Voltage Dependence Of Activation Research Articles

Two Novel Variants in the CHRNA2 and SCN2A Genes in Italian Patients with Febrile Seizures

Background: Febrile seizures (FSs) are the most common form of epilepsy in children aged between six months and five years. The exact cause is unknown, but several studies have demonstrated the importance of genetic predisposition, with increasing involvement of receptors and ion channels. The present study aims to identify novel pathogenic variants in Italian patients with FSs. Methods: We performed targeted panel sequencing in a cohort of 21 patients with FSs. In silico analysis was performed to predict the pathogenic role of the resulting variants. Results: We found two novel variants segregating in two families with FSs: c.1021C>G (p.Leu341Val) in the CHRNA2 gene and c.140A>G (p.Glu47Gly) in SCN2A. Conclusions: The c.1021C>G (p.Leu341Val) variant leads to a codon change of highly conserved leucine to valine at position 341 and is located in segments M3 of the subunit, which is important for channel gating. The c.140A>G (p.Glu47Gly) variant causes a substitution of glutamic acid with glycine at position 47 of the protein, which is highly conserved across the species. Moreover, it is located in the N-terminal domain, a region commonly affected in ASD, which impacts the inactivation kinetics and voltage dependence of steady-state activation. Further analyses are needed to better explain the role of CHRNA2 and SCN2A in the development of febrile seizures.

Empagliflozin treatment rescues abnormally reduced Na+ currents in ventricular cardiomyocytes from dystrophin-deficient mdx mice

Abstract Background Cardiac arrhythmias represent a major cause of death in Duchenne muscular dystrophy (DMD), a devastating muscle disease caused by dystrophin deficiency. An important source for arrhythmia vulnerability in DMD patients is impaired ventricular impulse conduction, which predisposes for ventricular asynchrony, and the development of reentrant circuits. Using the dystrophin-deficient mdx mouse model for DMD, we and others previously reported that the absence of dystrophin results in a significant peak Na+ current (INa) loss both in ventricular cardiomyocytes of the working myocardium, and in Purkinje fibers, ventricular myocytes specialized for rapid electrical impulse conduction. This finding provided a mechanistic explanation for ventricular conduction defects and concomitant arrhythmias in the dystrophic heart. Purpose In the present study, we tested the hypothesis that empagliflozin (EMPA), an inhibitor of sodium/glucose cotransporter 2 in clinical use to treat type II diabetes and nondiabetic heart failure, rescues the loss of peak INa in dystrophin-deficient ventricular cardiomyocytes. Methods In a first set of experiments, we treated a cohort of mdx mice with 15 mg/kg body weight/day EMPA via the drinking water. After 4 weeks of treatment, ventricular cardiomyocytes were isolated from these mice, as well as from untreated wild-type and mdx control mice, via Langendorff heart perfusion and enzymatic digestion. Peak INa was measured using the whole cell patch clamp technique. In a second experimental approach, peak INa was compared after a 24 hour exposure of isolated mdx ventricular cardiomyocytes with 1 µM EMPA, 10 µM of the Na+-H+ exchanger 1 inhibitor cariporide, or 10 µM cariporide in combination with 1 µM EMPA. In a third set of experiments, we superfused freshly isolated mdx ventricular cardiomyocytes with 1 or 10 µM EMPA in order to examine the acute action of the drug on peak INa. Results Consistent with the literature, we found peak INa of mdx ventricular cardiomyocytes to be substantially decreased compared to wild-type. Peak INa of ventricular cardiomyocytes derived from mdx mice, which had received EMPA for 4 weeks, was restored to wild-type level. Moreover, incubation of isolated mdx ventricular cardiomyocytes with EMPA for 24 hours significantly increased their peak INa. This effect did not depend on Na+-H+ exchanger 1 inhibition by the drug. The voltage dependencies of INa activation and fast inactivation were neither modified by long-term treatment nor incubation with EMPA. Finally, acute application of EMPA did not influence peak INa. Conclusion Our findings indicate that EMPA treatment can correct abnormally reduced peak INa of dystrophin-deficient ventricular cardiomyocytes. Long-term EMPA administration may diminish arrhythmia vulnerability in DMD patients.

Inactivation induced by pathogenic Cav1.3 L-type Ca2+-channel variants enhances sensitivity for dihydropyridine Ca2+ channel blockers.

Pathogenic gain-of-function mutations in Cav1.3 L-type voltage-gated Ca2+-channels (CACNA1D) cause neurodevelopmental disorders with or without endocrine symptoms. We aimed to confirm a pathogenic gain-of function phenotype of CACNA1D de novo missense mutations A749T and L271H, and investigated the molecular mechanism causing their enhanced sensitivity for the Ca2+-channel blocker isradipine, a potential therapeutic for affected patients. Wildtype and mutant channels were expressed in tsA-201 cells and their gating analysed using whole-cell and single-channel patch-clamp recordings. The voltage-dependence of isradipine action was quantified using protocols inducing variable fractions of inactivated channels. The molecular basis for altered channel gating in the mutants was investigated using in silico modelling and molecular dynamics simulations. Both mutations were confirmed pathogenic due to characteristic shifts of voltage-dependent activation and inactivation towards negative potentials (~20 mV). At negative holding potentials both mutations showed significantly higher isradipine sensitivity compared to wildtype. The affinity for wildtype and mutant channels increased with channel inactivation as predicted by the modulated receptor hypothesis (30- to 40-fold). The IC50 was indistinguishable for wildtype and mutants when >50% of channels were inactivated. Mutations A749T and L271H induce pathogenic gating changes. Like wildtype, isradipine inhibition is strongly voltage-dependent. Our data explains their apparent higher drug sensitivity at a given negative voltage by the availability of more inactivated channels due to their more negative inactivation voltage range. Low nanomolar isradipine concentrations will only inhibit Cav1.3 channels in neurons during prolonged depolarized states without selectivity for mutant channels.

A Rich Conformational Palette Underlies Human CaV2.1-Channel Availability.

Depolarization-evoked opening of CaV2.1 (P/Q-type) Ca2+-channels triggers neurotransmitter release, while voltage-dependent inactivation (VDI) limits channel availability to open, contributing to synaptic plasticity. The mechanism of CaV2.1 response to voltage is unclear. Using voltage-clamp fluorometry and kinetic modeling, we optically tracked and physically characterized the structural dynamics of the four CaV2.1 voltage-sensor domains (VSDs). VSD-I seems to directly drive opening and convert between two modes of function, associated with VDI. VSD-II is apparently voltage-insensitive. VSD-III and VSD-IV sense more negative voltages and undergo voltage-dependent conversion uncorrelated with VDI. Auxiliary -subunits regulate VSD-I-to-pore coupling and VSD conversion kinetics. CaV2.1 VSDs are differentially sensitive to voltage changes brief and long-lived. Specifically the voltage-dependent conformational changes of VSD-I are linked to synaptic release and plasticity.

Exploring the interplay between extracellular pH and Dronedarone's pharmacological effects on cardiac function

Dronedarone (DRN) is a clinically used drug to mitigate arrhythmias with multichannel block properties, including the sodium channel Nav1.5. Extracellular acidification is known to change the pharmacological properties of several antiarrhythmic drugs. Here, we explore how modification in extracellular pH (pHe) shapes the pharmacological profile of DRN upon Nav1.5 sodium current (INa) and in the ex vivo heart preparation. Embryonic human kidney cells (HEK293T/17) were used to transiently express the human isoform of Nav1.5 α-subunit. Patch-Clamp technique was employed to study INa. Neurotoxin-II (ATX-II) was used to induce the late sodium current (INaLate). Additionally, ex vivo Wistar male rat preparations in the Langendorff system were utilized to study electrocardiogram (ECG) waves. DRN preferentially binds to the closed state inactivation mode of Nav1.5 at pHe 7.0. The recovery from INa inactivation was delayed in the presence of DRN in both pHe 7.0 and 7.4, and the use-dependent properties were distinct at pHe 7.0 and 7.4. However, the potency of DRN upon the peak INa, the voltage dependence for activation, and the steady-state inactivation curves were not altered in both pHe tested. Also, the pHe did not change the ability of DRN to block INaLate. Lastly, DRN in a concentration and pH dependent manner modulated the QRS complex, QT and RR interval in clinically relevant concentration. Thus, the pharmacological properties of DRN upon Nav1.5 and ex vivo heart preparation partially depend on the pHe. The pHe changed the biological effect of DRN in the heart electrical function in relevant clinical concentration.

The significance of electrical signals in maturing spermatozoa for phosphoinositide regulation through voltage-sensing phosphatase

Voltage-sensing phosphatase (VSP) exhibits voltage-dependent phosphatase activity toward phosphoinositides. VSP generates a specialized phosphoinositide environment in mammalian sperm flagellum. However, the voltage-sensing mechanism of VSP in spermatozoa is not yet characterized. Here, we found that VSP is activated during sperm maturation, indicating that electric signals in immature spermatozoa are essential. Using a heterologous expression system, we show the voltage-sensing property of mouse VSP (mVSP). The voltage-sensing threshold of mVSP is approximately −30 mV, which is sensitive enough to activate mVSP in immature spermatozoa. We also report several knock-in mice in which we manipulate the voltage-sensitivity or electrochemical coupling of mVSP. Notably, the V312R mutant, with a minor voltage-sensitivity change, exhibits abnormal sperm motility after, but not before, capacitation. Additionally, the V312R mutant shows a significant change in the acyl-chain profile of phosphoinositide. Our findings suggest that electrical signals during sperm maturation are crucial for establishing the optimal phosphoinositide environment in spermatozoa.

Diverse biophysical mechanisms in voltage-gated sodium channel Nav1.4 variants associated with myotonia.

Mutations in SCN4A gene encoding Nav1.4 channel α-subunit, are known to cause neuromuscular disorders such as myotonia or paralysis. Here, we study the effect of two amino acid replacements, K1302Q and G1306E, in the DIII-IV loop of the channel, corresponding to mutations found in patients with myotonia. We combine clinical, electrophysiological, and molecular modeling data to provide a holistic picture of the molecular mechanisms operating in mutant channels and eventually leading to pathology. We analyze the existing clinical data for patients with the K1302Q substitution, which was reported for adults with or without myotonia phenotypes, and report two new unrelated patients with the G1306E substitution, who presented with severe neonatal episodic laryngospasm and childhood-onset myotonia. We provide a functional analysis of the mutant channels by expressing Nav1.4 α-subunit in Xenopus oocytes in combination with β1 subunit and recording sodium currents using two-electrode voltage clamp. The K1302Q variant exhibits abnormal voltage dependence of steady-state fast inactivation, being the likely cause of pathology. K1302Q does not lead to decelerated fast inactivation, unlike several other myotonic mutations such as G1306E. For both mutants, we observe increased window currents corresponding to a larger population of channels available for activation. To elaborate the structural rationale for our experimental data, we explore the contacts involving K/Q1302 and E1306 in the AlphaFold2 model of wild-type Nav1.4 and Monte Carlo-minimized models of mutant channels. Our data provide the missing evidence to support the classification of K1302Q variant as likely pathogenic and may be used by clinicians.

Functional properties of a disease mutation for migraine in Kv2.1/6.4 channels

Voltage-gated potassium (Kv) channels are integral to cellular excitability, impacting the resting membrane potential, repolarization, and shaping action potentials in neurons and cardiac myocytes. Structurally, Kv channels are homo or heterotetramers comprising four α-subunits, each with six transmembrane segments (S1–S6). Silent Kv (KvS), includes Kv5.1, Kv6.1–6.4, Kv8.1–8.2, and Kv9.1–9.3, they do not form functional channels on their own but modulate the properties of heteromeric channels. Recent studies have identified the Kv6.4 subunit as a significant modulator within heteromeric channels, such as Kv2.16.4. The Kv2.16.4 heteromer exhibits altered biophysical properties, including a shift in voltage-dependent inactivation and a complex activation. Current genetic studies in migraine patients have revealed a single missense mutation in the Kv6.4 gene. The single missense mutation, L360P is in the highly conserved S4–S5 linker region. This study aims to demonstrate the biophysical effects of the L360P mutation in Kv2.1 6.4 channels with a fixed 2:2 stoichiometry, using monomeric (Kv2.1/6.4) and tandem dimer (Kv2.1_6.4) configurations. Our findings suggest that the L360P mutation significantly impacts the function and regulation of Kv2.1/6.4 channels, providing insights into the molecular mechanisms underlying channel dysfunction in migraine pathology.

A positively charged residue at the Kv1.1 T1 interface is critical for voltage-dependent activation and gating kinetics.

Within the tetramerization domain (T1) of most voltage-gated potassium channels (Kv) are highly conserved charged residues that line the T1-T1 interface. We investigated the Kv1.1 residue R86 located at the narrowest region of the T1 interface. A Kv1.1 R86Q mutation was reported in a child diagnosed with lower limb dyskinesia (Set KK, Ghosh D, Huq AHM, Luat AF. Mov Disord Clin Pract 4: 784-786, 2017). The child did not present with episodic ataxia 1 (EA1) symptoms typically associated with Kv1.1 loss-of-function mutations. We characterized the electrophysiological outcome of the R86Q substitution by expressing Kv1.1 in Xenopus laevis oocytes. Mutated α-subunits were able to form functional channels that pass delayed rectifier currents. Oocytes that expressed only mutated α-subunits produced a significant reduction in Kv1.1 current and showed a positive shift in voltage dependence of activation. In addition, there was substantially slower activation and faster deactivation implying a reduction in the time the channel is in its open state. Oocytes co-injected with both mutated and wild-type cRNA in equal amounts, to mimic the heterozygous condition of the disease, showed a decrease in current amplitude at -10 mV, a positive shift in activation voltage-dependence and faster deactivation kinetics when compared with the wild-type channel. These findings indicate that T1 plays a role in Kv1.1's voltage-dependent activation and in its kinetics of activation and deactivation.NEW & NOTEWORTHY This is the first Kv1.1 study to characterize the electrophysiological and structural phenotype of a tetramerization (T1) domain mutation. Surprisingly, the mutated α-subunits were able to tetramerize, albeit with different gating kinetics and voltage dependence. This novel finding points to a clear role of T1 in the channel's voltage dependence and gating. Mimicking the heterozygous condition resulted in milder alterations in channel function when compared with previously reported mutations. This is in agreement with the child's milder symptoms.

A Feature Selection-Incorporated Simulation Study to Reveal the Effect of Calcium Ions on Cardiac Repolarization Alternans during Myocardial Ischemia

(1) Background: The main factors and their interrelationships contributing to cardiac repolarization alternans (CRA) remain unclear. This study aimed to elucidate the calcium (Ca2+)-related mechanisms underlying myocardial ischemia (MI)-induced CRA. (2) Materials and Methods: CRA was induced using S1 stimuli for pacing in an in silico ventricular model with MI. The standard deviations of nine Ca2+-related subcellular parameters among heartbeats from 100 respective nodes with and without alternans were chosen as features, including the maximum systole and end-diastole and corresponding differences in the Ca2+ concentration in the intracellular region([Ca2+]i) and junctional sarcoplasmic reticulum ([Ca2+]jsr), as well as the maximum opening of the L-type Ca2+ current (ICaL) voltage-dependent activation gate (d-gate), maximum closing of the inactivation gate (ff-gate), and the gated channel opening time (GCOT). Feature selection was applied to determine the importance of these features. (3) Results: The major parameters affecting CRA were the differences in [Ca2+]i at end-diastole, followed by the extent of d-gate activation and GCOT among beats. (4) Conclusions: MI-induced CRA is primarily characterized by functional changes in Ca2+ re-uptake, leading to alternans of [Ca2+]i and subsequent alternans of ICaL-dependent properties. The combination of computational simulation and machine learning shows promise in researching the underlying mechanisms of cardiac electrophysiology.

Characterization of ClC-1 chloride channels in zebrafish: a new model to study myotonia.

The function of the chloride channel ClC-1 is crucial for the control of muscle excitability. Thus, reduction of ClC-1 functions by CLCN1 mutations leads to myotonia congenita. Many different animal models have contributed to understanding the myotonia pathophysiology. However, these models do not allow in vivo screening of potentially therapeutic drugs, as the zebrafish model does. In this work, we identified and characterized the two zebrafish orthologues (clc-1a and clc-1b) of the ClC-1 channel. Both channels are mostly expressed in the skeletal muscle as revealed by RT-PCR, western blot, and electrophysiological recordings of myotubes, and clc-1a is predominantly expressed in adult stages. Characterization in Xenopus oocytes shows that the zebrafish channels display similar anion selectivity and voltage dependence to their human counterparts. However, they show reduced sensitivity to the inhibitor 9-anthracenecarboxylic acid (9-AC), and acidic pH inverts the voltage dependence of activation. Reduction of clc-1a/b expression hampers spontaneous and mechanically stimulated movement, which could be reverted by expression of human ClC-1 but not by some ClC-1 containing myotonia mutations. Treatment of clc-1-depleted zebrafish with mexiletine, a typical drug used in human myotonia, improves the motor behaviour. Our work extends the repertoire of ClC channels to evolutionary structure-function studies and proposes the zebrafish clcn1 crispant model as a simple tool to find novel therapies for myotonia. KEY POINTS: We have identified two orthologues of ClC-1 in zebrafish (clc-1a and clc-1b) which are mostly expressed in skeletal muscle at different developmental stages. Functional characterization of the activity of these channels reveals many similitudes with their mammalian counterparts, although they are less sensitive to 9-AC and acidic pH inverts their voltage dependence of gating. Reduction of clc-1a/b expression hampers spontaneous and mechanically stimulated movement which could be reverted by expression of human ClC-1. Myotonia-like symptoms caused by clc-1a/b depletion can be reverted by mexiletine, suggesting that this model could be used to find novel therapies for myotonia.

Zinc inhibits the voltage-gated proton channel HCNL1

Voltage-gated ion channels allow ion flux across biological membranes in response to changes in the membrane potential. HCNL1 is a recently discovered voltage-gated ion channel that selectively conducts protons through its voltage-sensing domain (VSD), reminiscent of the well-studied depolarization-activated Hv1 proton channel. However, HCNL1 is activated by hyperpolarization, allowing the influx of protons, which leads to an intracellular acidification in zebrafish sperm. Zinc ions (Zn2+) are important cofactors in many proteins and essential for sperm physiology. Proton channels such as Hv1 and Otopetrin1 are inhibited by Zn2+. We investigated the effect of Zn2+ on heterologously expressed HCNL1 channels using electrophysiological and fluorometric techniques. Extracellular Zn2+ inhibits HCNL1 currents with an apparent half-maximal inhibition (IC50) of 26 μM. Zn2+ slows voltage-dependent current kinetics, shifts the voltage-dependent activation to more negative potentials, and alters hyperpolarization-induced conformational changes of the voltage sensor. Our data suggest that extracellular Zn2+ inhibits HCNL1 currents by multiple mechanisms, including modulation of channel gating. Two histidine residues located at the extracellular side of the VSD might weakly contribute to Zn2+ coordination: mutants with either histidine replaced with alanine show modest shifts of the IC50 values to higher concentrations. Interestingly, Zn2+ inhibits HCNL1 at even lower concentrations from the intracellular side (IC50 ≈ 0.5 μM). A histidine residue at the intracellular end of S1 (position 50) is important for Zn2+ binding: much higher Zn2+ concentrations are required to inhibit the mutant HCNL1-H50A (IC50 ≈ 106 μM). We anticipate that Zn2+ will be a useful ion to study the structure-function relationship of HCNL1 as well as the physiological role of HCNL1 in zebrafish sperm.

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

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

Voltage dependence of M2 muscarinic receptor antagonists and allosteric modulators

Muscarinic receptors are G protein-coupled receptors (GPCRs) that play a role in various physiological functions. Previous studies have shown that these receptors, along with other GPCRs, are voltage-sensitive; both their affinity toward agonists and their activation are regulated by membrane potential. To our knowledge, whether the effect of antagonists on these receptors is voltage-dependent has not yet been studied. In this study, we used Xenopus oocytes expressing the M2 muscarinic receptor (M2R) to investigate this question. Our results indicate that the potencies of two M2R antagonists, atropine and scopolamine, are voltage-dependent; they are more effective at resting potential than under depolarization. In contrast, the M2R antagonist AF-DX 386 did not exhibit voltage-dependent potency.Furthermore, we discovered that the voltage dependence of M2R activation by acetylcholine remains unchanged in the presence of two allosteric modulators, the negative modulator gallamine and the positive modulator LY2119620. These findings enhance our understanding of GPCRs’ voltage dependence and may have pharmacological implications.

Dimerization is required for the glycosylation of S1-S2 linker of sea urchin voltage-gated proton channel Hv1

Multimerization of ion channels is essential for establishing the ion-selective pathway and tuning the gating regulated by membrane potential, second messengers, and temperature. Voltage-gated proton channel, Hv1, consists of voltage-sensor domain and coiled-coil domain. Hv1 forms dimer, whereas voltage-dependent channel activity is self-contained in monomer unlike many ion channels, which assemble to form ion-conductive pathways among multiple subunits. Dimerization of Hv1 is necessary for cooperative gating, but other roles of dimerization in physiological aspects are still largely unclear. In this study, we show that dimerization of Hv1 takes place in ER. Sea urchin Hv1 (Strongylocentrotus purpuratus Hv1: SpHv1) was glycosylated in the consensus sequence for N-linked glycosylation within the S1-S2 extracellular loop. However, glycosylation was not observed in the monomeric SpHv1 that lacks the coiled-coil domain. A version of mHv1 in which the S1-S2 loop was replaced by that of SpHv1 showed glycosylation and its monomeric form was not glycosylated. Tandem dimer of monomeric SpHv1 underwent glycosylation, suggesting that dimerization of Hv1 is required for glycosylation. Moreover, when monomeric Hv1 has a dilysine motif in the C-terminal end, which is known to act as a retrieval signal from Golgi to ER, prolonging the time of residency in ER, it was glycosylated. Overall, our results suggest that monomeric SpHv1 does not stay long in ER, thereby escaping glycosylation, while the dimerization causes the proteins to stay longer in ER. Thus, the findings highlight the novel significance of dimerization of Hv1: regulation of biogenesis and maturation of the proteins in intracellular compartments.

Requirement of β subunit for the reduced voltage-gated Na+ current of a Brugada syndrome patient having novel double missense mutation (p.A385T/R504T) of SCN5A.

Mutations within the SCN5A gene, which encodes the α-subunit 5 (NaV1.5) of the voltage-gated Na+ channel, have been linked to three distinct cardiac arrhythmia disorders: long QT syndrome type 3, Brugada syndrome (BrS), and cardiac conduction disorder. In this study, we have identified novel missense mutations (p.A385T/R504T) within SCN5A in a patient exhibiting overlap arrhythmia phenotypes. This study aims to elucidate the functional consequences of SCN5A mutants (p.A385T/R504T) to understand the clinical phenotypes. Whole-cell patch-clamp technique was used to analyze the NaV1.5 current (INa) in HEK293 cells transfected with the wild-type and mutant SCN5A with or without SCN1B co-expression. The amplitude of INa was not altered in mutant SCN5A (p.A385T/R504T) alone. Furthermore, a rightward shift of the voltage-dependent inactivation and faster recovery from inactivation was observed, suggesting a gain-of-function state. Intriguingly, the coexpression of SCN1B with p.A385T/R504T revealed significant reduction of INa and slower recovery from inactivation, consistent with the loss-of-function in Na+ channels. The SCN1B dependent reduction of INa was also observed in a single mutation p.R504T, but p.A385T co-expressed with SCN1B showed no reduction. In contrast, the slower recovery from inactivation with SCN1B was observed in A385T while not in R504T. The expression of SCN1B is indispensable for the electrophysiological phenotype of BrS with the novel double mutations; p.A385T and p.R504T contributed to the slower recovery from inactivation and reduced current density of NaV1.5, respectively.

Nav1.2 channel mutations preventing fast inactivation lead to SCN2A encephalopathy

Abstract SCN2A gene-related early-infantile developmental and epileptic encephalopathy (EI-DEE) is a rare and severe disorder that manifests in early infancy. SCN2A mutations affecting the fast inactivation gating mechanism can result in altered voltage dependence and incomplete inactivation of the encoded neuronal Nav1.2 channel and lead to abnormal neuronal excitability. In this study, we evaluated clinical data of seven missense Nav1.2 variants associated with DEE and performed molecular dynamics simulations, patch-clamp electrophysiology, and dynamic clamp real-time neuronal modelling to elucidate the molecular and neuron-scale phenotypic consequences of the mutations. The N1662D mutation almost completely prevented fast inactivation without affecting activation. The comparison of wild-type and N1662D channel structures suggested that the ambifunctional hydrogen bond formation between residues N1662 and Q1494 is essential for fast inactivation. Fast inactivation could also be prevented with engineered Q1494A or Q1494L Nav1.2 channel variants, whereas Q1494E or Q1494 K variants resulted in incomplete inactivation and persistent current. Molecular dynamics simulations revealed a reduced affinity of the hydrophobic IFM-motif to its receptor site with N1662D and Q1494L variants relative to wild-type. These results demonstrate that the interactions between N1662 and Q1494 underpin the stability and the orientation of the inactivation gate and are essential for the development of fast inactivation. Six DEE-associated Nav1.2 variants, with mutations mapped to channel segments known to be implicated in fast inactivation were also evaluated. Remarkably, the L1657P variant also prevented fast inactivation and produced biophysical characteristics that were similar to those of N1662D, whereas the M1501 V, M1501T, F1651C, P1658S, and A1659 V variants resulted in biophysical properties that were consistent with gain-of-function and enhanced action potential firing of hybrid neurons in dynamic action potential clamp experiments. Paradoxically, low densities of N1662D or L1657P currents potentiated action potential firing, whereas increased densities resulted in sustained depolarization. Our results provide novel structural insights into the molecular mechanism of Nav1.2 channel fast inactivation and inform treatment strategies for SCN2A-related EI-DEE. The contribution of non-inactivating Nav1.2 channels to neuronal excitability may constitute a distinct cellular mechanism in the pathogenesis of SCN2A-related DEE.

Glycine to valine substitution in the short intracellular linkers of domain II enhances I1011M-mediated sodium channel resistance to Type I pyrethroids, but not Type II pyrethroids

Pyrethroids are widely used against agricultural pests and human disease vectors due to their broad insecticidal spectrum, fast action, and low mammalian toxicity. Unfortunately, overuse of pyrethroids has led to knockdown resistance (kdr) caused by mutations in voltage-gated sodium channels. Mutation I1011M was repeatedly detected in numerous pyrethroid-resistant Aedes aegypti populations from Latin American and Brazil. In addition, mutation G923V was first reported to coexist with I1011M in permethrin/DDT-resistant Ae. aegypti, whether G923V enhances the I1011M-mediated pyrethroid resistance in sodium channels remains unclear. In this study, we introduced mutations G923V and I1011M alone or in combination into the pyrethroid-sensitive sodium channel AaNav1–1 and examined the effects of these mutations on gating properties and pyrethroid sensitivity. We found mutations I1011M and G923V + I1011M shifted the voltage dependence of activation in the depolarizing direction, and none of mutations affect the voltage-dependence of inactivation. G923V and G923V + I1011M mutations reduced the channel sensitivity to both Type I and Type II pyrethroids. However, I1011M alone conferred resistance to Type I pyrethroids, not to Type II pyrethroids. Interestingly, significant synergism effects on Type I pyrethroids were observed between mutations G923V and I1011M. The effects of all mutations on channel sensitivity to DDT were identical with those to Type I pyrethroids. Our results confirm the molecular basis of resistance mediated by mutations G923V and I1011M and may contribute to develop molecular markers for monitoring pest resistance to pyrethroids.

Electrocontractile remodeling of isolated cardiomyocytes induced during early-stage hypercholesterolemia.

Hypercholesterolemia is one of the most important risk factors for cardiovascular diseases. However, it is mostly associated with vascular dysfunction and atherosclerotic lesions, while evidence of direct effects of hypercholesterolemia on cardiomyocytes and heart function is still incomplete and controversial. In this study, we assessed the direct effects of hypercholesterolemia on heart function and the electro-contractile properties of isolated cardiomyocytes. After 5 weeks, male Swiss mice fed with AIN-93 diet added with 1.25% cholesterol (CHO), developed an increase in total serum cholesterol levels and cardiomyocytes cholesterol content. These changes led to altered electrocardiographic records, with a shortening of the QT interval. Isolated cardiomyocytes displayed a shortening of the action potential duration with increased rate of depolarization, which was explained by increased IK, reduced ICa.L and altered INa voltage-dependent inactivation. Also, reduced diastolic [Ca2+]i was found with preserved adrenergic response and cellular contraction function. However, contraction of isolated hearts is impaired in isolated CHO hearts, before and after ischemia/reperfusion, although CHO heart was less susceptible to arrhythmic contractions. Overall, our results demonstrate that early hypercholesterolemia-driven increase in cellular cholesterol content is associated with direct modulation of the heart and cardiomyocytes' excitability, Ca2+ handling, and contraction.

Regulating neuronal excitability: The role of S-palmitoylation in NaV1.7 activity and voltage sensitivity.

S-palmitoylation, a reversible lipid post-translational modification, regulates the functions of numerous proteins. Voltage-gated sodium channels (NaVs), pivotal in action potential generation and propagation within cardiac cells and sensory neurons, can be directly or indirectly modulated by S-palmitoylation, impacting channel trafficking and function. However, the role of S-palmitoylation in modulating NaV1.7, a significant contributor to pain pathophysiology, has remained unexplored. Here, we addressed this knowledge gap by investigating if S-palmitoylation influences NaV1.7 channel function. Acyl-biotin exchange assays demonstrated that heterologously expressed NaV1.7 channels are modified by S-palmitoylation. Blocking S-palmitoylation with 2-bromopalmitate resulted in reduced NaV1.7 current density and hyperpolarized steady-state inactivation. We identified two S-palmitoylation sites within NaV1.7, both located in the second intracellular loop, which regulated different properties of the channel. Specifically, S-palmitoylation of cysteine 1126 enhanced NaV1.7 current density, while S-palmitoylation of cysteine 1152 modulated voltage-dependent inactivation. Blocking S-palmitoylation altered excitability of rat dorsal root ganglion neurons. Lastly, in human sensory neurons, NaV1.7 undergoes S-palmitoylation, and the attenuation of this post-translational modification results in alterations in the voltage-dependence of activation, leading to decreased neuronal excitability. Our data show, for the first time, that S-palmitoylation affects NaV1.7 channels, exerting regulatory control over their activity and, consequently, impacting rodent and human sensory neuron excitability. These findings provide a foundation for future pharmacological studies, potentially uncovering novel therapeutic avenues in the modulation of S-palmitoylation for NaV1.7 channels.