Eugenol and lidocaine inhibit voltage-gated Na+ channels from dorsal root ganglion neurons with different mechanisms.

Eugenol (EUG) is a bioactive monoterpenoid used as an analgesic, preservative, and flavoring agent. Our new data show EUG as a voltage-gated Na+ channel (VGSC) inhibitor, comparable but not identical to lidocaine (LID). EUG inhibits both total and only TTX-R voltage-activated Na+ currents (INa) recorded from VGSCs naturally expressed on dorsal root ganglion sensory neurons in rats. Inhibition is quick, fully reversible, and dose-dependent. Our biophysical and pharmacological analyses showed that EUG and LID inhibit VGSCs with different mechanisms. EUG inhibits VGSCs with a dose-response relationship characterized by a Hill coefficient of 2, while this parameter for the inhibition by LID is 1. Furthermore, in a different way from LID, EUG modified the voltage dependence of both the VGSC activation and inactivation processes and the recovery from fast inactivated states and the entry to slow inactivated states. In addition, we suggest that EUG, but not LID, interacts with VGSC pre-open-closed states, according to our data.

Multisite Validation of a Functional Assay to Adjudicate SCN5A Brugada Syndrome-Associated Variants.

BACKGROUND: Brugada syndrome is an inheritable arrhythmia condition that is associated with rare, loss-of-function variants in SCN5A. Interpreting the pathogenicity of SCN5A missense variants is challenging, and ≈79% of SCN5A missense variants in ClinVar are currently classified as variants of uncertain significance. Automated patch clamp technology enables high-throughput functional studies of ion channel variants and can provide evidence for variant reclassification. METHODS: An in vitro SCN5A-Brugada syndrome automated patch clamp assay was generated and independently studied at Vanderbilt University Medical Center and Victor Chang Cardiac Research Institute. The assay was calibrated according to ClinGen Sequence Variant Interpretation recommendations using high-confidence variant controls (n=49). Normal and abnormal ranges of function were established based on the distribution of benign variant assay results. Odds of pathogenicity values were derived from the experimental results according to ClinGen Sequence Variant Interpretation recommendations. The calibrated assay was then used to study SCN5A variants of uncertain significance observed in 4 families with Brugada syndrome and other arrhythmia phenotypes associated with SCN5A loss-of-function. RESULTS: Variant channel parameters generated independently at the 2 research sites showed strong correlations, including peak INa density (R2=0.86). The assay accurately distinguished benign controls (24/25 concordant variants) from pathogenic controls (23/24 concordant variants). Odds of pathogenicity values yielded 0.042 for normal function and 24.0 for abnormal function, corresponding to strong evidence for both American College of Medical Genetics and Genomics/Association for Molecular Pathology benign and pathogenic functional criteria (BS3 and PS3, respectively). Application of the assay to 4 clinical SCN5A variants of uncertain significance revealed loss-of-function for 3/4 variants, enabling reclassification to likely pathogenic. CONCLUSIONS: This validated high-throughput assay provides clinical-grade functional evidence to aid the classification of current and future SCN5A-Brugada syndrome variants of uncertain significance.

Deconvoluting and derisking QRS complex widening to improve cardiac safety profile of novel plasmepsin X antimalarials.

Quinoline-related antimalarial drugs have been associated with cardiotoxicity risk, in particular QT prolongation and QRS complex widening. In collaboration with Medicines for Malaria Venture (MMV), we discovered novel plasmepsin X (PMX) inhibitors for malaria treatment. The first lead compounds tested in anesthetized guinea pigs (GP) induced profound QRS widening, although exhibiting weak inhibition of NaV1.5-mediated currents in standard patch clamp assays. To understand the mechanism(s) underlying QRS widening to identify further compounds devoid of such liability, we established a set of in vitro models including CaV1.2, NaV1.5 rate-dependence and NaV1.8 patch clamp assays, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM), and Langendorff-perfused isolated GP hearts. Six compounds were tested in all models including anesthetized GP, and 8 additional compounds were tested in vitro only. All compounds tested in anesthetized GP and isolated hearts showed a similar cardiovascular profile, consisting of QRS widening, bradycardia, negative inotropy, hypotension, and for some, QT prolongation. However, a left shift of the concentration-response curves was noted from in vitro to in vivo GP data. When comparing in vitro models, there was a good consistency between decrease in sodium spike amplitude in hiPSC-CM and QRS widening in isolated hearts. Patch clamp assay results showed that the QRS widening observed with PMX inhibitors is likely multifactorial, primarily due to NaV1.8 and NaV1.5 rate-dependent sodium blockade and/or calcium channel-mediated mechanisms. In conclusion, early de-risking of QRS widening using a set of different in vitro assays allowed to identify novel PMX inhibitors with improved cardiac safety profile.

Unveiling Tst3, a Multi-Target Gating Modifier Scorpion α Toxin from Tityus stigmurus Venom of Northeast Brazil: Evaluation and Comparison with Well-Studied Ts3 Toxin of Tityus serrulatus.

Studies on the interaction sites of peptide toxins and ion channels typically involve site-directed mutations in toxins. However, natural mutant toxins exist among them, offering insights into how the evolutionary process has conserved crucial sequences for activities and molecular target selection. In this study, we present a comparative investigation using electrophysiological approaches and computational analysis between two alpha toxins from evolutionarily close scorpion species of the genus Tityus, namely, Tst3 and Ts3 from T. stigmurus and T. serrulatus, respectively. These toxins exhibit three natural substitutions near the C-terminal region, which is directly involved in the interaction between alpha toxins and Nav channels. Additionally, we characterized the activity of the Tst3 toxin on Nav1.1-Nav1.7 channels. The three natural changes between the toxins did not alter sensitivity to Nav1.4, maintaining similar intensities regarding their ability to alter opening probabilities, delay fast inactivation, and induce persistent currents. Computational analysis demonstrated a preference for the down conformation of VSD4 and a shift in the conformational equilibrium towards this state. This illustrates that the sequence of these toxins retained the necessary information, even with alterations in the interaction site region. Through electrophysiological and computational analyses, screening of the Tst3 toxin on sodium isoform revealed its classification as a classic α-NaTx with a broad spectrum of activity. It effectively delays fast inactivation across all tested isoforms. Structural analysis of molecular energetics at the interface of the VSD4-Tst3 complex further confirmed this effect.

Dynamic Changes in Ion Channels during Myocardial Infarction and Therapeutic Challenges.

In different areas of the heart, action potential waveforms differ due to differences in the expressions of sodium, calcium, and potassium channels. One of the characteristics of myocardial infarction (MI) is an imbalance in oxygen supply and demand, leading to ion imbalance. After MI, the regulation and expression levels of K+, Ca2+, and Na+ ion channels in cardiomyocytes are altered, which affects the regularity of cardiac rhythm and leads to myocardial injury. Myocardial fibroblasts are the main effector cells in the process of MI repair. The ion channels of myocardial fibroblasts play an important role in the process of MI. At the same time, a large number of ion channels are expressed in immune cells, which play an important role by regulating the in- and outflow of ions to complete intracellular signal transduction. Ion channels are widely distributed in a variety of cells and are attractive targets for drug development. This article reviews the changes in different ion channels after MI and the therapeutic drugs for these channels. We analyze the complex molecular mechanisms behind myocardial ion channel regulation and the challenges in ion channel drug therapy.

Nanomedicine and voltage-gated sodium channel blockers in pain management: a game changer or a lost cause?

Pain, a complex and debilitating condition affecting millions globally, is a significant concern, especially in the context of post-operative recovery. This comprehensive review explores the complexity of pain and its global impact, emphasizing the modulation of voltage-gated sodium channels (VGSC or NaV channels) as a promising avenue for pain management with the aim of reducing reliance on opioids. The article delves into the role of specific NaV isoforms, particularly NaV 1.7, NaV 1.8, and NaV 1.9, in pain process and discusses the development of sodium channel blockers to target these isoforms precisely. Traditional local anesthetics and selective NaV isoform inhibitors, despite showing varying efficacy in pain management, face challenges in systemic distribution and potential side effects. The review highlights the potential of nanomedicine in improving the delivery of local anesthetics, toxins and selective NaV isoform inhibitors for a targeted and sustained release at the site of pain. This innovative strategy seeks to improve drug bioavailability, minimize systemic exposure, and optimize therapeutic outcomes, holding significant promise for secure pain management and enhancing the quality of life for individuals recovering from surgical procedures or suffering from chronic pain.

Amiloride versus furosemide for the treatment of edema in patients with nephrotic syndrome: A pilot study (AMILOR).

AIM: In rodent models of nephrotic syndrome (NS), edema formation was prevented by blockade of the epithelial sodium channel ENaC with amiloride. However, apart from case reports, there is no evidence favoring ENaC blockade in patients with NS. METHODS: The monocentric randomized controlled AMILOR study investigated the antiedematous effect of amiloride (starting dose 5 mg/day, max. 15 mg/day) in comparison to standard therapy with the loop diuretic furosemide (40 mg/day, max. 120 mg/day) over 16 days. Overhydration (OH) was measured by bioimpedance spectroscopy (BCM, Fresenius). Depending on the OH response, diuretic dose was adjusted on days 2, 5, 8 and 12, and if necessary, hydrochlorothiazide (HCT) was added from d8 (12.5 mg/day, max. 25 mg/day). The primary endpoint was the decrease in OH on d8. The study was terminated prematurely due to insufficient recruitment and a low statistical power due to a low actual effect size. RESULTS: Median baseline OH was +26.4 (interquartile range 15.5-35.1)% extracellular water (ECW) in the amiloride arm and + 27.9 (24.1-29.4)% ECW in the furosemide arm and decreased by 1.95 (0.80-6.40) and 5.15 (0.90-8.30)% ECW after 8 days, respectively, and by 10.10 (1.30-14.40) and 7.40 (2.80-10.10)% ECW after 16 days, respectively. OH decrease on d8 and d16 was not significantly different between both arms. CONCLUSION: The AMILOR study is the first randomized controlled pilot study suggesting a similar antiedematous effect as furosemide. Further studies are required to better define the role of amiloride in NS (EudraCT 2019-002607-18).

New focus on cardiac voltage-gated sodium channel ß1 and ß1B: Novel targets for treating and understanding arrhythmias?

Voltage-gated sodium channels (VGSCs) are transmembrane protein complexes that are vital to the generation and propagation of action potentials in nerve and muscle fibers. The canonical VGSC is generally conceived as a heterotrimeric complex formed by two classes of membrane-spanning subunit-an α-subunit (pore forming) and two ß-subunits (non-pore forming). NaV1.5 is the main sodium channel α-subunit of mammalian ventricle, with lower amounts of other α-subunits, including NaV1.6, being present. There are four ß-subunits, ß1-ß4, encoded by four genes, SCN1B-SCN4B, each of which are expressed in cardiac tissues. Recent studies suggest that in addition to assignments in channel gating and trafficking, products of Scn1b may have novel roles in conduction of action potential in the heart and intracellular signaling. This includes evidence that the ß-subunit extracellular Amino-terminal domain facilitates adhesive interactions in intercalated discs and that its Carboxyl-terminal region is a substrate for a regulated intramembrane proteolysis (RIP) signaling pathway-with a Carboxyl-terminal peptide generated by ß1 RIP trafficked to the nucleus and altering transcription of various genes, including NaV1.5. In addition to ß1, the Scn1b gene encodes for an alternative splice variant, ß1B, which contains an identical extracellular adhesion domain to ß1, but has a unique Carboxyl-terminus. Whilst ß1B is generally understood to be a secreted variant, evidence indicates that when co-expressed with NaV1.5, it is maintained at the cell membrane, suggesting potential unique roles for this understudied protein. In this review, we focus on what is known on the two ß-subunit variants encoded by Scn1b in heart, with particular focus on recent findings and the questions raised by this new information. We also explore data that indicate ß1 and ß1B may be attractive targets for novel anti-arrhythmic therapeutics.

Development and characterization of the mode-of-action of inhibitory and agonist peptides targeting the voltage-gated sodium channel SCN1B beta-subunit.

Cardiac arrhythmia treatment is a clinical challenge necessitating safer and more effective therapies. Recent studies have highlighted the role of the perinexus, an intercalated disc nanodomain enriched in voltage-gated sodium channels including both Nav1.5 and ß1 subunits, adjacent to gap junctions. These findings offer insights into action potential conduction in the heart. A 19-amino acid SCN1B (ß1/ß1B) mimetic peptide, ßadp1, disrupts VGSC beta subunit-mediated adhesion in cardiac perinexii, inducing arrhythmogenic changes. We aimed to explore ßadp1's mechanism and develop novel SCN1B mimetic peptides affecting ß1-mediated adhesion. Using patch clamp assays in neonatal rat cardiomyocytes and electric cell substrate impedance sensing (ECIS) in ß1-expressing cells, we observed ßadp1 maintained inhibitory effects for up to 5 h. A shorter peptide (LQLEED) based on the carboxyl-terminus of ßadp1 mimicked this inhibitory effect, while dimeric peptides containing repeated LQLEED sequences paradoxically promoted intercellular adhesion over longer time courses. Moreover, we found a link between these peptides and ß1-regulated intramembrane proteolysis (RIP) - a signaling pathway effecting gene transcription including that of VGSC subunits. ßadp1 increased RIP continuously over 48 h, while dimeric agonists acutely boosted RIP for up to 6 h. In the presence of DAPT, an RIP inhibitor, ßadp1's effects on ECIS-measured intercellular adhesion was reduced, suggesting a relationship between RIP and the peptide's inhibitory action. In conclusion, novel SCN1B (ß1/ß1B) mimetic peptides are reported with the potential to modulate intercellular VGSC ß1-mediated adhesion, potentially through ß1 RIP. These findings suggest a path towards the development of anti-arrhythmic drugs targeting the perinexus.

Beyond the Norm: A Report of a Rare Case of Sodium Channel 8 Alpha (SCN8A) Gene-Related Epilepsy Unveiled in a Nine-Year-Old Child.

Sodium channel 8 alpha (SCN8A) mutations encompass a spectrum of epilepsy phenotypes with diverse clinical manifestations, posing diagnostic challenges. We present a case of a nine-year-old male with SCN8A gene-associated developmental and epileptic encephalopathies (DEEs), characterized by generalized tonic-clonic seizures (GTCS) since infancy. Despite treatment with multiple antiepileptic drugs (AEDs), including phenytoin, valproate, levetiracetam, carbamazepine, and clobazam, seizure control remained elusive, prompting genetic testing. Whole exome sequencing confirmed a heterozygous mutation (p.Phe210Ser) in SCN8A exon 6, indicative of DEE-13. Functional studies revealed a gain-of-function mechanism in SCN8A variants, resulting in heightened ion channel activity and altered voltage dependence of activation. Despite treatment adjustments, the patient's seizures persisted until topiramate was introduced, offering partial relief. SCN8A, encoding Nav1.6 sodium channels, modulates neuronal excitability, with mutations leading to increased persistent currents and hyperexcitability. Early seizure onset and developmental delays are hallmarks of SCN8A-related DEE. This case highlights the significance of genetic testing in refractory epilepsy management, guiding personalized treatment strategies. Sodium channel blockers like phenytoin and carbamazepine are often first-line therapies, while topiramate presents as a potential adjunctive option in SCN8A-related DEE. Overall, this case underscores the diagnostic and therapeutic complexities of managing SCN8A-related epileptic encephalopathy, emphasizing the importance of long-term monitoring and personalized treatment approaches for optimizing outcomes in refractory epilepsy.

Conotoxins targeting voltage-gated sodium ion channels.

Voltage-gated sodium (NaV) channels are intimately involved in the generation and transmission of action potentials, and dysfunction of these channels may contribute to nervous system diseases such as epilepsy, neuropathic pain, psychosis, autism and cardiac arrhythmia. Many venom peptides selectively act on NaV channels. These include conotoxins, which are neurotoxins secreted by cone snails for prey capture or self-defense, but which are also valuable pharmacological tools for the identification and/or treatment of human diseases. Typically, conotoxins contain two or three disulfide bonds and these internal cross-braces contribute to conotoxins having compact, well-defined structures and high stability. Of the conotoxins containing three disulfide bonds some selectively target mammalian NaV channels and can block, stimulate, or modulate these channels. Such conotoxins have great potential to serve as pharmacological tools for studying the functions and characteristics of NaV channels or as drug leads for neurological diseases related to NaV channels. Accordingly, discovering or designing conotoxins targeting NaV channels with high potency and selectivity is important. The amino acid sequences, disulfide bond connectivity, and three-dimensional structures are key factors that affect the biological activity of conotoxins, and targeted synthetic modifications of conotoxins can greatly improve their activity and selectivity. This review examines NaV channel-targeted conotoxins, focusing on their structures, activities and designed modifications, with a view towards expanding their applications. Significance Statement NaV channels are crucial in various neurological diseases. Some conotoxins selectively target NaV channels, causing either blockade or activation, thus enabling their use as pharmacological tools for studying the channels' characteristics and functions. Conotoxins also have promising potential to be developed as drug leads. The disulfide bonds in these peptides are important for stabilizing their structures, thus leading to enhanced specificity and potency. Together, conotoxins targeting NaV channels have both immediate research value and promising future application prospects.

Molecular and cellular context influences SCN8A variant function.

Pathogenic variants in SCN8A, which encodes the voltage-gated sodium (NaV) channel NaV1.6, associate with neurodevelopmental disorders, including developmental and epileptic encephalopathy. Previous approaches to determine SCN8A variant function may be confounded by use of a neonatally expressed, alternatively spliced isoform of NaV1.6 (NaV1.6N) and engineered mutations rendering the channel tetrodotoxin (TTX) resistant. We investigated the impact of SCN8A alternative splicing on variant function by comparing the functional attributes of 15 variants expressed in 2 developmentally regulated splice isoforms (NaV1.6N, NaV1.6A). We employed automated patch clamp recording to enhance throughput, and developed a neuronal cell line (ND7/LoNav) with low levels of endogenous NaV current to obviate the need for TTX-resistance mutations. Expression of NaV1.6N or NaV1.6A in ND7/LoNav cells generated NaV currents with small, but significant, differences in voltage dependence of activation and inactivation. TTX-resistant versions of both isoforms exhibited significant functional differences compared with the corresponding WT channels. We demonstrated that many of the 15 disease-associated variants studied exhibited isoform-dependent functional effects, and that many of the studied SCN8A variants exhibited functional properties that were not easily classified as either gain- or loss-of-function. Our work illustrates the value of considering molecular and cellular context when investigating SCN8A variants.

Impacts of DCM-linked gating pore currents on the electrophysiological characteristics of hiPSC-CM monolayers.

BACKGROUND: Variants of the SCN5A gene, which encodes the NaV1.5 cardiac sodium channel, have been linked to arrhythmic disorders associated with dilated cardiomyopathy (DCM). However, the precise pathological mechanisms remain elusive. The present study aimed to elucidate the pathophysiological consequences of the DCM-linked Nav1.5/R219H variant, which is known to generate a gating pore current, using patient-specific human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) cultured in monolayers. METHODS: Ventricular- and atrial-like hiPSC-CM monolayers were generated from DCM patients carrying the R219H SCN5A variant as well as from healthy control individuals. CRISPR-corrected hiPSC-CMs served as isogenic controls. Simultaneous optical mapping of action potentials (APs) and calcium transients (CaTs) was employed to measure conduction velocities (CVs) and AP durations (APDs) and served as markers of electrical excitability. Calcium handling was evaluated by assessing CaT uptake (half-time to peak), recapture (tau of decay), and durations (TD50 and TD80). A multi-electrode array (MEA) analysis was conducted on hiPSC-CM monolayers to measure field potential (FP) parameters, including corrected Fridericia FP durations (FPDc). RESULTS: Our results revealed that CVs were significantly reduced by more than 50 % in both ventricular- and atrial-like hiPSC-CM monolayers carrying the R219H variant compared to the control group. APDs were also prolonged in the R219H group compared to the control and CRISPR-corrected groups. CaT uptake, reuptake, and duration were also markedly delayed in the R219H group compared to the control and CRISPR-corrected groups in both the ventricular- and the atrial-like hiPSC-CM monolayers. Lastly, the MEA data revealed a notably prolonged FPDc in the ventricular- and atrial-like hiPSC-CMs carrying the R219H variant compared to the control and isogenic control groups. CONCLUSIONS: These findings highlight the impact of the gating pore current on AP propagation and calcium homeostasis within a functional syncytium environment and offer valuable insights into the potential mechanisms underlying DCM pathophysiology.

Peripherally targeted analgesia via AAV-mediated sensory neuron-specific inhibition of multiple pronociceptive sodium channels.

This study reports that targeting intrinsically disordered regions of the voltage-gated sodium channel 1.7 (NaV1.7) protein facilitates discovery of sodium channel inhibitory peptide aptamers (NaViPA) for adeno-associated virus-mediated (AAV-mediated), sensory neuron-specific analgesia. A multipronged inhibition of INa1.7, INa1.6, INa1.3, and INa1.1 - but not INa1.5 and INa1.8 - was found for a prototype and named NaViPA1, which was derived from the NaV1.7 intracellular loop 1, and is conserved among the TTXs NaV subtypes. NaViPA1 expression in primary sensory neurons (PSNs) of dorsal root ganglia (DRG) produced significant inhibition of TTXs INa but not TTXr INa. DRG injection of AAV6-encoded NaViPA1 significantly attenuated evoked and spontaneous pain behaviors in both male and female rats with neuropathic pain induced by tibial nerve injury (TNI). Whole-cell current clamp of the PSNs showed that NaViPA1 expression normalized PSN excitability in TNI rats, suggesting that NaViPA1 attenuated pain by reversal of injury-induced neuronal hypersensitivity. IHC revealed efficient NaViPA1 expression restricted in PSNs and their central and peripheral terminals, indicating PSN-restricted AAV biodistribution. Inhibition of sodium channels by NaViPA1 was replicated in the human iPSC-derived sensory neurons. These results summate that NaViPA1 is a promising analgesic lead that, combined with AAV-mediated PSN-specific block of multiple TTXs NaVs, has potential as a peripheral nerve-restricted analgesic therapeutic.

Unplugging lateral fenestrations of NALCN reveals a hidden drug binding site within the pore region.

The sodium (Na+) leak channel (NALCN) is a member of the four-domain voltage-gated cation channel family that includes the prototypical voltage-gated sodium and calcium channels (NaVs and CaVs, respectively). Unlike NaVs and CaVs, which have four lateral fenestrations that serve as routes for lipophilic compounds to enter the central cavity to modulate channel function, NALCN has bulky residues (W311, L588, M1145, and Y1436) that block these openings. Structural data suggest that occluded fenestrations underlie the pharmacological resistance of NALCN, but functional evidence is lacking. To test this hypothesis, we unplugged the fenestrations of NALCN by substituting the four aforementioned residues with alanine (AAAA) and compared the effects of NaV, CaV, and NALCN blockers on both wild-type (WT) and AAAA channels. Most compounds behaved in a similar manner on both channels, but phenytoin and 2-aminoethoxydiphenyl borate (2-APB) elicited additional, distinct responses on AAAA channels. Further experiments using single alanine mutants revealed that phenytoin and 2-APB enter the inner cavity through distinct fenestrations, implying structural specificity to their modes of access. Using a combination of computational and functional approaches, we identified amino acid residues critical for 2-APB activity, supporting the existence of drug binding site(s) within the pore region. Intrigued by the activity of 2-APB and its analogues, we tested compounds containing the diphenylmethane/amine moiety on WT channels. We identified clinically used drugs that exhibited diverse activity, thus expanding the pharmacological toolbox for NALCN. While the low potencies of active compounds reiterate the pharmacological resistance of NALCN, our findings lay the foundation for rational drug design to develop NALCN modulators with refined properties.

Preictal dysfunctions of inhibitory interneurons paradoxically lead to their rebound hyperactivity and to low-voltage-fast onset seizures in Dravet syndrome.

Epilepsies have numerous specific mechanisms. The understanding of neural dynamics leading to seizures is important for disclosing pathological mechanisms and developing therapeutic approaches. We investigated electrographic activities and neural dynamics leading to convulsive seizures in patients and mouse models of Dravet syndrome (DS), a developmental and epileptic encephalopathy in which hypoexcitability of GABAergic neurons is considered to be the main dysfunction. We analyzed EEGs from DS patients carrying a SCN1A pathogenic variant, as well as epidural electrocorticograms, hippocampal local field potentials, and hippocampal single-unit neuronal activities in Scn1a+/- and Scn1aRH/+ DS mice. Strikingly, most seizures had low-voltage-fast onset in both patients and mice, which is thought to be generated by hyperactivity of GABAergic interneurons, the opposite of the main pathological mechanism of DS. Analyzing single-unit recordings, we observed that temporal disorganization of the firing of putative interneurons in the period immediately before the seizure (preictal) precedes the increase of their activity at seizure onset, together with the entire neuronal network. Moreover, we found early signatures of the preictal period in the spectral features of hippocampal and cortical field potential of Scn1a mice and of patients' EEG, which are consistent with the dysfunctions that we observed in single neurons and that allowed seizure prediction. Therefore, the perturbed preictal activity of interneurons leads to their hyperactivity at the onset of generalized seizures, which have low-voltage-fast features that are similar to those observed in other epilepsies and are triggered by hyperactivity of GABAergic neurons. Preictal spectral features may be used as predictive seizure biomarkers.

XOB: A novel phenylalkylamine antagonist of 5-HT2A receptors and voltage-gated sodium channels.

Bipolar disorder impacts millions of patients in the United States but the mechanistic understanding of its pathophysiology and therapeutics is incomplete. Atypical antipsychotic serotonin2A (5-HT2A) receptor antagonists, such as quetiapine and olanzapine, and mood-stabilizing voltage-gated sodium channel (VGSC) blockers, such as lamotrigine, carbamazepine, and valproate, show therapeutic synergy and are often prescribed in combination for the treatment of bipolar disorder. Combination therapy is a complex task for clinicians and patients, often resulting in unexpected difficulties with dosing, drug tolerances, and decreased patient compliance. Thus, an unmet need for bipolar disorder treatment is to develop a therapeutic agent that targets both 5-HT2A receptors and VGSCs. Towards this goal, we developed a novel small molecule that simultaneously antagonizes 5-HT2A receptors and blocks sodium current. The new compound, N-(4-bromo-2,5-dimethoxyphenethyl)-6-(4-phenylbutoxy)hexan-1-amine (XOB) antagonizes 5-HT-stimulated, Gq-mediated, calcium flux at 5-HT2A receptors at low micromolar concentrations while displaying negligible affinity and activity at 5-HT1A, 5-HT2B, and 5-HT2C receptors. At similar concentrations, XOB administration inhibits sodium current in heterologous cells and results in reduced action potential (AP) firing and VGSC-related AP properties in mouse prefrontal cortex layer V pyramidal neurons. Thus, XOB represents a new, proof-of-principle tool that can be used for future preclinical investigations and therapeutic development. This polypharmacology approach of developing a single molecule to act upon two targets, which are currently independently targeted by combination therapies, may lead to safer alternatives for the treatment of psychiatric disorders that are increasingly being found to benefit from the simultaneous targeting of multiple receptors. Significance Statement We synthesized a novel small molecule (XOB) that simultaneously antagonizes two key therapeutic targets of bipolar disorder, 5-HT2A receptors and voltage-gated sodium channels (VGSCs), in heterologous cells, and inhibits the intrinsic excitability of mouse prefrontal cortex layer V pyramidal neurons in brain slices. XOB represents a valuable new proof-of-principle tool for future preclinical investigations and provides a novel molecular approach to the pharmacological treatment of complex neuropsychiatric disease, which often requires a combination of therapeutics for sufficient patient benefit.

Exploring the mechanism of Nav1.3 in the ION-CCI rat model based on the TLR4/TRAF6/NF-κB pathway.

BACKGROUND: Trigeminal neuralgia (TN) is a common and difficult-to-treat neuropathic pain disorder in clinical practice. Previous studies have shown that Toll-like receptor 4 (TLR4) modulates the activation of the NF-κB pathway to affect neuropathic pain in rats. Voltage-gated sodium channels (VGSCs) are known to play an important role in neuropathic pain electrical activity. OBJECTIVE: To investigate whether TLR4 can regulate Nav1.3 through the TRAF6/NF-κB p65 pathway after infraorbital nerve chronic constriction injury (ION-CCI). STUDY DESIGN: ION-CCI modeling was performed on SD (Sprague Dawley) rats. To verify the success of the modeling, we need to detect the mechanical pain threshold and ATF3. Then, detecting the expression of TLR4, TRAF6, NF-κB p65, p-p65, and Nav1.3 in rat TG. Subsequently, investigate the role of TLR4/TRAF6/NF-κB pathway in ION-CCI model by intrathecal injections of LPS-rs (TLR4 antagonist), C25-140 (TRAF6 inhibitor), and PDTC (NF-κB p65 inhibitor). RESULTS: ION-CCI surgery decreased the mechanical pain threshold of rats and increased the expression of ATF3, TLR4, TRAF6, NF-κB p-p65 and Nav1.3, but there was no difference in NF-κB p65 expression. After inject antagonist or inhibitor of the TLR4/TRAF6/NF-κB pathway, the expression of Nav1.3 was decreased and mechanical pain threshold was increased. CONCLUSION: In the rat model of ION-CCI, TLR4 in the rat trigeminal ganglion regulates Nav1.3 through the TRAF6/NF-κB p65 pathway, and TLR4 antagonist alleviates neuropathic pain in ION-CCI rats.

Age-dependent small fiber neuropathy: Mechanistic insights from animal models.

Small fiber neuropathy (SFN) is a common and debilitating disease in which the terminals of small diameter sensory axons degenerate, producing sensory loss, and in many patients neuropathic pain. While a substantial number of cases are attributable to diabetes, almost 50% are idiopathic. An underappreciated aspect of the disease is its late onset in most patients. Animal models of human genetic mutations that produce SFN also display age-dependent phenotypes suggesting that aging is an important contributor to the risk of development of the disease. In this review we define how particular sensory neurons are affected in SFN and discuss how aging may drive the disease. We also evaluate how animal models of SFN can define disease mechanisms that will provide insight into early risk detection and suggest novel therapeutic interventions.