Voltage-gated Sodium Research Articles

The status of pyrethroid resistance mutation frequencies in Varroa destructor populations in the most important beekeeping areas of Türkiye

The Varroa destructor (hereafter referred to as Varroa) is a major pest of honeybees that is generally controlled using pyrethroid-based acaricides. However, resistance to these insecticides has become a growing problem, driven by the acquisition of knockdown resistance (kdr) mutations in the mite’s voltage-gated sodium channel (vgsc) gene. Resistance mutations in the vgsc gene, such as the L925V mutation, can confer resistance to pyrethroids like flumethrin and tau-fluvalinate. Monitoring genotypic resistance through molecular mutation screening is crucial for tracking and mitigating resistance spread. In this study, the frequency of resistance mutations in the vgsc was examined using a Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) approach in Varroa populations sampled throughout the Mediterranean, Aegean, and Black Sea regions of Türkiye. Considering all the samples analyzed, the results demonstrated a mean resistance allele frequency of 83.29%, indicating a relatively high frequency of resistant alleles. We observed 94.58%, 85.71%, and 69.58% resistant allele frequencies in populations sampled from the Mediterranean, Aegean, and Black Sea regions, respectively, in our study. The results of our investigation demonstrated substantial regional variations in the frequencies of resistant alleles among Varroa populations throughout Türkiye, with notably elevated resistance levels observed in the Mediterranean and Aegean regions. Due to the significant resistance mutation frequency differences between both provinces and regions, long-term monitoring of resistance alleles and the planning of regional control strategies are required for effective control of this pest.

Genetic Variants in the SCN9A Gene are Detected in a Minority of Erythromelalgia Patients.

Gain-of-function variants in the voltage-gated sodium channel Nav1.7, encoded by the SCN9A gene, have previously been identified in patients with erythromelalgia, a clinical diagnosis defined by intermittent attacks of painful, hot, swollen, and red skin, predominantly involving the hands and feet. Symptoms are induced or aggravated by warming and relieved by cooling. In primary erythromelalgia there is no known underlying disease. This study investigated the frequency of SCN9A variants in a cohort of primary erythromelalgia patients collected at a single centre, and examined the clinical signs and symptoms associated with identified variants. One hundred patients with possible erythromelalgia were collected prospectively and evaluated by clinical examination. Thirty-five patients fulfilling the clinical criteria of primary erythromelalgia were screened for variants in SCN9A. Five were found to carry likely causal variants, including a variant found in 2 related individuals and a variant not previously described in patients with erythromelalgia. The clinical findings differed significantly between the patients. Overall, in this cohort only 4/34 (11.7%) of unrelated patients had erythromelalgia likely caused by gain-of-function variants in SCN9A. Variants in SCN9A are therefore likely to cause or contribute to primary erythromelalgia in only a small proportion of patients.

Neonatal but not Juvenile Gene Therapy Reduces Seizures and Prolongs Lifespan in SCN1B-Dravet Syndrome Mice.

Dravet syndrome (DS) is a developmental and epileptic encephalopathy (DEE) that begins in the first year of life. While most cases of DS are caused by variants in SCN1A, variants in SCN1B, encoding voltage-gated sodium channel β1 subunits, are also linked to DS or to the more severe early infantile DEE. Both disorders fall under the OMIM term DEE52. Scn1b null mice model DEE52, with spontaneous generalized seizures and death in 100% of animals in the third postnatal week. Scn1b null cortical parvalbumin-positive interneurons and pyramidal neurons are hypoexcitable. The goal of this study was to develop a proof-of-principle gene replacement strategy for DEE52. We tested an adeno-associated viral vector encoding β1 subunit cDNA (AAV-Navβ1) in Scn1b null mice. We demonstrated that AAV-Navβ1 drives β1 protein expression in excitatory and inhibitory neurons in mouse brain. Bilateral intracerebroventricular administration of AAV-Navβ1 in Scn1b null mice at postnatal day (P) 2, but not at P10, reduced spontaneous seizure severity and duration, prolonged life span, prevented hyperthermia-induced seizures, and restored cortical neuron excitability. AAV-Navβ1 administration to WT mice resulted in β1 overexpression in brain but no obvious adverse effects. This work lays the foundation for future development of a gene therapeutic strategy for SCN1B-linked DEE patients. .

Nav1.8, an analgesic target for nonpsychotomimetic phytocannabinoids

Pain impacts billions of people worldwide, but treatment options are limited and have a spectrum of adverse effects. The search for safe and nonaddictive pain treatments has led to a focus on key mediators of nociceptor excitability. Voltage-gated sodium (Nav) channels in the peripheral nervous system-Nav1.7, Nav1.8, and Nav1.9-play crucial roles in pain signaling. Among these, Nav1.8 has shown promise due to its rapid recovery from inactivation and role in repetitive firing, with recent clinical studies providing proof-of-principal that block of Nav1.8 can reduce pain in humans. We report here that three nonpsychotomimetic cannabinoids-cannabidiol (CBD), cannabigerol (CBG), and cannabinol (CBN)-effectively inhibit Nav1.8, suggesting their potential as analgesic compounds. In particular, CBG shows significant promise due to its ability to effectively inhibit excitability of peripheral sensory neurons. These findings highlight the therapeutic potential of cannabinoids, particularly CBG, as agents that may attenuate pain via block of Nav1.8, warranting further in vivo studies.

Defining the Polycystin Pharmacophore Through HTS & Computational Biophysics.

Polycystins (PKD2, PKD2L1) are voltage-gated and Ca2+-modulated members of the transient receptor potential (TRP) family of ion channels. Loss of PKD2L1 expression results in seizure-susceptibility and autism-like features in mice, whereas variants in PKD2 cause autosomal dominant polycystic kidney disease. Despite decades of evidence clearly linking their dysfunction to human disease and demonstrating their physiological importance in the brain and kidneys, the polycystin pharmacophore remains undefined. Contributing to this knowledge gap is their resistance to drug screening campaigns, which are hindered by these channels' unique subcellular trafficking to organelles such as the primary cilium. PKD2L1 is the only member of the polycystin family to form constitutively active ion channels on the plasma membrane when overexpressed. HEK293 cells stably expressing PKD2L1 F514A were pharmacologically screened via high-throughput electrophysiology to identify potent polycystin channel modulators. In-silico docking analysis and mutagenesis were used to define the receptor sites of screen hits. Inhibition by membrane-impermeable QX-314 was used to evaluate PKD2L1's binding site accessibility. Screen results identify potent PKD2L1 antagonists with divergent chemical core structures and highlight striking similarities between the molecular pharmacology of PKD2L1 and voltage-gated sodium channels. Docking analysis, channel mutagenesis, and physiological recordings identify an open-state accessible lateral fenestration receptor within the pore, and a mechanism of inhibition that stabilizes the PKD2L1 inactivated state. Outcomes establish the suitability of our approach to expand our chemical knowledge of polycystins and delineates novel receptor moieties for the development of channel-specific antagonists in TRP channel research.

Cenobamate as add-on treatment for SCN8A developmental and epileptic encephalopathy.

Developmental and epileptic encephalopathies(DEEs) caused by pathogenic variants in SCN8A are associated with difficult-to-treat and early-onset seizures, developmental delay/intellectual disability, impaired quality of life, and increased risk of early mortality. High doses of sodium channel blockers are typically used to treat SCN8A-DEE caused by gain-of-function (GoF) variants. However, seizures are often drug resistant, and only a few patients achieve seizure freedom. In this retrospective study, the effect of cenobamate was assessed in patients with SCN8A-DEE. Across multiple centers and through collaborations with SCN8A patient advocacy organizations, patients with SCN8A-DEE treated with cenobamate for ≥6 months were identified. Data were obtained from patients' caregivers or treating physicians through a (Research Electronic Data Capture) REDCap survey. The functional effect of the SCN8A variants was obtained from the literature or assessed by prediction tools. Twelve patients (3-25 years of age (median 8 years), 9 females) with presumed GoF SCN8A variants were treated with cenobamate for a mean period of 17 months (range 6-42 months). Countable motor seizures were meaningfully reduced in 10 of 12 patients (83%). Six patients experienced a seizure reduction above 70%, of which two achieved seizure freedom. In addition, two patients achieved a reduction in seizures ranging between 50% and 70%. An increase in seizure-free days per patient was also reported. Rescue medication was decreased in six of seven patients (85%) in need. Furthermore, 80% of patients reported non-seizure-related improvements, which included increased alertness, better sleep, and improved muscle tone. Adverse effects were reported by 50% of patients, and half resolved spontaneously or through the reduction of concomitant antiseizure medications. Our data suggest that cenobamate is a promising and safe treatment for SCN8A-DEE, even during early childhood. As a potential precision approach to treatment, cenobamate significantly reduced seizure burden and improved non-seizure-related symptoms. These positive outcomes may also be achieved in patient cohorts with GoF variants in other voltage-gated sodium channel genes.

Target site mutations and metabolic detoxification of insecticides in continental populations of Cimex lectularius and Cimex hemipterus (Hemiptera: Cimicidae).

In recent decades, the common and the tropical bed bugs have experienced a resurgence in many parts of the world. The evolution of insecticide resistance in bed bug populations is considered a significant factor contributing to this resurgence. We analyzed samples of Cimex lectularius L. and Cimex hemipterus (F.) from Europe (Spain 41, Switzerland 2, the Czech Republic 1), Asia (Hong Kong 34), North America (USA 14, Mexico 3), and South America (Colombia 3) to assess the prevalence and mechanisms of insecticide resistance. We identified specimens morphologically and barcoded them by sequencing the mitochondrial Cytochrome c oxidase subunit I (COI) and the 16S ribosomal RNA (16S rRNA) genes. Additionally, we screened segments of the voltage-gated sodium channel (VGSC) and the nicotinic acetylcholine receptor (nAChR) genes for point mutations associated with insecticide resistance and measured the activity of detoxifying enzymes. All samples from North America and Europe were identified as C. lectularius, whereas specimens from Hong Kong were C. hemipterus. Out of 64 C. lectularius samples tested for knockdown resistance (kdr) mutations, 90.6% contained at least 1 known mutation. All 35 C. hemipterus samples exhibited kdr mutations. A new mutation was identified in the pyrethroid target site in both common (F1524C) and tropical (F1450C) bed bugs. No resistance-associated mutations in the nAChR gene were found. Several populations that exhibited kdr mutations also showed elevated activity of detoxifying enzymes. The high frequency of kdr-associated mutations in bed bug populations from Spain and Hong Kong limits the efficacy of pyrethroids for their control.

Overexpression of multiple cytochrome P450 genes with and without knockdown resistance mutations confers high resistance to deltamethrin in Culex quinquefasciatus

BackgroundThe cytochrome P450s-mediated metabolic resistance and the target site insensitivity caused by the knockdown resistance (kdr) mutation in the voltage-gated sodium channel (vgsc) gene were the main mechanisms conferring resistance to deltamethrin in Culex quinquefasciatus from Thailand. This study aimed to investigate the expression levels of cytochrome P450 genes and detect mutations of the vgsc gene in deltamethrin-resistant Cx. quinquefasciatus populations in Thailand.MethodsTwo field-collected strains of Cx. quinquefasciatus, Cq_SP and Cq_NiH, were selected with deltamethrin to generate the resistant strains Cq_SP-R and Cq_NiH-R, respectively. Bioassays were tested on larvae and adults of each strain according to WHO methods. Eight cytochrome P450 genes were analyzed for the expression level using quantitative real time-PCR. The cDNA of mosquitoes was amplified and sequenced for four fragments of vgsc gene. The kdr L1014F mutation and the haplotype of the CYP9M10 gene were detected in survivor and dead mosquitoes after exposure to the deltamethrin WHO test paper. Statistical analyses were performed using Fisher’s exaction test.ResultsBioassay tests revealed a significantly higher resistance level in Cq_SP-R than in Cq_NiH-R strains in both larvae and adults. All eight cytochrome P450 genes were significantly overexpressed in larvae of Cq_NiH-R strain compared to the parent and susceptible Cq_Sus strains. The CYP6AA7 and CYP9J34 genes had the highest expression ratios, exceeding 24-fold in Cq_NiH-R larvae. In Cq_SP-R strain, the CYP4H34 and CYP9J34 genes were overexpressed in both stages. The kdr L1014F mutation was found in Cq_SP-R and its parent Cq_SP strains with a significantly higher mutant allele frequency in the survivor mosquitoes than in dead mosquitoes (P < 0.0001). The V240M and novel L925F mutations were found only in Cq_SP-R strain. Heterozygous genotype for the D-Cu( +)/Cu(–) of CYP9M10 gene was detected in Cq_NiH and Cq_NiH-R strains but other strains were mostly homozygous for the Cu(–)/Cu(–).ConclusionsOverexpression of multiple cytochrome P450 genes alone has a relatively minor impact on resistance. The combined mechanisms of cytochrome P450- and kdr-mediated resistance result in significantly higher resistance to deltamethrin in Cx. quinquefasciatus. This study supports sustainable public health initiatives in Thailand to address the evolving challenges of insecticide resistance.

Marine-derived bioactive compounds for neuropathic pain: pharmacology and therapeutic potential.

Neuropathic pain, a challenging condition often associated with diabetes, trauma, or chemotherapy, impairs patients' quality of life. Current treatments often provide inconsistent relief and notable adverse effects, highlighting the urgent need for safer and more effective alternatives. This review investigates marine-derived bioactive compounds as potential novel therapies for neuropathic pain management. Marine organisms, including fungi, algae, cone snails, sponges, soft corals, tunicates, and fish, produce a diverse range of secondary metabolites with significant pharmacological properties. These include peptides (e.g., conopeptides, piscidin 1), non-peptides (e.g., guanidinium toxins, astaxanthin, docosahexaenoic acid, fucoidan, apigenin, fumagillin, aaptamine, flexibilide, excavatolide B, capnellenes, austrasulfones, lemnalol), and crude extracts (e.g., Spirulina platensis, Dunaliella salina, Cliothosa aurivilli). These compounds exhibit diverse mechanisms of action, such as modulating ion channels (e.g., transient receptor potential channels, voltage-gated sodium, calcium, and potassium channels, and G protein-coupled inwardly rectifying potassium channels), interacting with cell-surface receptors (e.g., nicotinic acetylcholine, NMDA, kainate, GABAB​, and neurotensin receptors), inhibiting norepinephrine transporters, reducing oxidative stress, and attenuating neuroinflammation. These effects collectively contribute to alleviating nerve degeneration and symptoms of neuropathic pain, including hyperalgesia, allodynia, and associated psychomotor disturbances. Marine-derived bioactive compounds represent promising alternatives to conventional neuropathic pain treatments, to advance their development and assess their integration into neuropathic pain management strategies.

Differential encoding of mammalian proprioception by voltage-gated sodium channels.

Animals requiring purposeful movement for survival are endowed with mechanoreceptors, called proprioceptors, that provide essential sensory feedback from muscles and joints to spinal cord circuits, which modulates motor output. Despite the essential nature of proprioceptive signaling in daily life, the mechanisms governing proprioceptor activity are poorly understood. Here, we identified nonredundant roles for two voltage-gated sodium channels (NaVs), NaV1.1 and NaV1.6, in mammalian proprioception. Deletion of NaV1.6 in somatosensory neurons (NaV1.6cKO mice) causes severe motor deficits accompanied by loss of proprioceptive transmission, which contrasts with our previous findings using similar mouse models to target NaV1.1 (NaV1.1cKO). In NaV1.6cKO animals, we observed impairments in proprioceptor end-organ structure and a marked reduction in skeletal muscle myofiber size that were absent in NaV1.1cKO mice. We attribute the differential contributions of NaV1.1 and NaV1.6 to distinct cellular localization patterns. Collectively, we provide evidence that NaVs uniquely shape neural signaling within a somatosensory modality.

Elucidating the roles of voltage sensors in NaV1.9 activation and inactivation through a spider toxin.

The gating process of voltage-gated sodium (NaV) channels is extraordinary intrinsic and involves numerous factors, such as voltage-sensing domain (VSD), the N-terminus and C-terminus, and the auxiliary subunits. To date, the gating mechanism of NaV channel has not been clearly elucidated. NaV1.9 has garnered significant attention due to its slow gating kinetics. Due to the challenges of NaV1.9 heterologous expression, research on its gating mechanism is relatively limited. Whether there are any differences in the functions of the four VSDs in NaV1.9 compared to those in other subtypes remains an open question. Here, we employed the established chimera method to transplant the S3b-S4 motif from the VSDIV of the toxin-sensitive donor channel (NaV1.9) into the receptor channel (NaV1.9/1.8 DIV S3b-S4 chimera). This modification imparted animal toxin sensitivity to the other three VSDs. Our results demonstrate that all four VSDs of NaV1.9 are involved in channel opening, VSDIII and VSDIV are primarily involved in regulating fast inactivation, and VSDII also regulates the steady-state inactivation of channels. These findings provide a new insight into the gating mechanism of NaV1.9.

Arising amitraz and pyrethroids resistance mutations in the ectoparasitic Varroa destructor mite in Canada

The ectoparasitic mite Varroa destructor remains a great threat for the beekeeping industry, for example contributing to excessive winter colony loss in Canada. For decades, beekeepers have sequentially used the registered synthetic varroacides tau-fluvalinate, coumaphos, amitraz, and flumethrin, leading to the risk of resistance evolution in the mites. In addition to the widespread resistance to coumaphos and pyrethroids, a decline in amitraz efficacy has recently been reported in numerous beekeeping regions in Canada. The goals of this study were to assess the evolution of resistance to amitraz in Canadian mite populations and to evaluate the presence and incidence of mutations previously associated with resistance to amitraz and pyrethroids in V. destructor. Our bioassay results confirmed the presence of amitraz-resistant mites in the population of Alberta. These phenotypic results were complemented by targeted genotyping of the octopamine receptor gene Octβ2R which revealed the presence of the mutation Y215H in 90% of tested apiaries with local allele frequencies ranging from 5 to 95%. The phenotypic resistance showed a significant correlation with the presence of this mutation across apiaries. In parallel, the L925I and L925M mutations in the voltage-gated sodium channel were identified in 100% of the tested apiaries with frequencies ranging from 33 to 97%, suggesting that resistance to pyrethroids remains widespread. These results support the notion that the practice of relying on a single treatment for a prolonged period can increase rates of resistance to current varroacides. Our findings suggest the need for large-scale resistance monitoring via genotyping to provide timely information to beekeepers and regulators. This will enable them to make an effective management plan, including rotation of available treatments to suppress or at least delay the evolution of resistance in V. destructor populations.

Interneuron FGF13 regulates seizure susceptibility via a sodium channel-independent mechanism.

Developmental and epileptic encephalopathies (DEEs), a class of devastating neurological disorders characterized by recurrent seizures and exacerbated by disruptions to excitatory/inhibitory balance in the brain, are commonly caused by mutations in ion channels. Disruption of, or variants in, FGF13 were implicated as causal for a set of DEEs, but the underlying mechanisms were clouded because FGF13 is expressed in both excitatory and inhibitory neurons, FGF13 undergoes extensive alternative splicing producing multiple isoforms with distinct functions, and the overall roles of FGF13 in neurons are incompletely cataloged. To overcome these challenges, we generated a set of novel cell-type-specific conditional knockout mice. Interneuron-targeted deletion of Fgf13 led to perinatal mortality associated with extensive seizures and impaired the hippocampal inhibitory/excitatory balance while excitatory neuron-targeted deletion of Fgf13 caused no detectable seizures and no survival deficits. While best studied as a voltage-gated sodium channel (Nav) regulator, we observed no effect of Fgf13 ablation in interneurons on Navs but rather a marked reduction in K+ channel currents. Re-expressing different Fgf13 splice isoforms could partially rescue deficits in interneuron excitability and restore K+ channel current amplitude. These results enhance our understanding of the molecular mechanisms that drive the pathogenesis of Fgf13-related seizures and expand our understanding of FGF13 functions in different neuron subsets.

Pharmacology and Mechanism of Action of Suzetrigine, a Potent and Selective NaV1.8 Pain Signal Inhibitor for the Treatment of Moderate to Severe Pain.

There is a high unmet need for safe and effective non-opioid medicines to treat moderate to severe pain without risk of addiction. Voltage-gated sodium channel 1.8 (NaV1.8) is a genetically and pharmacologically validated pain target that is selectively expressed in peripheral pain-sensing neurons and not in the central nervous system (CNS). Suzetrigine (VX-548) is a potent and selective inhibitor of NaV1.8, which has demonstrated clinical efficacy and safety in multiple acute pain studies. Our study was designed to characterize the mechanism of action of suzetrigine and assess both nonclinical and clinical data to test the hypothesis that selective NaV1.8 inhibition translates into clinical efficacy and safety, including lack of addictive potential. Preclinical pharmacology and mechanism of action studies were performed in vitro using electrophysiology and radiolabeled binding methods in cells recombinantly expressing human NaV channels, human proteins, and primary human dorsal root ganglion (DRG) sensory neurons. Safety and addictive potential assessments included in vitro secondary pharmacology studies, nonclinical repeat-dose toxicity and dependence studies in rats and/or monkeys, and a systematic analysis of adverse event data generated from 2447 participants from phase 3 acute pain studies of suzetrigine. Suzetrigine is selective against all other NaV subtypes (≥ 31,000-fold) and 180 other molecular targets. Suzetrigine inhibits NaV1.8 by binding to the protein's second voltage sensing domain (VSD2) to stabilize the closed state of the channel. This novel allosteric mechanism results in tonic inhibition of NaV1.8 and reduces pain signals in primary human DRG sensory neurons. Nonclinical and clinical safety assessments with suzetrigine demonstrate no adverse CNS, cardiovascular or behavioral effects and no evidence of addictive potential or dependence. The comprehensive pharmacology assessment presented here indicates that suzetrigine represents the first in a new class of non-opioid analgesics that are selective NaV1.8 pain signal inhibitors acting in the peripheral nervous system to safely treat pain without addictive potential.

First detection of the S989P+V1016G+D1763Y haplotype and expansion of voltage-gated sodium channel mutations in Aedes aegypti in Taiwan in 2016-2023.

Aedes aegypti transmits various arthropod-borne diseases such as dengue, posing a significant burden to public health in tropical and subtropical regions. Pyrethroid-based control strategies are effective in managing this vector; however, the development of insecticide resistance has hindered these efforts. Hence, long-term monitoring of insecticide resistance in mosquito populations is crucial for effective vector and disease control. In this study, we identified insecticide resistance due to a voltage-gated sodium channel (vgsc) mutation in Ae. aegypti in Taiwan between 2016 and 2023. In total, 1,761 field-caught Ae. aegypti samples from Tainan, Kaohsiung, and Pingtung were genotyped. The frequencies of S989P, V1016G, T1520I, F1534C, and D1763Y amino acid variants increased over the surveillance period. A T1520I mutation was detected for the first time and has since rapidly spread throughout Taiwan. The triple-mutant haplotype PGTFY was first documented in Ae. aegypti. Moreover, the unmutated haplotype vanished in Taiwan, suggesting that the vgsc mutations were fixed in local populations of Ae. aegypti. Five resistance-associated genotypes, SVTCD/SVTCD, SGTFY/PGTFD, SVTCD/SGTFY, PGTFD/PGTFD, and SVTCD/PGTFD, exhibited an increased frequency and accounted for 76% of the total field population. We also detected the resistant genotype SVICD/PGTFD, and its frequency increased 13-fold in the field between 2016 and 2023. Moreover, we also observed that mutations differed geographically, with S989P mainly found in Kaohsiung and V1016G in Kaohsiung and Pingtung. The frequency of T1520I was noticeably higher in Kaohsiung, and D1763Y occurred mainly in Tainan. The emergence and expansion of mutations along with the disappearance of wild-type mosquitoes in Taiwan underscores the threat of resistance and difficulty of mosquito control in Taiwan as well as globally. This study determined the insecticide resistance status of Ae. aegypti in Taiwan, and the findings will be helpful for resistance monitoring in areas where pyrethroids are used to control Ae. aegypti.

Isoform-specific N-linked glycosylation of NaV channel α-subunits alters β-subunit binding sites.

Voltage-gated sodium channel α-subunits (NaV1.1-1.9) initiate and propagate action potentials in neurons and myocytes. The NaV β-subunits (β1-4) have been shown to modulate α-subunit properties. Homo-oligomerization of β-subunits on neighboring or opposing plasma membranes has been suggested to facilitate cis or trans interactions, respectively. The interactions between several NaV channel isoforms and β-subunits have been determined using cryogenic electron microscopy (cryo-EM). Interestingly, the NaV cryo-EM structures reveal the presence of N-linked glycosylation sites. However, only the first glycan moieties are typically resolved at each site due to the flexibility of mature glycan trees. Thus, existing cryo-EM structures may risk de-emphasizing the structural implications of glycans on the NaV channels. Herein, molecular modeling and all-atom molecular dynamics simulations were applied to investigate the conformational landscape of N-linked glycans on NaV channel surfaces. The simulations revealed that negatively charged sialic acid residues of two glycan sites may interact with voltage-sensing domains. Notably, two NaV1.5 isoform-specific glycans extensively cover the α-subunit region that, in other NaV channel α-subunit isoforms, corresponds to the binding site for the β1- (and likely β3-) subunit immunoglobulin (Ig) domain. NaV1.8 contains a unique N-linked glycosylation site that likely prevents its interaction with the β2 and β4-subunit Ig-domain. These isoform-specific glycans may have evolved to facilitate specific functional interactions, for example, by redirecting β-subunit Ig-domains outward to permit cis or trans supraclustering within specialized cellular compartments such as the cardiomyocyte perinexal space. Further experimental work is necessary to validate these predictions.

Multiple gating processes associated with the distal end of the S6 segment of domain II in the Nav channels.

Voltage-gated sodium (Nav) channels are transmembrane proteins that play crucial roles in the initiation and propagation of action potentials (APs) in excitable tissues such as the heart, muscles, and nerves. The distal ends of the four domain S6 segments of Nav channels contain hydrophobic residues, which form an intracellular gate. This gate allows Nav channels to control ion flux in excitable cells by opening and closing. However, the mechanism of the distal end of Domain II (DII) S6 segment in channel gating remains unclear. In this study, using whole-cell patch clamp recording, we systematically investigated the biophysical characteristics of various mutants L811 site (located at the distal end of the DII S6 segment) of Nav1.9 and the corresponding L796P mutant of Nav1.4. We found that the mutations significantly shifted the activation and inactivation curves, slowed the fast inactivation, accelerated the slow inactivation and use-dependent slow inactivation, and L811P altered the ion selectivity of the channel. Therefore, our findings suggest that the distal end of the DII S6 segment in Nav channels plays a pivotal role in regulating multiple gating processes.

Targeting Nav1.7 and Nav1.8 with a PIKfyve inhibitor to Reverse inflammatory and neuropathic pain

PIKfyve (1-phosphatidylinositol 3-phosphate 5-kinase), a lipid kinase, plays an important role in generating phosphatidylinositol (3,5)-bisphosphate (PI(3,5)P2). SGC-PIKFYVE-1, a potent and selective inhibitor of PIKfyve, has been used as a chemical probe to explore pathways dependent on PIKfyve activity. Based on reported changes in membrane dynamics and ion transport in response to PIKfyve inhibition, we hypothesized that pharmacological inhibition of PIKfyve could modulate pain. Acute treatment with SGC-PIKFYVE-1 (10 µM) inhibited voltage-gated sodium currents through the inhibition of Nav1.7 and Nav1.8 channels, without affecting voltage-gated calcium or potassium currents in sensory neurons. Additionally, systemic administration of SGC-PIKFYVE-1 (30 mg/kg) alleviated mechanical and cold sensitivity induced by neuropathic or inflammatory pain in both male and female mice, without causing motor impairments. Although other functions of PIKfyve are well characterized, its role in inhibiting chronic pain has not been fully elucidated. Our study provides proof-of-concept for this alternative approach to pain management. Collectively, these results highlight the inhibitory effects of PIKfyve as a promising avenue for further exploration in chronic pain treatment.

Compensatory changes after spinal cord injury in aremyelination deficient mouse model.

The development of therapeutic strategies to reduce impairments following spinal cord injury (SCI) motivates an active area of research, because there are no effective therapies. One strategy is to address injury-induced demyelination of spared axons by promoting endogenous or exogenous remyelination. However, previously, we showed that new myelin was not necessary to regain hindlimb stepping following moderate thoracic spinal cord contusion in 3-month-old mice. The present analysis investigated two potential mechanisms by which animals can re-establish locomotion in the absence of remyelination: compensation through intact white matter and conduction through spared axons. We induced a severe contusion injury to reduce the spared white matter rim in the remyelination deficient model, with no differences in recovery between remyelination deficient animals and injured littermate controls. We investigated the nodal properties of the axons at the lesion and found that in the remyelination deficient model, axons express the Nav1.2 voltage-gated sodium channel, a sub-type not typically expressed at mature nodes of Ranvier. In a moderate contusion injury, conduction velocities through the lesions of remyelination deficient animals were similar to those in animals with the capacity to remyelinate after injury. Detailed gait analysis and kinematics reveal subtle differences between remyelination deficient animals and remyelination competent controls, but no worse deficits. It is possible that upregulation of Nav1.2 channels may contribute to establishing conduction through the lesion. This conduction could contribute to compensation and regained motor function in mouse models of SCI. Such compensatory mechanism may have implications for interpreting efficacy results for remyelinating interventions in mice and the development of therapies for improving recovery following SCI.

Identification of Lauric Acid as a Potent Sodium Channel NaV1.5 Blocker from Compound Chinese Medicine Wenxin Keli.

The major cardiac voltage-gated sodium channel NaV1.5 (INa) is essential for cardiac action potential initiation and subsequent propagation. Compound Chinese medicine Wenxin Keli (WXKL) has been shown to suppress arrhythmias and heart failure. However, its active components have not been fully elucidated. This study focused on identifying the active inhibitor of INa in WXKL and exploring their mode of action in electrophysiological conduction. A chemical fraction library was constructed from an aqueous extract of WXKL and screened using an automated patch-clamping system in cells stably expressing the NaV1.5 gene SCN5A. Candidate fractions with INa-inhibition activity were analyzed by HPLC-ESI-IT-TOF-MS and GC-MS to identify the ingredients. NaV1.5 blocker molecules identified by single-cell electrocardiogram were tested in hiPSC-derived cardiomyocytes. We evaluated the SCN5A inhibitory potential of Wenxin Keli effective monomer employing molecular docking and molecular dynamics simulation approaches. A primary screen of the WXKL chemical library identified five fractions that significantly inhibited the NaV1.5 channel, with one of them rich in poly-saturated fatty acids. Molecular structural characterization revealed the presence of lauric acid, myristic acid, palmitic acid, and stearic acid in the active subfraction. Electrophysiological characterization demonstrated lauric acid (LA) as the most effective monomer for INa-inhibition with an IC50 at 27.40 ± 12.78 μM. LA shifted the steady-state inactivation of INa to more negative potentials and decreased the amplitude of extracellular field potential in hiPSC-derived cardiomyocytes. We demonstrate for the first time that naturally poly-saturated fatty acid, lauric acid, as a potential novel INa blocker. Molecular docking and molecular dynamics simulation suggested that LA binds to the NaV1.5 protein, with a significant binding affinity forming interactions with functionally essential residues and blocks the inward flow of Na+. Mechanistically, lauric acid acts on the fast inactivation of NaV1.5 alter electrophysiology conduction of hiPSC-derived cardiomyocytes and contribute to the antiarrhythmic effect of WXKL. Lauric acid is a potent blocker for sodium channel NaV1.5 and alleviates arrhythmia via inhibiting INa.