK3Ββ significantly decreased both Na+ currents and intrinsic firing compared to controls specifically through Nav1.6, while GSK3Ββ over‐expression or pharmacological disinhibition produced opposing phenotypes. Multiple lines of evidence indicate that hippocampal hyperactivity is present in patients with MCI and early‐stage AD and it is observed that reduction of hippocampal hyperactivity in MCI patients improves cognition and memory performance. We hypothesize that small‐molecule modulation of the Nav1.6:GSK3β PPI interface can counteract hippocampal hyperexcitability and could be used to treat early phases of AD, preventing the onset of cognitive decline.MethodHEK293 cells co‐transfected with WT or mutant Nav1.6 and GSK3β were utilized to interrogate the Nav1.6:GSK3β PPI and screen a series of 50 validated hit compounds using split‐luciferase complementation (LCA). Whole‐cell patch‐clamp electrophysiology was performed in heterologous cells and acute slice preparations to validate GSK3β‐mediated regulation of Nav1.6 activity and evaluate lead compound efficacy.ResultMutagenesis screening revealed putative interaction sites of the Nav1.6:GSK3β PPI. Counter‐screening of 50 validated hits revealed 5 compounds that inhibit Nav1.6:GSK3β complex assembly at low micromolar concentrations (Figure 1). Compound 5671063, identified from the counter‐screen, exhibits functional modulation of Nav1.6 activity in heterologous cells (Figure 2).ConclusionThe Nav1.6:GSK3β complex is a critical mediator of neuronal activity and represents a novel target for treatment of pathological hippocampal hyperexcitability associated with cognitive decline.

Local Anesthetic Like Inhibition of the Cardiac Na+ Channel Nav1.5 by Chloroquine and Hydroxychloroquine.

Chloroquine (CQ) and its derivate hydroxychloroquine (HCQ) are successfully deployed for different diseases beyond the prophylaxis and treatment of malaria. Both substances exhibit antiviral properties and have been proposed for prophylaxis and treatment of COVID-19 caused by SARS-CoV-2. CQ and HCQ cause similar adverse events including life-threatening cardiac arrhythmia generally based on QT-prolongation, which is one of the most reported adverse events for both agents associated with the treatment of COVID-19. Various drugs known to induce QT-prolongation have been proven to exert local anesthetic (LA)-like properties regarding their impact on the cardiac Na+ channel Nav1.5. Inhibition of Nav1.5 is considered as the primary mechanism of cardiotoxicity caused by LAs. However, the mechanism of the arrhythmogenic effects of CQ and HCQ related to Nav1.5 has not yet been fully investigated. Therefore, the exact mechanism of how CQ and HCQ affect the sodium currents generated by Nav1.5 need to be further elucidated. This in vitro study aims to investigate the effects of CQ and HCQ on Nav1.5-generated sodium currents to identify possible LA-like mechanisms that might contribute to their arrhythmogenic properties. The effects of CQ and HCQ on Nav1.5-generated sodium currents by HEK-293 cells expressing either wild-type human Nav1.5 or mutant Nav1.5 F1760A are measured using the whole-cell patch-clamp technique. Both agents induce a state-dependent inhibition of Nav1.5. Furthermore, CQ and HCQ produce a use-dependent block of Nav1.5 and a shift of fast and slow inactivation. Results of experiments investigating the effect on the LA-insensitive mutant Nav1.5-F1760A indicate that both agents at least in part employ the proposed LA-binding site of Nav1.5 to induce inhibition. This study demonstrated that CQ and HCQ exert LA-typical effects on Nav1.5 involving the proposed LA binding site, thus contributing to their arrhythmogenic properties.

Periodic paralysis due to cumulative effects of rare variants in SCN4A with small functional alterations.

Mutations in the SCN4A gene encoding a voltage-gated sodium channel (Nav1.4) cause hyperkalemic periodic paralysis (HyperPP) and hypokalemic periodic paralysis (HypoPP). Typically, both HyperPP and HypoPP are considered as monogenic disorders caused by a missense mutation with a large functional effect. However, a few cases with atypical periodic paralysis phenotype have been caused by multiple mutations in ion-channel genes expressed in skeletal muscles. In this study we investigated the underlying pathogenic mechanisms in such cases. We clinically assessed two families: proband 1 with HyperPP and proband 2 with atypical periodic paralysis with hypokalemia. Genetic analyses were performed by next-generation sequencing and conventional Sanger sequencing, followed by electrophysiological analyses of the mutant Nav1.4 channels expressed in human embryonic kidney 293T (HEK293T) cells using the whole-cell patch-clamp technique. In proband 1, K880del was identified in the SCN4A gene. In proband 2, K880del and a novel mutation, R1639H, were identified in the same allele of the SCN4A gene. Functional analyses revealed that the K880del in SCN4A has a weak functional effect on hNav1.4, increasing the excitability of the sarcolemma, which could represent a potential pathogenic factor. Although R1639H alone did not reveal functional changes strong enough to be pathogenic, Nav1.4 with both K880del and R1639H showed enhanced activation compared with K880del alone, indicating that R1639H may modify the hNav1.4 channel function. A cumulative effect of variants with small functional alterations may be considered as the underpinning oligogenic pathogenic mechanisms for the unusual phenotype of periodic paralysis.

CaMKII Inhibition Attenuates Distinct Gain-of-Function Effects Produced by Mutant Nav1.6 Channels and Reduces Neuronal Excitability.

Aberrant Nav1.6 activity can induce hyperexcitability associated with epilepsy. Gain-of-function mutations in the SCN8A gene encoding Nav1.6 are linked to epilepsy development; however, the molecular mechanisms mediating these changes are remarkably heterogeneous and may involve post-translational regulation of Nav1.6. Because calcium/calmodulin-dependent protein kinase II (CaMKII) is a powerful modulator of Nav1.6 channels, we investigated whether CaMKII modulates disease-linked Nav1.6 mutants. Whole-cell voltage clamp recordings in ND7/23 cells show that CaMKII inhibition of the epilepsy-related mutation R850Q largely recapitulates the effects previously observed for WT Nav1.6. We also characterized a rare missense variant, R639C, located within a regulatory hotspot for CaMKII modulation of Nav1.6. Prediction software algorithms and electrophysiological recordings revealed gain-of-function effects for R639C mutant channel activity, including increased sodium currents and hyperpolarized activation compared to WT Nav1.6. Importantly, the R639C mutation ablates CaMKII phosphorylation at a key regulatory site, T642, and, in contrast to WT and R850Q channels, displays a distinct response to CaMKII inhibition. Computational simulations demonstrate that modeled neurons harboring the R639C or R850Q mutations are hyperexcitable, and simulating the effects of CaMKII inhibition on Nav1.6 activity in modeled neurons differentially reduced hyperexcitability. Acute CaMKII inhibition may represent a promising mechanism to attenuate gain-of-function effects produced by Nav1.6 mutations.

Human iPSC Modeling of Genetic Febrile Seizure Reveals Aberrant Molecular and Physiological Features Underlying an Impaired Neuronal Activity.

Mutations in SCN1A gene, encoding the voltage-gated sodium channel (VGSC) NaV1.1, are widely recognized as a leading cause of genetic febrile seizures (FS), due to the decrease in the Na+ current density, mainly affecting the inhibitory neuronal transmission. Here, we generated induced pluripotent stem cells (iPSCs)-derived neurons (idNs) from a patient belonging to a genetically well-characterized Italian family, carrying the c.434T > C mutation in SCN1A gene (hereafter SCN1AM145T). A side-by-side comparison of diseased and healthy idNs revealed an overall maturation delay of SCN1AM145T cells. Membranes isolated from both diseased and control idNs were injected into Xenopus oocytes and both GABA and AMPA currents were successfully recorded. Patch-clamp measurements on idNs revealed depolarized action potential for SCN1AM145T, suggesting a reduced excitability. Expression analyses of VGSCs and chloride co-transporters NKCC1 and KCC2 showed a cellular “dysmaturity” of mutated idNs, strengthened by the high expression of SCN3A, a more fetal-like VGSC isoform, and a high NKCC1/KCC2 ratio, in mutated cells. Overall, we provide strong evidence for an intrinsic cellular immaturity, underscoring the role of mutant NaV1.1 in the development of FS. Furthermore, our data are strengthening previous findings obtained using transfected cells and recordings on human slices, demonstrating that diseased idNs represent a powerful tool for personalized therapy and ex vivo drug screening for human epileptic disorders.

Kinetic Alterations in Resurgent Sodium Currents of Mutant Nav1.4 Channel in Two Patients Affected by Paramyotonia Congenita

Simple SummaryParamyotonia congenita (PMC) is a rare muscle disorder that causes myotonia and weakness of facial and limb muscles. The electromyography in PMC shows continual spontaneous, high-frequency spike potentials in skeletal muscles. Genetic mutations in the Nav1.4 channel that cause hyperexcitability of muscle fibers are responsible for PMC. However, the genotype–phenotype relationship is highly diversified, and the molecular pathology remains unclear. Here, we investigated the electrophysiology in the Nav1.4 channel with mutations, p.V781I and p.A1737T, which were found in two Taiwanese patients. We identified the distinct changes in gating mechanisms altered by mutations which may underlie the clinical phenotype.Paramyotonia congenita (PMC) is a rare skeletal muscle disorder characterized by muscle stiffness upon repetitive exercise and cold exposure. PMC was reported to be caused by dominant mutations in the SCN4A gene encoding the α subunit of the Nav1.4 channel. Recently, we identified two missense mutations of the SCN4A gene, p.V781I and p.A1737T, in two PMC families. To evaluate the changes in electrophysiological properties caused by the mutations, both mutant and wild-type (WT) SCN4A genes were expressed in CHO-K1 and HEK-293T cells. Then, whole-cell patch-clamp recording was employed to study the altered gating of mutant channels. The activation curve of transient current showed a hyperpolarizing shift in both mutant Nav1.4 channels as compared to the WT channel, whereas there was a depolarizing shift in the fast inactivation curve. These changes confer to an increase in window current in the mutant channels. Further investigations demonstrated that the mutated channel proteins generate significantly larger resurgent currents as compared to the WT channel and take longer to attain the peak of resurgent current than the WT channel. In conclusion, the current study demonstrates that p.V781I and p.A1737T mutations in the Nav1.4 channel increase both the sustained and the resurgent Na+ current, leading to membrane hyperexcitability with a lower firing threshold, which may influence the clinical phenotype.

A SCN8A variant associated with severe early onset epilepsy and developmental delay: Loss- or gain-of-function?

SCN8A, encoding the voltage-gated sodium channel subunit NaV1.6, has been associated with a wide spectrum of neuropsychiatric disorders. Missense variants in SCN8A which increase the channel activity can cause a severe developmental and epileptic encephalopathy (DEE). One DEE variant (p.(Arg223Gly)) was described to cause a predominant loss-of-function (LOF) mechanism when expressed in neuroblastoma cells, which is not consistent with the genotype-phenotype correlations in this gene. To resolve this discrepancy and understand the pathophysiological mechanism of this variant, we performed comprehensive electrophysiological studies in both neuroblastoma cells and primary hippocampal neuronal cultures. Although we also found that p.(Arg223Gly) significantly decreased Na+ current density and enhanced fast inactivation compared to the wild type (WT) channel in transfected neuroblastoma cells (both LOF mechanisms), it also caused a strong hyperpolarizing shift of steady-state activation and accelerated the recovery from fast inactivation (both gain-of-function (GOF) mechanisms). In cultured neurons transfected with mutant vs. WT NaV1.6 channels, we found more depolarized resting membrane potentials and a decreased rheobase leading to enhanced action potential firing. We conclude that SCN8A p.(Arg223Gly) leads to a net GOF resulting in neuronal hyperexcitability and a higher firing rate, fitting with the central role of GOF mechanisms in DEE.

Hyperexcitability and Pharmacological Responsiveness of Cortical Neurons Derived from Human iPSCs Carrying Epilepsy-Associated Sodium Channel Nav1.2-L1342P Genetic Variant.

With the wide adoption of genomic sequencing in children having seizures, an increasing number of SCN2A genetic variants have been revealed as genetic causes of epilepsy. Voltage-gated sodium channel Nav1.2, encoded by gene SCN2A, is predominantly expressed in the pyramidal excitatory neurons and supports action potential (AP) firing. One recurrent SCN2A genetic variant is L1342P, which was identified in multiple patients with epileptic encephalopathy and intractable seizures. However, the mechanism underlying L1342P-mediated seizures and the pharmacogenetics of this variant in human neurons remain unknown. To understand the core phenotypes of the L1342P variant in human neurons, we took advantage of a reference human-induced pluripotent stem cell (hiPSC) line from a male donor, in which L1342P was introduced by CRISPR/Cas9-mediated genome editing. Using patch-clamping and microelectrode array (MEA) recordings, we revealed that cortical neurons derived from hiPSCs carrying heterozygous L1342P variant have significantly increased intrinsic excitability, higher sodium current density, and enhanced bursting and synchronous network firing, suggesting hyperexcitability phenotypes. Interestingly, L1342P neuronal culture displayed a degree of resistance to the anticonvulsant medication phenytoin, which recapitulated aspects of clinical observation of patients carrying the L1342P variant. In contrast, phrixotoxin-3 (PTx3), a Nav1.2 isoform-specific blocker, can potently alleviate spontaneous and chemically-induced hyperexcitability of neurons carrying the L1342P variant. Our results reveal a possible pathogenic underpinning of Nav1.2-L1342P mediated epileptic seizures and demonstrate the utility of genome-edited hiPSCs as an in vitro platform to advance personalized phenotyping and drug discovery.SIGNIFICANCE STATEMENT A mounting number of SCN2A genetic variants have been identified from patients with epilepsy, but how SCN2A variants affect the function of human neurons contributing to seizures is still elusive. This study investigated the functional consequences of a recurring SCN2A variant (L1342P) using human iPSC-derived neurons and revealed both intrinsic and network hyperexcitability of neurons carrying a mutant Nav1.2 channel. Importantly, this study recapitulated elements of clinical observations of drug-resistant features of the L1342P variant, and provided a platform for in vitro drug testing. Our study sheds light on cellular mechanism of seizures resulting from a recurring Nav1.2 variant, and helps to advance personalized drug discovery to treat patients carrying pathogenic SCN2A variant.

Elementary mechanisms of calmodulin regulation of NaV1.5 producing divergent arrhythmogenic phenotypes

In cardiomyocytes, NaV1.5 channels mediate initiation and fast propagation of action potentials. The Ca2+-binding protein calmodulin (CaM) serves as a de facto subunit of NaV1.5. Genetic studies and atomic structures suggest that this interaction is pathophysiologically critical, as human mutations within the NaV1.5 carboxy-terminus that disrupt CaM binding are linked to distinct forms of life-threatening arrhythmias, including long QT syndrome 3, a "gain-of-function" defect, and Brugada syndrome, a "loss-of-function" phenotype. Yet, how a common disruption in CaM binding engenders divergent effects on NaV1.5 gating is not fully understood, though vital for elucidating arrhythmogenic mechanisms and for developing new therapies. Here, using extensive single-channel analysis, we find that the disruption of Ca2+-free CaM preassociation with NaV1.5 exerts two disparate effects: 1) a decrease in the peak open probability and 2) an increase in persistent NaV openings. Mechanistically, these effects arise from a CaM-dependent switch in the NaV inactivation mechanism. Specifically, CaM-bound channels preferentially inactivate from the open state, while those devoid of CaM exhibit enhanced closed-state inactivation. Further enriching this scheme, for certain mutant NaV1.5, local Ca2+ fluctuations elicit a rapid recruitment of CaM that reverses the increase in persistent Na current, a factor that may promote beat-to-beat variability in late Na current. In all, these findings identify the elementary mechanism of CaM regulation of NaV1.5 and, in so doing, unravel a noncanonical role for CaM in tuning ion channel gating. Furthermore, our results furnish an in-depth molecular framework for understanding complex arrhythmogenic phenotypes of NaV1.5 channelopathies.

The Schizophrenia Variant V1282F in SCN2A Causes Functional Impairment of NaV1.2

Neuropsychiatric disorders such as schizophrenia are challenging to treat due to the biological complexity of the disease and the lack of knowledge of the underlying pathophysiology. Whole exome and genome sequencing studies have identified disease-linked rare variants in patients with large effect size. Here, we functionally characterize the schizophrenia linked variant V1282F in SCN2A, encoding the sodium channel Nav1.2. This variant was introduced into isogenic lines of hiPSCs using CRISPR/CAS9 genome editing tools. hiPSCs were then differentiated into cortical neurons to understand how the variant and gene may be contributing to disease. We observed a significant (~25%) decrease in sodium current in the V1282F neurons compared to control neurons, suggesting the mutation is causing a loss-of-channel function. These results were supported by recordings in recombinant cells overexpressing either the mutant or wildtype Nav1.2, with the mutant channel having significantly (~75%) lower current amplitude than wildtype. We hypothesize that this phenotype may contribute to disease either through the direct loss of neuronal activity or through subsequent abnormal neurodevelopment.

Changes in Resurgent Sodium Current Contribute to the Hyperexcitability of Muscles in Patients with Paramyotonia Congenita.

Paramyotonia congenita (PMC) is a rare hereditary skeletal muscle disorder. The major symptom, muscle stiffness, is frequently induced by cold exposure and repetitive exercise. Mutations in human SCN4A gene, which encodes the α-subunit of Nav1.4 channel, are responsible for PMC. Mutation screening of SCN4A gene from two PMC families identified two missense mutations, p.T1313M and p.R1448H. To elucidate the electrophysiological abnormalities caused by the mutations, the p.T1313M, p.R1448H, and wild-type (WT) SCN4A genes were transient expressed on Chinese hamster ovary (CHO-K1) cells. The detailed study on the gating defects of the mutant channels using the whole-cell patch clamping technique was performed. The mutant Nav1.4 channels impaired the basic gating properties with increasing sustained and window currents during membrane depolarization and facilitated the genesis of resurgent currents during repolarization. The mutations caused a hyperpolarization shift in the fast inactivation and slightly enhanced the slow inactivation with an increase in half-maximal inactivation voltage. No differences were found in the decay kinetics of the tail current between mutant and WT channels. In addition to generating the larger resurgent sodium current, the time to peak in the mutant channels was longer than that in the WT channels. In conclusion, our results demonstrated that the mutations p.T1313M and p.R1448H in Nav1.4 channels can enhance fast inactivation, slow inactivation, and resurgent current, revealing that subtle changes in gating processes can influence the clinical phenotype.

Abstract MP113: Protein Hyperacetylation Mediates Bradyarrhythmia in Mice With Mitochondrial Disease

Background: Mice with germline mutation of the mitochondrial respiratory complex I subunit Ndufs4 exhibit progressive neurodegenerative phenotypes resembling Leigh syndrome (LS). Cardiomyocyte-specific Ndufs4 -/- mice demonstrate protein hyperacetylation and accelerated heart failure in response to pressure overload. Methods and Results: LS mice developed bradyarrhythmia with heart rate <400 bpm. EKG showed sick sinus syndrome (SSS) and high degree AV block. Supplementation of Nicotinamide Riboside (NR), which increased cellular NAD + , significantly reduced arrhythmia events and restored normal sinus rhythm, as shown by confocal scanning of sinoatrial nodal tissue dissected from LS mouse hearts. Immunoprecipitation and immunohistochemistry using antibody specific for K1479 of Nav 1.5 showed hyperacetylation of LS SA nodal tissue, which was attenuated by NR. HEK293-cells deficient in mitochondrial complex I subunit Ndufs2 had higher DCFDA (ROS) and lower TMRM intensity (mitochondrial membrane potential) compared with Ndufs4 knock-out cells. Ndufs2-knock-out HEK293 cells transfected with wild-type Nav 1.5 showed hyperacetylation of K1479 of Nav 1.5 . Patch clamp showed significantly reduced sodium current (I Na ) in Ndufs2 deficient HEK293 cells. NR prevented the decrease of I Na in Ndufs2 KO cells. HEK293 cells expressing mutant Nav 1.5 constructs mimicking deacetylation (K1479R) had increased I Na compared with HEK293 expressing WT Nav 1.5, suggesting that acetylation status affect Nav1.5 function. Arrythmia was noted in conduction tissue specific HCN4-Ndufs4 -/- mice, suggested a cell autonomous effect leading to arrhythmia. These findings indicate that mitochondrial complex I deficiency lead to hyperacetylation of Nav 1.5 , reduction of I Na , which subsequently cause bradyarrhythmia and this can be reversed by NR. Using echocardiography, we demonstrated significant diastolic dysfunction (decreased E’/A’, prolonged isovolemic relaxation time) in LS mice, which was ameliorated by NR. The calcium transient amplitude and decay rate, an indicator of ryanodine receptor and SERCA2 functions, was significantly decreased by ~15% in LS hearts and this was mitigated by NR, concomitant with reversal of SERCA2 hyperacetylation. Conclusions: Despite normal cardiac structure and systolic function, LS mice showed SSS, high degree AV block and diastolic dysfunction, which is mediated by hyperacetylation of Nav 1.5 and SERCA2, respectively. NR significantly reversed both arrhythmia and diastolic dysfunction. Our study demonstrates novel mechanistic link between mitochondrial complex I deficiency and the function of Nav 1.5 and SERCA2.

A Study among the Genotype, Functional Alternations, and Phenotype of 9 SCN1A Mutations in Epilepsy Patients

Mutations in the voltage-gated sodium channel Nav1.1 (SCN1A) are linked to various epileptic phenotypes with different severities, however, the consequences of newly identified SCN1A variants on patient phenotype is uncertain so far. The functional impact of nine SCN1A variants, including five novel variants identified in this study, was studied using whole-cell patch-clamp recordings measurement of mutant Nav1.1 channels expressed in HEK293T mammalian cells. E78X, W384X, E1587K, and R1596C channels failed to produce measurable sodium currents, indicating complete loss of channel function. E788K and M909K variants resulted in partial loss of function by exhibiting reduced current density, depolarizing shifts of the activation and hyperpolarizing shifts of the inactivation curves, and slower recovery from inactivation. Hyperpolarizing shifts of the activation and inactivation curves were observed in D249E channels along with slower recovery from inactivation. Slower recovery from inactivation was observed in E78D and T1934I with reduced current density in T1934I channels. Various functional effects were observed with the lack of sodium current being mainly associated with severe phenotypes and milder symptoms with less damaging channel alteration. In vitro functional analysis is thus fundamental for elucidation of the molecular mechanisms of epilepsy, to guide patients’ treatment, and finally indicate misdiagnosis of SCN1A related epilepsies.

A distinct molecular mechanism by which phenytoin rescues a novel long QT 3 variant

BackgroundGenetic variants in SCN5A can result in channelopathies such as the long QT syndrome type 3 (LQT3), but the therapeutic response to Na+ channel blockers can vary. We previously reported a case of an infant with malignant LQT3 and a missense Q1475P SCN5A variant, who was effectively treated with phenytoin, but only partially with mexiletine. Here, we functionally characterized this variant and investigated possible mechanisms for the differential drug actions. MethodsWild-type or mutant Nav1.5 cDNAs were examined in transfected HEK293 cells with patch clamping and biochemical assays. We used computational modeling to provide insights into altered channel kinetics and to predict effects on the action potential. ResultsThe Q1475P variant in Nav1.5 reduced the current density and channel surface expression, characteristic of a trafficking defect. The variant also led to positive shifts in the voltage dependence of steady-state activation and inactivation, faster inactivation and recovery from inactivation, and increased the “late” Na+ current. Simulations of Nav1.5 gating with a 9-state Markov model suggested that transitions from inactivated to closed states were accelerated in Q1475P channels, leading to accumulation of channels in non-inactivated closed states. Simulations with a human ventricular myocyte model predicted action potential prolongation with Q1475P, compared with wild type, channels. Patch clamp data showed that mexiletine and phenytoin similarly rescued some of the gating defects. Chronic incubation with mexiletine, but not phenytoin, rescued the Nav1.5-Q1475P trafficking defect, thus increasing mutant channel expression. ConclusionsThe gain-of-function effects of Nav1.5-Q1475P predominate to cause a malignant long QT phenotype. Phenytoin partially corrects the gating defect without restoring surface expression of the mutant channel, whereas mexiletine restores surface expression of the mutant channel, which may explain the lack of efficacy of mexiletine when compared to phenytoin. Our data makes a case for experimental studies before embarking on a one-for-all therapy of arrhythmias.