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The voltage-gated sodium (Nav) channel subtype Nav1.7 plays a critical role in pain signaling, making it an important drug target. A number of peptide toxins from cone snails (conotoxins) bind to the extracellular vestibule of the Nav channel pore and block ion conduction. While the known conotoxins have variable selectivity among Nav channel subtypes, they form potential scaffolds for engineering of selective and potent channel inhibitors. Here we studied the molecular interactions between μ-conotoxin KIIIA (KIIIA) and the human Nav1.7 channel (hNav1.7). Using the cryo-electron microscopy (cryo-EM) structure of the electric eel Nav1.4 channel as a template we developed a structural model of hNav1.7 with Rosetta computational modeling. We performed in silico docking of KIIIA using RosettaDock and identified residues forming specific pairwise contacts between KIIIA and hNav1.7. Pairwise interactions were experimentally validated using mutant cycle analysis. Comparison with a recently published cryo-EM structure of the KIIIA-hNav1.2 channel complex revealed key similarities and differences between channel subtypes with potential implications for the molecular mechanism of toxin block. Our integrative approach, combining high-resolution structural data with computational modeling and experimental validation, will be useful for engineering of molecular probes to study Nav channels function and for rational design of novel biologics to treat chronic pain, cardiac arrhythmias, and epilepsy.

Introduction: Diabetes is a common metabolic disease, which leads to diabetic peripheral neuropathy. Peripheral neuron damage result in Nav1.3 elevations. Exercise training has beneficial role in diabetes management and peripheral neuropathy. Alpha lipoic acid (ALA) is a powerful biological antioxidant. However, the role of exercise training and ALA on Nav1.3 are not well understood. The aim of the present study was to investigate the effect of training with different intensity and Alpha lipoic acid supplement on soleus muscle Nav1.3 protein in rats with type 2 diabetes. Thirty-five male Wistar rats were randomly divided into seven groups: healthy control, diabetic, complementary diabetic, intensive exercise diabetic, moderate exercise diabetic, intensive exercise + supplemental diabetic, moderate exercise + complementary diabetic. Methods: In this experimental study, 35 male Wistar rats were randomly divided into seven groups: healthy control, diabetic (D), complementary (alpha lipoic acid) diabetic (ALA), diabetic high intensity training (HIT), diabetic moderate intensity training (MIT), diabetes HIT+ALA (ALA + HIT), diabetic MIT + ALA (ALA + MIT). Rats were diabetic by intra-peritoneal injection of STZ. The HIT and MIT protocols were performed five days a week for six weeks. HIIT included 10 bouts of four minutes (running at 85–90% of maximum speed) and MIT 13 bouts of four minutes (running at 65–70% of maximum speed). ALA was administered orally 20 mg/kg once a day by gavage. Nav1.3 protein levels were measured by immunohistochemistry method. Statistical operations were performed with SPSS version 16 software. One-way analysis of variance and Tukey were used to analyze the data. Results: The level of Nav1.3 increased significantly in diabetic group compared to the control (p≤0.0001). Moreover, HIT (p=0.0015), MIT p=0.0056), ALA+HIT (p≤0.0001) and ALA+MIT (p≤0.0001) decreased significantly Nav1.3 compared to the diabetic group. Conclusion: HIT and MIT can reduce the expression of NaV1.3 in soleus muscle in diabetic rats. ALA combined with exercise training can be more effective to reduce diabetic neuropathy.

With the wide adoption of genomic sequencing in children having seizures, an increasing number ofSCN2Agenetic variants have been revealed as genetic causes of epilepsy. Voltage-gated sodium channel Nav1.2, encoded by geneSCN2A, is predominantly expressed in the pyramidal excitatory neurons and supports action potential (AP) firing. One recurrentSCN2Agenetic variant is L1342P, which was identified in multiple patients with epileptic encephalopathy and intractable seizures. However, the mechanism underlying L1342P-mediated seizures and the pharmacogenetics of this variant in human neurons remain unknown. To understand the core phenotypes of the L1342P variant in human neurons, we took advantage of a reference human-induced pluripotent stem cell (hiPSC) line from a male donor, in which L1342P was introduced by CRISPR/Cas9-mediated genome editing. Using patch-clamping and microelectrode array (MEA) recordings, we revealed that cortical neurons derived from hiPSCs carrying heterozygous L1342P variant have significantly increased intrinsic excitability, higher sodium current density, and enhanced bursting and synchronous network firing, suggesting hyperexcitability phenotypes. Interestingly, L1342P neuronal culture displayed a degree of resistance to the anticonvulsant medication phenytoin, which recapitulated aspects of clinical observation of patients carrying the L1342P variant. In contrast, phrixotoxin-3 (PTx3), a Nav1.2 isoform-specific blocker, can potently alleviate spontaneous and chemically-induced hyperexcitability of neurons carrying the L1342P variant. Our results reveal a possible pathogenic underpinning of Nav1.2-L1342P mediated epileptic seizures and demonstrate the utility of genome-edited hiPSCs as anin vitroplatform to advance personalized phenotyping and drug discovery.SIGNIFICANCE STATEMENTA mounting number ofSCN2Agenetic variants have been identified from patients with epilepsy, but howSCN2Avariants affect the function of human neurons contributing to seizures is still elusive. This study investigated the functional consequences of a recurringSCN2Avariant (L1342P) using human iPSC-derived neurons and revealed both intrinsic and network hyperexcitability of neurons carrying a mutant Nav1.2 channel. Importantly, this study recapitulated elements of clinical observations of drug-resistant features of the L1342P variant, and provided a platform forin vitrodrug testing. Our study sheds light on cellular mechanism of seizures resulting from a recurring Nav1.2 variant, and helps to advance personalized drug discovery to treat patients carrying pathogenicSCN2Avariant.

Background Anosmia/hyposmia affects 13.3 million people in the U.S. alone according to the recent U.S. National Health and Nutrition Examination Survey (NHANES). Hundreds of thousands more people with persistent olfactory dysfunction will be added to this number due to the COVID-19 pandemic. Patients with loss-of-function mutations in SCN9A, the gene encoding NaV1.7, experience anosmia in addition to congenital insensitivity to pain. Tsp1a is a recently discovered peptide that inhibits NaV1.7 with high potency and selectivity. In this study, we examined whether a fluorescently tagged version of Tsp1a could be used to visualize normal and damaged mouse olfactory nerves. Methods Athymic nude mice were intravenously injected with Tsp1a-IR800. As a control, mice were injected with PBS only, and as a blocking control were injected with combination of Tsp1a and Tsp1a-IR800. All mice were imaged in-vivo and epifluorescence images were acquired using an IVIS Spectrum animal imaging system. Semiquantitative analysis of the Tsp1a-IR800 signal was conducted by measuring the average radiant efficiency in the region of the olfactory epithelium/bulb (ROEB). Methimazole was used to chemically ablate the olfactory epithelium. We performed a food buried test to correlate the level of anosmia with the level of radiance efficiency. Results The area of olfactory epithelium/bulb was clearly visible in epifluorescence in-vivo images of mice receiving the imaging agent. The radiant efficiency was significantly less in both mice injected with PBS and in mice injected with the blocking formulation. The mice after olfactory ablation had a significantly reduced radiant efficiency compared with normal mice. Moreover, there was a statistically significant and inverse correlation between the time required for the mouse to find buried food and the radiant efficiency. We also performed immunohistochemistry using NaV1.7 antibody. Mice after olfactory ablation as well as COVID-19-infected mice had significantly lower expression of NaV1.7 on the level of olfactory epithelium/bulb. Conclusion We show that the fluorescent imaging of mouse olfactory epithelium/bulb is possible, suggesting that labeled Tsp1a tracers may serve as the first objective diagnostic tool of smell disorders, including those caused by COVID-19.

[Image: see text] The human nociceptor-specific voltage-gated sodium channel 1.7 (hNa(V)1.7) is critical for sensing various types of somatic pain, but it appears not to play a primary role in acute visceral pain. However, its role in chronic visceral pain remains to be determined. We used assay-guided fractionation to isolate a novel hNa(V)1.7 inhibitor, Tsp1a, from tarantula venom. Tsp1a is 28-residue peptide that potently inhibits hNa(V)1.7 (IC(50) = 10 nM), with greater than 100-fold selectivity over hNa(V)1.3–hNa(V)1.6, 45-fold selectivity over hNa(V)1.1, and 24-fold selectivity over hNa(V)1.2. Tsp1a is a gating modifier that inhibits Na(V)1.7 by inducing a hyperpolarizing shift in the voltage-dependence of channel inactivation and slowing recovery from fast inactivation. NMR studies revealed that Tsp1a adopts a classical knottin fold, and like many knottin peptides, it is exceptionally stable in human serum. Remarkably, intracolonic administration of Tsp1a completely reversed chronic visceral hypersensitivity in a mouse model of irritable bowel syndrome. The ability of Tsp1a to reduce visceral hypersensitivity in a model of irritable bowel syndrome suggests that pharmacological inhibition of hNa(V)1.7 at peripheral sensory nerve endings might be a viable approach for eliciting analgesia in patients suffering from chronic visceral pain.

The voltage-gated sodium channel Nav1.7 continues to be a high-profile target for the treatment of various pain afflictions due to its strong human genetic validation. While isoform selective molecules have been discovered and advanced into the clinic, to date, this target has yet to bear fruit in the form of marketed therapeutics for the treatment of pain. Lead optimization efforts over the past decade have focused on selectivity over Nav1.5 due to its link to cardiac side effects as well as the translation of preclinical efficacy to man. Inhibition of Nav1.6 was recently reported to yield potential respiratory side effects preclinically, and this finding necessitated a modified target selectivity profile. Herein, we report the continued optimization of a novel series of arylsulfonamide Nav1.7 inhibitors to afford improved selectivity over Nav1.6 while maintaining rodent oral bioavailability through the use of a novel multiparameter optimization (MPO) paradigm. We also report in vitro-in vivo correlations from Nav1.7 electrophysiology protocols to preclinical models of efficacy to assist in projecting clinical doses. These efforts produced inhibitors such as compound 19 with potency against Nav1.7, selectivity over Nav1.5 and Nav1.6, and efficacy in behavioral models of pain in rodents as well as inhibition of rhesus olfactory response indicative of target modulation.

How genetic loss of the sodium channel NaV1.7 results in painlessness is puzzling. MacDonald et al. (2021) demonstrate that instead of impairing peripheral excitability, NaV1.7 channels at central terminals of pain-sensing afferents play a pivotal role in the balance between pain and analgesia.

See Silagi and Segal (doi:10.1093/brain/awab196) for a scientific commentary on this article. The chemotherapy drug paclitaxel induces an increase in Nav1.7 channel expression in sensory neurons, enhancing their excitability. Akin et al. use live imaging to visualize trafficking and surface localization of Nav1.7 channels in sensory axons after exposure to paclitaxel, and reveal a role for inflammation in this process., The microtubule-stabilizing chemotherapy drug paclitaxel (PTX) causes dose-limiting chemotherapy-induced peripheral neuropathy (CIPN), which is often accompanied by pain. Among the multifaceted effects of PTX is an increased expression of sodium channel Nav1.7 in rat and human sensory neurons, enhancing their excitability. However, the mechanisms underlying this increased Nav1.7 expression have not been explored, and the effects of PTX treatment on the dynamics of trafficking and localization of Nav1.7 channels in sensory axons have not been possible to investigate to date. In this study we used a recently developed live imaging approach that allows visualization of Nav1.7 surface channels and long-distance axonal vesicular transport in sensory neurons to fill this basic knowledge gap. We demonstrate concentration and time-dependent effects of PTX on vesicular trafficking and membrane localization of Nav1.7 in real-time in sensory axons. Low concentrations of PTX increase surface channel expression and vesicular flux (number of vesicles per axon). By contrast, treatment with a higher concentration of PTX decreases vesicular flux. Interestingly, vesicular velocity is increased for both concentrations of PTX. Treatment with PTX increased levels of endogenous Nav1.7 mRNA and current density in dorsal root ganglion neurons. However, the current produced by transfection of dorsal root ganglion neurons with Halo-tag Nav1.7 was not increased after exposure to PTX. Taken together, this suggests that the increased trafficking and surface localization of Halo-Nav1.7 that we observed by live imaging in transfected dorsal root ganglion neurons after treatment with PTX might be independent of an increased pool of Nav1.7 channels. After exposure to inflammatory mediators to mimic the inflammatory condition seen during chemotherapy, both Nav1.7 surface levels and vesicular transport are increased for both low and high concentrations of PTX. Overall, our results show that PTX treatment increases levels of functional endogenous Nav1.7 channels in dorsal root ganglion neurons and enhances trafficking and surface distribution of Nav1.7 in sensory axons, with outcomes that depend on the presence of an inflammatory milieu, providing a mechanistic explanation for increased excitability of primary afferents and pain in CIPN.

Nav1.7 is an extensively investigated target for pain with a strong genetic link in humans, yet in spite of this effort, it remains challenging to identify efficacious, selective, and safe inhibitors. Here, we disclose the discovery and preclinical profile of GDC-0276 (1) and GDC-0310 (2), selective Nav1.7 inhibitors that have completed Phase 1 trials. Our initial search focused on close-in analogues to early compound 3. This resulted in the discovery of GDC-0276 (1), which possessed improved metabolic stability and an acceptable overall pharmacokinetics profile. To further derisk the predicted human pharmacokinetics and enable QD dosing, additional optimization of the scaffold was conducted, resulting in the discovery of a novel series of N-benzyl piperidine Nav1.7 inhibitors. Improvement of the metabolic stability by blocking the labile benzylic position led to the discovery of GDC-0310 (2), which possesses improved Nav selectivity and pharmacokinetic profile over 1.

The aim of the present study was to observe the changes of TTX-R, Nav1.8, and Nav1.9 Na+ currents in MSU-induced gouty arthritis mice, and to explore the possibility of Nav1.8 and Nav1.9 channels as potential targets for gout pain treatment. Acute gouty arthritis was induced by monosodium urate (MSU) in mice. Swelling degree was evaluated by measuring the circumference of the ankle joint. Mechanical allodynia was assessed by applying the electronic von Frey. Na+ currents were recorded by patch-clamp techniques in acute isolated dorsal root ganglion (DRG) neurons. MSU treatment significantly increased the swelling degree of ankle joint and decreased the mechanical pain threshold. The amplitude of TTX-R Na+ current was significantly increased and reached its peak on the 4th day after injection of MSU. For TTX-R Na+ channel subunits, Nav1.8 current density was significantly increased, but Nav1.9 current density was markedly decreased after MSU treatment. MSU treatment shifted the steady-state activation curves of TTX-R Na+ channel, Nav1.8 and Nav1.9 channels, and the inactivation curves of TTX-R Na+ channel and Nav1.8 channels to the depolarizing direction, but did not affect the inactivation curve of Nav1.9 channel. Compared with the normal group, the recovery of Nav1.8 channel was faster, while that of Nav1.9 channel was slower. The recovery of TTX-R Na+ channel remained unchanged after MSU treatment. Additionally, MSU treatment increased DRG neurons excitability by reducing action potential threshold. Nav1.8 channel, not Nav1.9 channel, may be involved in MSU-induced gout pain by increasing nerve excitability.

A fundamental mechanism that drives the propagation of electrical signals in the nervous system is the activation of voltage-gated sodium channels. The sodium channel subtype Nav1.7 is critical for the transmission of pain-related signaling, with gain-of-function mutations in Nav1.7 resulting in various painful pathologies. Loss-of-function mutations cause complete insensitivity to pain and anosmia in humans that otherwise have normal nervous system function, rendering Nav1.7 an attractive target for the treatment of pain. Despite this, no Nav1.7 selective therapeutic has been approved for use as an analgesic to date. Here we present a summary of research that has focused on engineering peptides found in spider venoms to produce Nav1.7 selective antagonists. We discuss the progress that has been made on various scaffolds from different venom families and highlight the challenges that remain in the effort to produce a Nav1.7 selective, venom-based analgesic.

Voltage-gated sodium channel NaV1.8 regulates transmission of pain signals to the brain. While NaV1.8 has the potential to serve as a drug target, the molecular mechanisms that shape NaV1.8 gating are not completely understood, particularly mechanisms that couple activation to inactivation. Interactions between toxin producing animals and their predators provide a novel approach for investigating NaV structure-function relationships. Arizona bark scorpions produce Na+ channel toxins that initiate pain signaling. However, in predatory grasshopper mice, toxins inhibit NaV1.8 currents and block pain signals. A screen of synthetic peptide toxins predicted from bark scorpion venom showed that peptide NaTx36 inhibited Na+ current recorded from a recombinant grasshopper mouse NaV1.8 channel (OtNaV1.8). Toxin NaTx36 hyperpolarized OtNaV1.8 activation, steady-state fast inactivation, and slow inactivation. Mutagenesis revealed that the first gating charge in the domain I (DI) S4 voltage sensor and an acidic amino acid (E) in the DII SS2 – S6 pore loop are critical for the inhibitory effects of NaTx36. Computational modeling showed that a DI S1 – S2 asparagine (N) stabilizes the NaTx36 – OtNaV1.8 complex while residues in the DI S3 – S4 linker and S4 voltage sensor form electrostatic interactions that allow a toxin glutamine (Q) to contact the first S4 gating charge. Surprisingly, the models predicted that NaTx36 contacts amino acids in the DII S5 – SS1 pore loop instead of the SS2 – S6 loop; the DII SS2 – S6 loop motif (QVSE) alters the conformation of the DII S5 – SS1 pore loop, enhancing allosteric interactions between toxin and the DII S5 – SS1 pore loop. Few toxins have been identified that modify NaV1.8 gating. Moreover, few toxins have been described that modify sodium channel gating via the DI S4 voltage sensor. Thus, NaTx36 and OtNaV1.8 provide tools for investigating the structure-activity relationship between channel activation and inactivation gating, and the connection to alternative pain phenotypes.

The voltage-gated sodium channel Nav1.8 mediates the tetrodotoxin-resistant (TTX-R) Na+ current in nociceptive primary sensory neurons, which has an important role in the transmission of painful st...

The voltage-gated sodium channels play a key role in the generation and propagation of the cardiac action potential. Emerging data indicate that the Nav1.8 channel, encoded by the SCN10A gene, is a modulator of cardiac conduction and variation in the gene has been associated with arrhythmias such as atrial fibrillation (AF) and Brugada syndrome (BrS). The voltage gated sodium channels contain a calmodulin (CaM)-binding IQ domain involved in channel slow inactivation, we here investigated the role of CaM regulation of Nav1.8 channel function, and showed that CaM enhanced slow inactivation of the Nav1.8 channel and hyperpolarized steady-state inactivation curve of sodium currents. The effects of CaM on the channel gating were disrupted in the Nav1.8 channel truncated IQ domain. We studied Nav1.8 IQ domain mutations associated with AF and BrS, and found that a BrS-linked mutation (R1863Q) reduced the CaM-induced hyperpolarization shift, AF-linked mutations (R1869C and R1869G) disrupted CaM-induced enhanced inactivation, and effects of CaM on both development and recovery from slow inactivation were attenuated in all pathogenic mutations. Our findings indicate a role of CaM in the regulation of Nav1.8 channel function in cardiac arrhythmias.

In the primary sensory neuron, ouabain activates the dual mechanism that modulates the functional activity of NaV1.8 channels. Ouabain at endogenous concentrations (EO) triggers two different signaling cascades, in which the Na,K-ATPase/Src complex is the EO target and the signal transducer. The fast EO effect is based on modulation of the NaV1.8 channel activation gating device. EO triggers the tangential signaling cascade along the neuron membrane from Na,K-ATPase to the NaV1.8 channel. It evokes a decrease in effective charge transfer of the NaV1.8 channel activation gating device. Intracellular application of PP2, an inhibitor of Src kinase, completely eliminated the effect of EO, thus indicating the absence of direct EO binding to the NaV1.8 channel. The delayed EO effect probably controls the density of NaV1.8 channels in the neuron membrane. EO triggers the downstream signaling cascade to the neuron genome, which should result in a delayed decrease in the NaV1.8 channels’ density. PKC and p38 MAPK are involved in this pathway. Identification of the dual mechanism of the strong EO effect on NaV1.8 channels makes it possible to suggest that application of EO to the primary sensory neuron membrane should result in a potent antinociceptive effect at the organismal level.

Neuropathic pain is commonly associated with lesion or disease of the somatosensory system and often reflected as indicator of impaired life. Although the central nervous system is main regulator o...

The discovery of a novel class of state-dependent voltage-gated sodium channel (NaV)1.7 inhibitors is described. By the modification of amide or urethane bond in NaV1.7 blocker III, structure-activity relationship studies that led to the identification of novel NaV1.7 inhibitor 2i (DS01171986) were performed. Compound 2i exhibited state-dependent inhibition of NaV1.7 without NaV1.1, NaV1.5 or human ether-a-go-go related gene (hERG) liabilities at concentrations up to 100 μM. Further biological profiling successfully revealed that 2i possessed potent analgesic properties in a murine model of neuropathic pain (ED50: 3.4 mg/kg) with an excellent central nervous system (CNS) safety margin (> 600 fold).

The voltage-gated sodium channel Nav1.7 is a genetically validated target for pain; pharmacological blockers are promising as a new class of nonaddictive therapeutics. The search for Nav1.7 subtype selective inhibitors requires a reliable, scalable, and sensitive assay. Previously, we developed an all-optical electrophysiology (Optopatch) Spiking HEK platform to study activity-dependent modulation of Nav1.7 in a format compatible with high-throughput screening. In this study, we benchmarked the Optopatch Spiking HEK assay with an existing validated automated electrophysiology assay on the IonWorks Barracuda (IWB) platform. In a pilot screen of 3520 compounds, which included compound plates from a random library as well as compound plates enriched for Nav1.7 inhibitors, the Optopatch Spiking HEK assay identified 174 hits, of which 143 were confirmed by IWB. The Optopatch Spiking HEK assay maintained the high reliability afforded by traditional fluorescent assays and further demonstrated comparable sensitivity to IWB measurements. We speculate that the Optopatch assay could provide an affordable high-throughput screening platform to identify novel Nav1.7 subtype selective inhibitors with diverse mechanisms of action, if coupled with a multiwell parallel optogenetic recording instrument.

Hyperactivity in cochlear nucleus (CN) is one of the major neural correlates for tinnitus induction, yet the molecular factors that participate in the neuronal hyperexcitability remain unclear. The present study showed that acute and chronic administrations of salicylate were both capable of inducing reversible tinnitus in rats. The number of GAD 65/67-immunoreactive neurons in the AVCN and DCN was decreased, while the number of VGLUT 1/2-immunoreactive neurons in the AVCN and DCN was increased when rats were experiencing tinnitus, providing evidence for excitatory-inhibitory imbalance in CN is correlated with tinnitus. Interestingly, the expression level of Nav1.6, an important subtype of voltage-gated sodium channels was significantly increased in the DCN and AVCN of rats experiencing tinnitus, the up-regulation of Nav1.6 was returned to normal level following the disappearance of tinnitus. Double-labeling experiments revealed that Nav1.6 expression was down-regulated in the GAD 65/67-positive neurons in the DCN and AVCN of rats experiencing tinnitus. Notably, the percentage of co-localization of Nav1.6 and NeuN-labeling fusiform neurons was markedly increased in the DCN during tinnitus. These findings uncover the tinnitus-associated alteration in Nav1.6, a potential key contributor that can lead to hyperexcitability in CN and contribute to salicylate-induced tinnitus.

A highly potent, selective NaV1.7 inhibitor, DS-1971a, has been discovered. Exploration of the left-hand phenyl ring of sulfonamide derivatives (I and II) led to the discovery of novel series of cycloalkane derivatives with high NaV1.7 inhibitory potency in vitro. As the right-hand heteroaromatic ring affected the mechanism-based inhibition liability of CYP3A4, replacement of this moiety resulted in the generation of 4-pyrimidyl derivatives. Additionally, GSH adducts formation, which can cause idiosyncratic drug toxicity, was successfully avoided by this modification. An additional optimization led to the discovery of DS-1971a. In preclinical studies, DS-1971a demonstrated highly potent selective in vitro profile with robust efficacy in vivo. DS-1971a exhibited a favorable toxicological profile, which enabled multiple-dose studies of up to 600 mg bid or 400 mg tid (1200 mg/day) administered for 14 days to healthy human males. DS-1971a is expected to exert potent efficacy in patients with peripheral neuropathic pain, with a favorable safety profile.

Background: To verify the hypothesis that the nature of trigeminal neuralgia (TN) is an ectopic impulse induced by sodium channel modulated by cytokines, we conducted an animal study using the infr...

Carvacrol is the predominant monoterpene in essential oils from many aromatic plants. Several animal studies showing analgesic effects of carvacrol indicate potential of carvacrol as a new medication for patients with refractory pain. Voltage-gated sodium channels (Nav) are thought to have crucial roles in the development of inflammatory and neuropathic pain, but there is limited information about whether the analgesic mechanism of carvacrol involves Nav. We used whole-cell, two-electrode, voltage-clamp techniques to examine the effects of carvacrol on sodium currents in Xenopus oocytes expressing α subunits of Nav1.2, Nav1.3, Nav1.6, Nav1.7, and Nav1.8. Carvacrol dose-dependently suppressed sodium currents at a holding potential that induced half-maximal current. The half-maximal inhibitory concentration values for Nav1.2, Nav1.3, Nav1.6, Nav1.7, and Nav1.8 were 233, 526, 215, 367, and 824 μmol/L, respectively, indicating that carvacrol had more potent inhibitory effects towards Nav1.2 and Nav1.6 than Nav1.3, Nav1.7, and Nav1.8. Gating analysis showed a depolarizing shift of the activation curve and a hyperpolarizing shift of the inactivation curve in all five α subunits following carvacrol treatment. Furthermore, carvacrol exhibits a use-dependent block for all five α Nav subunits. These findings provide a better understanding of the mechanisms associated with the analgesic effect of carvacrol. Keywords: Carvacrol, Voltage-gated sodium channels, Analgesic mechanism, Analgesic effect, More potent inhibitory effects toward Nav1.2 and Nav1.6

Introduction Mutations of the voltage-gated sodium channel gene (SCN4A), which encodes Nav1.4, cause nondystrophic myotonia that occasionally is associated with severe apnea and laryngospasm. There are case reports of nondystrophic myotonia due to mutations in the C-terminal tail (CTerm) of Nav1.4, but the functional analysis is scarce. Methods We present two families with nondystrophic myotonia harboring a novel heterozygous mutation (E1702del) and a known heterozygous mutation (E1702K). Results The proband with E1702K exhibited repeated rhabdomyolysis, and the daughter showed laryngospasm and cyanosis. Functional analysis of the two mutations as well as another known heterozygous mutation (T1700_E1703del), all located on EF hand-like motif in CTerm, was conducted with whole-cell recording of heterologously expressed channel. All mutations displayed impaired fast inactivation. Discussion The CTerm of Nav1.4 is vital for regulating fast inactivation. The study highlights the importance of accumulating pathological mutations of Nav1.4 and their functional analysis data.

Neuropathic pain is clinically unsatisfactorily treated because of unclear mechanisms. The present study aims to explore the concrete mechanisms underlying the alleviation of resveratrol-activated silent information regulator 1 (Sirt1) to chronic constriction injury (CCI)-induced neuropathic pain. CCI surgery was conducted to the unilateral sciatic nerve of male Sprague-Dawley rats to induce neuropathic pain experimentally. Resveratrol with or without miR-182 antagomir were administered to CCI rats via intrathecal catheter. Behavioral tests including paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) were conducted to explore mechanical allodynia and thermal hyperalgesia. Western blot, qRT-PCR were used to detect the expression levels of Sirt1, miR-182, and Nav1.7 in CCI dorsal root ganglions (DRGs). CCI rats displayed lower PWT and PWL compared with the sham control. Also, the CCI DRGs displayed lower Sirt1 and miR-182 expression as well as higher Nav1.7 expression, which would be almost reversed by resveratrol treatment for 4 successive days. We also found that miR-182 expression inhibition erased the analgesia effect of resveratrol to CCI–induced neuropathic pain possibly through upregulating Nav1.7 expression. In summary, resveratrol alleviated CCI–induced neuropathic pain, possibly through activating Sirt1 to suppress Nav1.7 expression via upregulating miR-182 expression in CCI DRGs.

Corrigendum notice: Protective Effect of Interval Exercise Training with Different Intensity and Alpha-Lipoic Acid Supplement on Nav1.3 Protein in Soleus Muscle of Diabetic Rats Seyedeh Fatemeh Fatemi1, Seyed Abdollah Hashemvarzi1, Amin Farzaneh Hesari1 1Department of Exercise Physiology, Sari Branch, Islamic Azad University, Sari, Iran. *Corresponding author: Tel: 09111160278, email: hashemvarzi_tkd@yahoo.com Corrigendum notice: In the above article, which was published in the Volume 29, Issue of 8, November 2021, affiliations of all of the authors have been corrected.

The voltage-gated sodium NaV1.7 channel, critical for sensing pain, has been actively targeted by drug developers; however, there are currently no effective and safe therapies targeting NaV1.7. Here, we tested whether a different approach, indirect NaV1.7 regulation, could have antinociceptive effects in preclinical models. We found that preventing addition of small ubiquitin-like modifier (SUMO) on the NaV1.7-interacting cytosolic collapsin response mediator protein 2 (CRMP2) blocked NaV1.7 functions and had antinociceptive effects in rodents. In silico targeting of the SUMOylation site in CRMP2 (Lys374) identified200 hits, of which compound 194 exhibited selective in vitro and ex vivo NaV1.7 engagement. Orally administered 194 was not only antinociceptive in preclinical models of acute and chronic pain but also demonstrated synergy alongside other analgesics—without eliciting addiction, rewarding properties, or neurotoxicity. Analgesia conferred by 194 was opioid receptor dependent. Our results demonstrate that 194 is a first-in-class protein-protein inhibitor that capitalizes on CRMP2-NaV1.7 regulation to deliver safe analgesia in rodents.

It has been repeatedly proved that Nav1.8 tetrodotoxin (TTX)-resistant sodium currents are expressed in peripheral sensory neurons where they play important role in nociception. There are very few publications that show the presence of TTX-resistant sodium currents in central neurons. The aim of this study was to assess if functional Nav1.8 TTX-resistant sodium currents are expressed in prefrontal cortex pyramidal neurons. All recordings were performed in the presence of TTX in the extracellular solution to block TTX-sensitive sodium currents. The TTX-resistant sodium current recorded in this study was mainly carried by the Nav1.8 sodium channel isoform because the Nav1.9 current was inhibited by the -65 mV holding potential that we used throughout the study. Moreover, the sodium current that we recorded was inhibited by treatment with the selective Nav1.8 inhibitor A-803467. Confocal microscopy experiments confirmed the presence of the Nav1.8 α subunit in prefrontal cortex pyramidal neurons. Activation and steady state inactivation properties of TTX-resistant sodium currents were also assessed in this study and they were similar to activation and inactivation properties of TTX-resistant sodium currents expressed in dorsal root ganglia (DRG) neurons. Moreover, this study showed that carbamazepine (60 µM) inhibited the maximal amplitude of the TTX-resistant sodium current. Furthermore, we found that carbamazepine shifts steady state inactivation curve of TTX-resistant sodium currents toward hyperpolarization. This study suggests that the Nav1.8 TTX-resistant sodium channel is expressed not only in DRG neurons, but also in cortical neurons and may be molecular target for antiepileptic drugs such as carbamazepine.

Unmyelinated fibers in the central nervous system are known to exist in hippocampal mossy fibers, cerebellar parallel fibers and striatal projection fibers. Previously, we and others reported diffuse distribution of Nav1.2, a voltage-gated sodium channel α-subunit encoded by the SCN2A gene, on unmyelinated striatal projection fibers. Mutations in the SCN2A gene are associated with epilepsies and autism. In this study, we investigated the distribution of Nav1.2 on the unmyelinated fibers in the corpus callosum and stria terminalis by immunohistochemistry and immunoelectron microscopy analysis, suggesting that diffuse localization of Nav1.2 on mid-axonal regions can be a useful marker for unmyelinated fibers.

Pitt-Hopkins syndrome (PTHS) is a rare autism spectrum-like disorder characterized by intellectual disability, developmental delays, and breathing problems involving episodes of hyperventilation followed by apnea. PTHS is caused by functional haploinsufficiency of the gene encoding transcription factor 4 (Tcf4). Despite the severity of this disease, mechanisms contributing to PTHS behavioral abnormalities are not well understood. Here, we show that a Tcf4 truncation (Tcf4tr/+) mouse model of PTHS exhibits breathing problems similar to PTHS patients. This behavioral deficit is associated with selective loss of putative expiratory parafacial neurons and compromised function of neurons in the retrotrapezoid nucleus that regulate breathing in response to tissue CO2/H+. We also show that central Nav1.8 channels can be targeted pharmacologically to improve respiratory function at the cellular and behavioral levels in Tcf4tr/+ mice, thus establishing Nav1.8 as a high priority target with therapeutic potential in PTHS., Disordered breathing is a hallmark of Pitt-Hopkins syndrome (PTHS), yet little is known regarding how loss of Tcf4 (gene associated with PTHS) affects development and function of respiratory neurons. Here, the authors show that parafacial respiratory neurons are selectively disrupted in a mouse model of PTHS, and central Nav1.8 channels can be targeted to improve PTHS-associated behavior abnormalities.

For individual neurons to function appropriately within a network that is undergoing synaptic reorganization and refinement due to developmental or experience-dependent changes in circuit activity, they must homeostatically adapt their intrinsic excitability to maintain a consistent output despite the changing levels of synaptic input. This homeostatic plasticity of excitability is particularly important for the development of sensory circuits, where subtle deficits in neuronal and circuit function cause developmental disorders including autism spectrum disorder and epilepsy. Despite the critical importance of this process for normal circuit development, the molecular mechanism by which this homeostatic control of intrinsic excitability is regulated is not fully understood. Here, we demonstrate thatXenopusoptic tectal neurons express distinct fast, persistent and resurgent Na+currents. Here, we demonstrate thatXenopusoptic tectal neurons express distinct fast, persistent and resurgent Na+currents. These are regulated with developmental changes in synaptic input, and homeostatically in response to changes in visual input. We show that expression of the voltage-gated Na+channel subtype Nav1.6 is regulated with changes in intrinsic excitability, that blocking Nav1.6 channels is sufficient to decrease intrinsic excitability. Furthermore, that upregulation of Nav1.6 expression is necessary for experience-dependent increases in Na+currents and intrinsic excitability. Finally, by examining behaviors that rely on visual and multisensory integration, we extend these findings to show that tight regulation of Na+channel gene expression during a critical period of tectal circuit development is required for the normal functional development of the tectal circuitry.

Background Sleep-disordered breathing (SDB) is often associated with atrial fibrillation, but detailed mechanisms remain elusive. Interestingly, late Na current (late INa) has been shown to be increased in patients with SDB, while expression of cardiac Na channel NaV1.5 and peak Na current were decreased. Indeed, recent data demonstrated that enhanced NaV1.8-dependent late INa may also induce pro-arrhythmic activity. Purpose We tested whether Na-V1.8 expression and subsequent NaV1.8-dependent pro-arrhythmic activity are increased in patients with SDB. Methods We prospectively analysed 29 right atrial appendage biopsies of patients undergoing elective coronary artery bypass grafting. SDB was assessed using polygraphy in the preoperative night and an apnoea-hypopnea index (AHI) ≥15/h defined SDB. Micro-dissected atrial trabeculae were electrically field stimulated (at 1 Hz, 5 V for 50 ms, at 37°C) to elicit regular contractions. Trabecular arrhythmias were induced using 100 nM isoproterenol at [Ca]o of 3.5 mmol/L and pro-arrhythmic activity was scored from 0 (no arrhythmias) to 5 (salve). Sarcoplasmic reticulum Ca leak was estimated by the contractility after paused stimulation (at 2 Hz, normalized to before pause). To correlate functional and expression data for each individual patient, NaV1.8 mRNA expression was quantified in each trabeculum using qPCR. Results NaV1.8 mRNA expression was increased in patients with SDB, leading to a significant positive correlation with the severity of SDB (i.e. AHI, p=0.02, r2=0.22, Fig. 1A). Multivariate regression analysis revealed that this association was independent from age, sex, atrial fibrillation, heart failure, diabetes mellitus, and renal function (p=0.03, r2=0.35). Accordingly, selective NaV1.8 blockade with PF-01247324 (PF, 1 μM, 30 min) significantly improved post-pause contractility of isolated trabeculae from 1.69±0.31 to 2.95±0.54 in patients with SDB (p=0.001), whereas no significant improvement was observed in patients without SDB. This resulted in significant positive correlations between the PF-dependent improvement of post-pause contractility and both AHI (p=0.047, r2=0.19) and NaV1.8 mRNA expression (p=0.03, r2=0.17). Most importantly, we also observed a significant increase in arrhythmia severity in patients with SDB of 2.21±0.52 (vs. 1.00±0.49, p=0.03) that could be significantly reduced by selective NaV1.8 inhibition with PF to 0.25±0.18 (p=0.0008, Fig. 1B). In accordance, there was a significant positive correlation between arrhythmia severity and AHI (p=0.01, r2=0.28) that was abolished in the presence of PF (interaction analysis: p=0.ehab724.33141, r2=0.46). Conclusion In patients with SDB, enhanced NaV1.8 expression contribute to atrial pro-arrhythmic activity independent from comorbidities. Selective NaV1.8 inhibition may have therapeutic implications for patients with SDB. Funding Acknowledgement Type of funding sources: Other. Main funding source(s): Part of the study was supported by grants from Philips Respironics (Murrysville, PA 15668) and the Medical Faculty at the University of Regensburg. Figure 1

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

Frontiers in molecular neuroscience 14, 709228 (2021). doi:10.3389/fnmol.2021.709228 special issue: "Brain Disease Mechanisms", Published by Frontiers Research Foundation, Lausanne

Genetic variants in SCN2A, encoding the NaV1.2 voltage-gated sodium channel, are associated with a range of neurodevelopmental disorders with overlapping phenotypes. Some variants fit into a framework wherein gain-of-function missense variants that increase neuronal excitability lead to infantile epileptic encephalopathy, while loss-of-function variants that reduce neuronal excitability lead to developmental delay and/or autism spectrum disorder with or without co- morbid seizures. One unique case less easily classified using this binary paradigm is the de novo missense variant SCN2A p.K1422E, associated with infant-onset developmental delay, infantile spasms, and features of autism spectrum disorder. Prior structure-function studies demonstrated that K1422E substitution alters ion selectivity of NaV1.2, conferring Ca2+ permeability, lowering overall conductance, and conferring resistance to tetrodotoxin (TTX). Based on heterologous expression of K1422E, we developed a compartmental neuron model that predicted mixed effects on channel function and neuronal activity. We also generated Scn2aK1422E mice and characterized effects on neurons and neurological/neurobehavioral phenotypes. Dissociated neurons from heterozygous Scn2aK1422E/+ mice exhibited a novel TTX-resistant current with a reversal potential consistent with mixed ion permeation. Cortical slice recordings from Scn2aK1442E/+ tissue demonstrated impaired action potential initiation and larger Ca2+ transients at the axon initial segment during the rising phase of the action potential, suggesting mixed effects on channel function. Scn2aK1422E/+ mice exhibited rare spontaneous seizures, interictal EEG abnormalities, altered response to induced seizures, reduced anxiety-like behavior and alterations in olfactory-guided social behavior. Overall, Scn2aK1422E/+ mice present with phenotypes similar yet distinct from Scn2a knockout models, consistent with mixed effects of K1422E on NaV1.2 channel function.Significance StatementThe early-onset epilepsy variant SCN2A-p.K1422E displays unique biophysical properties in vitro. To model the impact of this rare variant, we generated Scn2aK1422E mice. Neurons from heterozygous Scn2aK1422E/+ mice showed functional deficits similar to the loss-of-function effects observed in the Scn2a haploinsufficiency model, as well as gain-of-function effects specific to the K1422E variant. There is also some overlap in neurobehavioral phenotypes between Scn2aK1422E/+ and Scn2a haploinsufficient mice. However, Scn2aK1422E/+ mice exhibited unique epilepsy-related phenotypes, including epileptiform events and seizures. Scn2aK1422E/+ mice serve as a useful platform to investigate phenotypic complexity of SCN2A-associated disorders.

Adult rats previously submitted to neonatal limited bedding (NLB), a model of early-life stress, display muscle mechanical hyperalgesia and nociceptor hyperexcitability, the underlying mechanism for which is unknown. Since voltage-gated sodium channel subtype 7 (NaV1.7) contributes to mechanical hyperalgesia in several preclinical pain models and is critical for nociceptor excitability, we explored its role in the muscle hyperalgesia exhibited by adult NLB rats. Western blot analyses demonstrated increased NaV1.7 protein expression in L4-L5 dorsal root ganglia (DRG) from adult NLB rats, and antisense oligodeoxynucleotide (AS ODN) targeting NaV1.7 alpha subunit mRNA attenuated the expression of NaV1.7 in DRG extracts. While this AS ODN did not affect nociceptive threshold in normal rats it significantly attenuated hyperalgesia in NLB rats. The selective NaV1.7 activator OD1 produced dose-dependent mechanical hyperalgesia that was enhanced in NLB rats, whereas the NaV1.7 blocker ProTx-II prevented OD1-induced hyperalgesia in control rats and ongoing hyperalgesia in NLB rats. AS ODN knockdown of extracellular signal-regulated kinase 1/2, which enhances NaV1.7 function, also inhibited mechanical hyperalgesia in NLB rats. Our results support the hypothesis that overexpression of NaV1.7 in muscle nociceptors play a role in chronic muscle pain induced by early-life stress, suggesting that NaV1.7 is a target for the treatment of chronic muscle pain. PERSPECTIVE: We demonstrate that early-life adversity, induced by exposure to inconsistent maternal care, produces chronic muscle hyperalgesia, which depends, at least in part, on increased expression of NaV1.7 in nociceptors.

Nav1.4 channelopathies due to SCN4A mutations can present with episodic attacks of myotonia triggered by fluctuation in the potassium level (potassium-aggravated myotonia). We report a case of potassium-aggravated myotonia due to Nav1.4-M1592V channelopathy with severe and long-lasting focal attacks of myotonia resembling dystonic posturing with diffuse muscle edema in the affected muscles in magnetic resonance imaging and almost constant presence of myotonic discharges in electromyography that can best be described as focal "status myotonicus".

The axon initial segment (AIS), the site of action potential initiation in neurons, is a critical determinant of neuronal excitability. Growing evidence indicates that appropriate recruitment of the AIS macrocomplex is essential for synchronized firing. However, disruption of the AIS structure is linked to the etiology of multiple disorders, including autism spectrum disorder (ASD), a condition characterized by deficits in social communication, stereotyped behaviors, and very limited interests. To date, a complete understanding of the molecular components that underlie the AIS in ASD has remained elusive. In this research, we examined the AIS structure in a BTBR T+Itpr3tf/J mouse model (BTBR), a valid model that exhibits behavioral, electrical, and molecular features of autism, and compared this to the C57BL/6J wild-type control mouse. Using Western blot studies and high-resolution confocal microscopy in the prefrontal frontal cortex (PFC), our data indicate disrupted expression of different isoforms of the voltage-gated sodium channels (NaV) at the AIS, whereas other components of AIS such as ankyrin-G and fibroblast growth factor 14 (FGF14) and contactin-associated protein 1 (Caspr) in BTBR were comparable to those in wild-type control mice. A Western blot assay showed that BTBR mice exhibited a marked increase in different sodium channel isoforms in the PFC compared to wild-type mice. Our results provide potential evidence for previously undescribed mechanisms that may play a role in the pathogenesis of autistic-like phenotypes in BTBR mice.

NaV1.8 channels play a crucial role in regulating the action potential in nociceptive neurons. A single nucleotide polymorphism in the human NaV1.8 gene SCN10A, A1073V (rs6795970, G>A), has been linked to the diminution of mechanical pain sensation as well as cardiac conduction abnormalities. Furthermore, studies have suggested that this polymorphism may result in a “loss-of-function” phenotype. In the present study, we performed genomic analysis of A1073V polymorphism presence in a cohort of patients undergoing sigmoid colectomy who provided information regarding perioperative pain and analgesic use. Homozygous carriers reported significantly reduced severity in postoperative abdominal pain compared with heterozygous and wild-type carriers. Homozygotes also trended toward using less analgesic/opiates during the postoperative period. We also heterologously expressed the wild-type and A1073V variant in rat superior cervical ganglion neurons. Electrophysiological testing demonstrated that the mutant NaV1.8 channels activated at more depolarized potentials compared with wild-type channels. Our study revealed that postoperative abdominal pain is diminished in homozygous carriers of A1073V and that this is likely due to reduced transmission of action potentials in nociceptive neurons. Our findings reinforce the importance of NaV1.8 and the A1073V polymorphism to pain perception. This information could be used to develop new predictive tools to optimize patient pain experience and analgesic use in the perioperative setting. NEW & NOTEWORTHY We present evidence that in a cohort of patients undergoing sigmoid colectomy, those homozygous for the NaV1.8 polymorphism (rs6795970) reported significantly lower abdominal pain scores than individuals with the homozygous wild-type or heterozygous genotype. In vitro electrophysiological recordings also suggest that the mutant NaV1.8 channel activates at more depolarizing potentials than the wild-type Na+ channel, characteristic of hypoactivity. This is the first report linking the rs6795970 mutation with postoperative abdominal pain in humans.

Purpose Several studies have indicated a potential role for SCN10A/NaV1.8 in modulating cardiac electrophysiology and arrhythmia susceptibility. However, by which mechanism SCN10A/NaV1.8 impacts on cardiac electrical function is still a matter of debate. To address this, we here investigated the functional relevance of NaV1.8 in atrial and ventricular cardiomyocytes (CMs), focusing on the contribution of NaV1.8 to the peak and late sodium current (INa) under normal conditions in different species. Methods The effects of the NaV1.8 blocker A-803467 were investigated through patch-clamp analysis in freshly isolated rabbit left ventricular CMs, human left atrial CMs and human-induced pluripotent stem cell-derived CMs (hiPSC-CMs). Results A-803467 treatment caused a slight shortening of the action potential duration (APD) in rabbit CMs and hiPSC-CMs, while it had no effect on APD in human atrial cells. Resting membrane potential, action potential (AP) amplitude, and AP upstroke velocity were unaffected by A-803467 application. Similarly, INa density was unchanged after exposure to A-803467 and NaV1.8-based late INa was undetectable in all cell types analysed. Finally, low to absent expression levels of SCN10A were observed in human atrial tissue, rabbit ventricular tissue and hiPSC-CMs. Conclusion We here demonstrate the absence of functional NaV1.8 channels in non-diseased atrial and ventricular CMs. Hence, the association of SCN10A variants with cardiac electrophysiology observed in, e.g. genome wide association studies, is likely the result of indirect effects on SCN5A expression and/or NaV1.8 activity in cell types other than CMs.

Voltage-gated sodium (NaV) channels play a fundamental role in normal neurological function, especially via the initiation and propagation of action potentials. The NaV1.1 subtype is found in inhibitory interneurons of the brain and it is essential for maintaining a balance between excitation and inhibition in neuronal networks. Heterozygous loss-of-function mutations of SCN1A, the gene encoding NaV1.1, underlie Dravet syndrome (DS), a severe pediatric epilepsy. We recently demonstrated that selective inhibition of NaV1.1 inactivation prevents seizures and premature death in a mouse model of DS. Thus, selective modulators of NaV1.1 might be useful therapeutics for treatment of DS as they target the underlying molecular deficit. Numerous scorpion-venom peptides have been shown to modulate the activity of NaV channels, but little is known about their activity at NaV1.1. Here we report the isolation, sequence, three-dimensional structure, recombinant production, and functional characterization of two peptidic modulators of NaV1.1 from venom of the buthid scorpion Hottentotta jayakari. These peptides, Hj1a and Hj2a, are potent agonists of NaV1.1 (EC50 of 17 and 32 nM, respectively), and they present dual α/β activity by modifying both the activation and inactivation properties of the channel. NMR studies of rHj1a indicate that it adopts a cystine-stabilized αβ fold similar to known scorpion toxins. Although Hj1a and Hj2a have only limited selectivity for NaV1.1, their unusual dual mode of action provides an alternative approach to the development of selective NaV1.1 modulators for the treatment of DS.

Vagal afferents innervating the small intestinal mucosa regulate feeding, gastrointestinal (GI) digestive, and immune functions. Their anatomical-functional characterization has been impeded by the inability to selectively label and manipulate them. Nav 1.8-Cre-tdTomato mice label 80% of nodose and dorsal root ganglia neurons. Here, the origin of these neuron's terminals and their distribution in the small intestinal mucosa were examined by quantitatively comparing tdTomato-labeled innervation in nonoperated (control), subdiaphragmatic vagotomy (VAGX), and sham-operated mice. Control mice exhibited a large proximal-to-distal decrease and a moderate mesentery-to-antimesentery decrease in villus innervation. VAGX reduced this innervation to a greater degree proximally (91-93%) than distally (65-72%), resulting in flat proximal-distal distributions. Therefore, estimates of vagal villus afferent distributions (control minus VAGX) paralleled control distributions, but were slightly reduced in magnitude. Compared with villus afferents, crypt innervation exhibited a muted proximal-to-distal decrease in control mice and a smaller loss after VAGX (45-48%). Sham-operated mice exhibited similar distributions of villus and crypt afferents as control mice, suggesting surgery did not contribute to the effects of VAGX. Most crypt and villus afferent terminals along the entire proximal-distal small intestinal axis had similar morphology to those previously reported in the proximal duodenum, but the density of terminal branches varied. Our findings suggest the majority of small intestinal mucosal innervation labeled in Nav 1.8-Cre-tdTomato mice is vagal in origin. Therefore, these mice will be valuable for studying vagal mucosal afferent morphology, interactions with other GI elements, plasticity, and function.

Dravet syndrome is a severe form of childhood epilepsy characterized by frequent temperature-sensitive seizures and delays in cognitive development. In the majority (80%) of cases, Dravet syndrome is caused by mutations in the SCN1A gene, encoding the voltage-gated sodium channel NaV1.1, which is abundant in the central nervous system. Dravet syndrome can be caused by either gain-of-function mutation or loss of function in NaV1.1, making it necessary to characterize each novel mutation. Here we use a combination of patch-clamp recordings and immunocytochemistry to characterize the first known NH2-terminal amino acid duplication mutation found in a patient with Dravet syndrome, M72dup. M72dup does not significantly alter rate of fast inactivation recovery or rate of fast inactivation onset at any measured membrane potential. M72dup significantly shifts the midpoint of the conductance voltage relationship to more hyperpolarized potentials. Most interestingly, M72dup significantly reduces peak current of NaV1.1 and reduces membrane expression. This suggests that M72dup acts as a loss-of-function mutation primarily by impacting the ability of the channel to localize to the plasma membrane. NEW & NOTEWORTHY Genetic screening of a patient with Dravet syndrome revealed a novel mutation in SCN1A. Of over 700 SCN1A mutations known to cause Dravet syndrome, M72dup is the first to be identified in the NH2-terminus of NaV1.1. We studied M72dup using patch-clamp electrophysiology and immunocytochemistry. M72dup causes a decrease in membrane expression of NaV1.1 and overall loss of function, consistent with the role of the NH2-terminal region in membrane trafficking of NaV1.1.

Twenty million Americans suffer from peripheral nerve injury caused by trauma and medical disorders, resulting in a broad spectrum of potentially debilitating side effects. In one out of four cases, patients identify surgery as the root cause of their nerve injury. Particularly during tumor resections or after traumatic injuries, tissue distortion and poor visibility can challenge a surgeon's ability to precisely locate and preserve peripheral nerves. Intuitively, surgical outcomes would improve tremendously if nerves could be highlighted using an exogeneous contrast agent. In clinical practice, however, the current standard of care-visual examination and palpation-remains unchanged. To address this unmet clinical need, we explored the expression of voltage-gated sodium channel Nav1.7 as an intraoperative marker for the peripheral nervous system. We show that expression of Nav1.7 is high in peripheral nerves harvested from both human and mouse tissue. We further show that modification of a Nav1.7-selective peptide, Hsp1a, can serve as a targeted vector for delivering a fluorescent sensor to the peripheral nervous system. Ex vivo, we observe a high signal-to-noise ratio for fluorescently labeled Hsp1a in both histologically prepared and fresh tissue. Using a surgical fluorescent microscope, we show in a simulated clinical scenario that the identification of mouse sciatic nerves is possible, suggesting that fluorescently labeled Hsp1a tracers could be used to discriminate nerves from their surrounding tissues in a routine clinical setting.

Hyperexcitability and alterations in neuronal networks contribute to cognitive impairment in Alzheimer’s Disease (AD). Voltage-gated sodium channels (NaV), which are crucial for regulating neuronal excitability, have been implicated in AD-related hippocampal hyperactivity and higher incidence of spontaneous non-convulsive seizures. Here, we show by using primary hippocampal neurons exposed to amyloid-β1–42 (Aβ1–42) oligomers and from Tg2576 mouse embryos, that the selective upregulation of NaV1.6 subtype contributes to membrane depolarization and to the increase of spike frequency, thereby resulting in neuronal hyperexcitability. Interestingly, we also found that NaV1.6 overexpression is responsible for the aberrant neuronal activity observed in hippocampal slices from 3-month-old Tg2576 mice. These findings identify the NaV1.6 channels as a determinant of the hippocampal neuronal hyperexcitability induced by Aβ1–42 oligomers. The selective blockade of NaV1.6 overexpression and/or hyperactivity might therefore offer a new potential therapeutic approach to counteract early hippocampal hyperexcitability and subsequent cognitive deficits in the early stages of AD.

Summary It is well known that nuclear factor-kappaB (NF-κB) regulates neuronal structures and functions by nuclear transcription. Here, we showed that phospho-p65 (p-p65), an active form of NF-κB subunit, reversibly interacted with Nav1.7 channels in the membrane of dorsal root ganglion (DRG) neurons of rats. The interaction increased Nav1.7 currents by slowing inactivation of Nav1.7 channels and facilitating their recovery from inactivation, which may increase the resting state of the channels ready for activation. In cultured DRG neurons TNF-α upregulated the membrane p-p65 and enhanced Nav1.7 currents within 5 min but did not affect nuclear NF-κB within 40 min. This non-transcriptional effect on Nav1.7 may underlie a rapid regulation of the sensibility of the somatosensory system. Both NF-κB and Nav1.7 channels are critically implicated in many physiological functions and diseases. Our finding may shed new light on the investigation into the underlying mechanisms., Graphical Abstract, Highlights • NF-κB p-p65 interacts with Nav1.7 in the membrane of DRG neurons • The interaction is reversible, depending on the cytoplasmic p-p65 content • Reducing cytoplasmic p-p65 rapidly attenuates the interaction and Nav1.7 currents • The rapid effect on Nav1.7 channels is independent of p-p65 nuclear translocation, Biological Sciences; Neuroscience; Molecular Neuroscience; Cellular Neuroscience

Dravet Syndrome is a severe childhood epileptic disorder caused by haploinsufficiency of the SCN1A gene encoding brain voltage-gated sodium channel NaV1.1. Symptoms include treatment-refractory epilepsy, cognitive impairment, autistic-like behavior, and premature death. The specific loci of NaV1.1 function in the brain that underlie these global deficits remain unknown. Here we specifically deleted Scn1a in the hippocampus using the Cre-Lox method in weanling mice. Local gene deletion caused selective reduction of inhibitory neurotransmission measured in dentate granule cells. Mice with local NaV1.1 reduction had thermally evoked seizures and spatial learning deficits, but they did not have abnormalities of locomotor activity or social interaction. Our results show that local gene deletion in the hippocampus can induce two of the most severe dysfunctions of Dravet Syndrome: Epilepsy and cognitive deficit. Considering these results, the hippocampus may be a potential target for future gene therapy for Dravet Syndrome.