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Voltage-gated sodium channel subtypes, Nav1.7, Nav1.8, and Nav1.9 are predominantly expressed in peripheral sensory neurons. Recent genetic studies have revealed that they are involved in pathological pain processing and that the blockade of Nav1.7, Nav1.8, or Nav1.9 will become a promising pharmacotherapy especially for neuropathic pain. A growing number of drug discovery programs have targeted either of the subtypes to obtain a selective inhibitor which can provide pain relief without affecting the cardiovascular and central nervous systems, though none of them has been approved yet. Here we describe the in vitro characteristics of ANP-230, a novel sodium channel blocker under clinical development. Surprisingly, ANP-230 was shown to block three pain-related subtypes, human Nav1.7, Nav1.8, and Nav1.9 with similar potency, but had only low inhibitory activity to human cardiac Nav1.5 channel and rat central Nav channels. The voltage clamp experiments using different step pulse protocols revealed that ANP-230 had a "tonic block" mode of action without state- and use-dependency. In addition, ANP-230 caused a depolarizing shift of the activation curve and decelerated gating kinetics in human Nav1.7-stably expressing cells. The depolarizing shift of activation curve was commonly observed in human Nav1.8-stably expressing cells as well as rat dorsal root ganglion neurons. These data suggested a quite unique mechanism of Nav channel inhibition by ANP-230. Finally, ANP-230 reduced excitability of rat dorsal root ganglion neurons in a concentration dependent manner. Collectively, these promising results indicate that ANP-230 could be a potent drug for neuropathic pain., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Tatsuya Kamei reports financial support was provided by Sumitomo Pharma Co Ltd. Takehiro Kudo reports financial support was provided by Sumitomo Pharma Co Ltd. Hana Yamane reports financial support was provided by Sumitomo Pharma Co Ltd. Fumiaki Ishibashi reports financial support was provided by Sumitomo Pharma Co Ltd. Yoshinori Takada reports financial support was provided by Sumitomo Pharma Co Ltd. Shigeyuki Honda reports financial support was provided by Sumitomo Pharma Co Ltd. Yasuyo Maezawa reports financial support was provided by Sumitomo Pharma Co Ltd. Kazuhito Ikeda reports financial support was provided by Sumitomo Pharma Co Ltd. Yoshihiro Oyamada reports financial support was provided by Sumitomo Pharma Co Ltd. Yoshihiro Oyamada reports a relationship with AlphaNavi Pharma Inc that includes: board membership. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The pathogenesis of major depressive disorder (MDD) involves lipid metabolism. Our earlier research also revealed that MDD patients had much lower total cholesterol (TC) concentrations than healthy controls (HCs). However, it is still unclear why TC decreased in MDD. Here, based on the Ingenuity Knowledge Base's ingenuity pathway analysis, we found that sodium voltage-gated channel alpha subunit 11A (SCN11A) might serve as a link between low lipid levels and MDD. We analyzed the TC levels and used ELISA kits to measure the levels of SCN11A in the serum from 139 MDD patients, and 65 HCs to confirm this theory and explore the potential involvement of SCN11A in MDD. The findings revealed that TC levels were considerably lower and SCN11A levels were remarkably increased in MDD patients than those in HCs, while they were significantly reversed in drug-treatment MDD patients than in drug-naïve MDD patients. There was no significant difference in SCN11A levels among MDD patients who used single or multiple antidepressants, and selective serotonin reuptake inhibitors or other antidepressants. Pearson correlation analysis showed that the levels of TC and SCN11A were linked with the Hamilton Depression Rating Scales score. A substantial association was also found between TC and SCN11A. Moreover, a discriminative model made up of SCN11A was discovered, which produced an area under a curve of 0.9571 in the training set and 0.9357 in the testing set. Taken together, our findings indicated that SCN11A may serve as a link between low lipid levels and MDD, and showed promise as a candidate biomarker for MDD., (© 2024. The Author(s).)

Skeletal muscle contraction triggers the exercise pressor reflex (EPR) to regulate the cardiovascular system response to exercise. During muscle contraction, substances are released that generate action potential activity in group III and IV afferents that mediate the EPR. Some of these substances increase afferent activity via G-protein-coupled receptor (GPCR) activation, but the mechanisms are incompletely understood. We were interested in determining if tetrodotoxin-resistant (TTX-R) voltage-dependent sodium channels (Na V ) were involved and investigated the effect of a mixture of such compounds (bradykinin, prostaglandin, norepinephrine, and ATP, called muscle metabolites). Using whole cell patch-clamp electrophysiology, we show that the muscle metabolites significantly increased TTX-R Na V currents. The rise time of this enhancement averaged ∼2 min, which suggests the involvement of a diffusible second messenger pathway. The effect of muscle metabolites on the current-voltage relationship, channel activation and inactivation kinetics support Na V 1.9 channels as the target for this enhancement. When applied individually at the concentration used in the mixture, only prostaglandin and bradykinin significantly enhanced Na V current, but the sum of these enhancements was <1/3 that observed when the muscle metabolites were applied together. This suggests synergism between the activated GPCRs to enhance Na V 1.9 current. When applied at a higher concentration, all four substances could enhance the current, which demonstrates that the GPCRs activated by each metabolite can enhance channel activity. The enhancement of Na V 1.9 channel activity is a likely mechanism by which GPCR activation increases action potential activity in afferents generating the EPR. NEW & NOTEWORTHY G-protein-coupled receptor (GPCR) activation increases action potential activity in muscle afferents to produce the exercise pressor reflex (EPR), but the mechanisms are incompletely understood. We provide evidence that Na V 1.9 current is synergistically enhanced by application of a mixture of metabolites potentially released during muscle contraction. The enhancement of Na V 1.9 current is likely one mechanism by which GPCR activation generates the EPR and the inappropriate activation of the EPR in patients with cardiovascular disease.

Mesenchyme homeobox protein 2 (MEOX2) is a transcription factor involved in mesoderm differentiation, including development of bones, muscles, vasculature and dermatomes. We have previously identified dysregulation of MEOX2 in fibroblasts from Congenital Insensitivity to Pain patients, and confirmed that btn, the Drosophila homologue of MEOX2, plays a role in nocifensive responses to noxious heat stimuli. To determine the importance of MEOX2 in the mammalian peripheral nervous system, we used a Meox2 heterozygous (Meox2 +/- ) mouse model to characterise its function in the sensory nervous system, and more specifically, in nociception. MEOX2 is expressed in the mouse dorsal root ganglia (DRG) and spinal cord, and localises in the nuclei of a subset of sensory neurons. Functional studies of the mouse model, including behavioural, cellular and electrophysiological analyses, showed altered nociception encompassing impaired action potential initiation upon depolarisation. Mechanistically, we noted decreased expression of Scn9a and Scn11a genes encoding Na v 1.7 and Na v 1.9 voltage-gated sodium channels respectively, that are crucial in subthreshold amplification and action potential initiation in nociceptors. Further transcriptomic analyses of Meox2 +/- DRG revealed downregulation of a specific subset of genes including those previously associated with pain perception, such as PENK and NPY. Based on these observations, we propose a novel role of MEOX2 in primary afferent nociceptor neurons for the maintenance of a transcriptional programme required for proper perception of acute and inflammatory noxious stimuli., (© 2022 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The aim of the present study was to observe the changes of TTX-R, Na v 1.8, and Na v 1.9 Na + currents in MSU-induced gouty arthritis mice, and to explore the possibility of Na v 1.8 and Na v 1.9 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, Na v 1.8 current density was significantly increased, but Na v 1.9 current density was markedly decreased after MSU treatment. MSU treatment shifted the steady-state activation curves of TTX-R Na + channel, Na v 1.8 and Na v 1.9 channels, and the inactivation curves of TTX-R Na + channel and Na v 1.8 channels to the depolarizing direction, but did not affect the inactivation curve of Na v 1.9 channel. Compared with the normal group, the recovery of Na v 1.8 channel was faster, while that of Na v 1.9 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. Na v 1.8 channel, not Na v 1.9 channel, may be involved in MSU-induced gout pain by increasing nerve excitability., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC part of Springer Nature.)

The primary studies have shown that scorpion analgesic peptide N58A has a significant effect on voltage-gated sodium channels (VGSCs) and plays an important role in neuropathic pain. The purpose of this study was to investigate the analgesic effect of N58A on trigeminal neuralgia (TN) and its possible mechanism. The results showed that N58A could significantly increase the threshold of mechanical pain and thermal pain and inhibit the spontaneous asymmetric scratching behavior of rats. Western blotting results showed that N58A could significantly reduce the protein phosphorylation level of ERK1/2, P38, JNK, and ERK5/CREB pathways and the expression of Nav1.8 and Nav1.9 proteins in a dose-dependent manner. The changes in current and kinetic characteristics of Nav1.8 and Nav1.9 channels in TG neurons were detected by the whole-cell patch clamp technique. The results showed that N58A significantly decreased the current density of Nav1.8 and Nav1.9 in model rats, and shifted the activation curve to hyperpolarization and the inactivation curve to depolarization. In conclusion, the analgesic effect of N58A on the chronic constriction injury of the infraorbital (IoN-CCI) model rats may be closely related to the regulation of the MAPK pathway and Nav1.8 and Nav1.9 sodium channels.

Mutations in the genes encoding for voltage-gated sodium channels cause profound sensory disturbances and other symptoms dependent on the distribution of a particular channel subtype in different organs. Humans with the gain-of-function mutation p.Leu811Pro in SCN11A (encoding for the voltage-gated Nav1.9 channel) exhibit congenital insensitivity to pain, pruritus, self-inflicted injuries, slow healing wounds, muscle weakness, Charcot-like arthropathies, and intestinal dysmotility. As already shown, knock-in mice (Scn11a+/L799P) carrying the orthologous mutation p.Leu799Pro replicate reduced pain sensitivity and show frequent tissue lesions. In the present study we explored whether Scn11a+/L799P mice develop also pruritus, muscle weakness, and changes in gastrointestinal transit time. Furthermore, we analyzed morphological and functional differences in nerves, skeletal muscle, joints and small intestine from Scn11a+/L799P and Scn11a+/+ wild type mice. Compared to Scn11a+/+ mice, Scn11a+/L799P mice showed enhanced scratching bouts before skin lesions developed, indicating pruritus. Scn11a+/L799P mice exhibited reduced grip strength, but no disturbances in motor coordination. Skeletal muscle fiber types and joint architecture were unaltered in Scn11a+/L799P mice. Their gastrointestinal transit time was unaltered. The small intestine from Scn11a+/L799P showed a small shift towards less frequent peristaltic movements. Similar proportions of lumbar dorsal root ganglion neurons from Scn11a+/L799P and Scn11a+/+ mice were calcitonin gene-related peptide (CGRP-) positive, but isolated sciatic nerves from Scn11a+/L799P mice exhibited a significant reduction of the capsaicin-evoked release of CGRP indicating reduced neurogenic inflammation. These data indicate important Nav1.9 channel functions in several organs in both humans and mice. They support the pathophysiological relevance of increased basal activity of Nav1.9 channels for sensory abnormalities (pain and itch) and suggest resulting malfunctions of the motor system and of the gastrointestinal tract. Scn11a+/L799P mice are suitable to investigate the role of Nav1.9, and to explore the pathophysiological changes and mechanisms which develop as a consequence of Nav1.9 hyperactivity., Competing Interests: Matthias Ebbinghaus was funded by Charles River Discovery Research Services Germany GmbH after the experimental part of this study was finished. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

The sodium channels Na v 1.7, Na v 1.8 and Na v 1.9 are critical for pain perception in peripheral nociceptors. Loss of function of Na v 1.7 leads to congenital insensitivity to pain in humans. Here we show that the spider peptide toxin called HpTx1, first identified as an inhibitor of K v 4.2, restores nociception in Na v 1.7 knockout (Na v 1.7-KO) mice by enhancing the excitability of dorsal root ganglion neurons. HpTx1 inhibits Na v 1.7 and activates Na v 1.9 but does not affect Na v 1.8. This toxin produces pain in wild-type (WT) and Na v 1.7-KO mice, and attenuates nociception in Na v 1.9-KO mice, but has no effect in Na v 1.8-KO mice. These data indicate that HpTx1-induced hypersensitivity is mediated by Na v 1.9 activation and offers pharmacological insight into the relationship of the three Na v channels in pain signalling.

Genetic and functional studies have confirmed an important role for the voltage-gated sodium channel Nav1.9 in human pain disorders. However, low functional expression of Nav1.9 in heterologous systems ( e.g. in human embryonic kidney 293 (HEK293) cells) has hampered studies of its biophysical and pharmacological properties and the development of high-throughput assays for drug development targeting this channel. The mechanistic basis for the low level of Nav1.9 currents in heterologous expression systems is not understood. Here, we implemented a multidisciplinary approach to investigate the mechanisms that govern functional Nav1.9 expression. Recombinant expression of a series of Nav1.9-Nav1.7 C-terminal chimeras in HEK293 cells identified a 49-amino-acid-long motif in the C terminus of the two channels that regulates expression levels of these chimeras. We confirmed the critical role of this motif in the context of a full-length channel chimera, Nav1.9-Ct49aa Nav1.7 , which displayed significantly increased current density in HEK293 cells while largely retaining the characteristic Nav1.9-gating properties. High-resolution live microscopy indicated that the newly identified C-terminal motif dramatically increases the number of channels on the plasma membrane of HEK293 cells. Molecular modeling results suggested that this motif is exposed on the cytoplasmic face of the folded C terminus, where it might interact with other channel partners. These findings reveal that a 49-residue-long motif in Nav1.9 regulates channel trafficking to the plasma membrane.

Previous studies have found that increased expression of Nav1.9 and protein kinase C (PKC) contributes to pain hypersensitivity in a couple of inflammatory pain models. Here we want to observe if PKC can regulate the expression of Nav1.9 in dorsal root ganglion (DRG) in rheumatoid arthritis (RA) pain model. A chronic knee joint inflammation model was produced by intra-articular injection of the complete Freund's adjuvant (CFA) in rats. Nociceptive behaviors including mechanical, cold, and heat hyperalgesia were examined. The expression of Nav1.9 and PKCα in DRG was detected by a quantitative polymerase chain reaction, Western blot, and immunofluorescence. The in vitro and in vivo effects of a PKC activator (phorbol 12-myristate 13-acetate [PMA]) and a PKC inhibitor (GF-109203X) on the expression of Nav1.9 were examined. Moreover, the effects of PKC modulators on nociceptive behaviors were studied. Increased mechanical, heat, and cold sensitivity was observed 3 to 14 days after CFA injection. Parallel increases in messenger RNA and protein expression of Nav1.9 and PKCα were found. Immunofluorescence experiments found that Nav1.9 was preferentially colocalized with IB4+DRG neurons in RA rats. In cultured DRG neurons, PMA increased Nav1.9 expression while GF-109203X prevented the effect of PMA. PMA increased Nav1.9 expression in naïve rats while GF-109203X decreased Nav1.9 expression in RA rats. In naïve rats, PMA caused mechanical and cold hyperalgesia. On the other hand, GF-109203X attenuated mechanical and cold hyperalgesia in RA-pain model. Nav1.9 might be upregulated by PKCα in DRG, which contributes to pain hypersensitivity in CFA-induced chronic knee joint inflammation model of RA pain., (© 2019 Wiley Periodicals, Inc.)

Inflammatory monocyte and macrophage subset accumulation during the inflammatory response that drives atherosclerosis can exacerbate the extent of atherosclerosis. It has been demonstrated that voltage-gated sodium channels (VGSCs) can regulate cell bioactivities in monocytes/macrophages. We hypothesized that blockade of mononuclear phagocyte VGSCs was atheroprotective through monocyte/macrophage subset modulation and macrophage proliferation suppression in atherosclerotic lesions. In this experimental study, when VGSCs were knocked down with RNA interference plasmid transfection in mouse peripheral blood monocytes and monocyte-macrophage lineage RAW264.7 cells in vitro, the biological characteristics of proliferation, phagocytosis, and migration in RAW264.7 cells declined. In addition, suppression of LPS-induced M1 polarization and facilitation of IL-4-induced M2 polarization were also observed. In an in vivo study, ApoE knockout (ApoE -/- ) mice were fed a standard chow diet (CD) or a western diet (WD). After feeding with phenytoin (PHT), no significant differences were detected in plasma lipids, and the anti-inflammatory phenotypes of both monocytes and macrophages were elevated and proinflammatory phenotypes declined. The local proliferation of macrophages was also distinctly suppressed, along with a significant reduction in atheromatous plaques. In conclusion, blockade of VGSCs in the mononuclear phagocyte system reduced atherosclerotic lesions, which may occur through altering monocyte/macrophage subsets and suppressing macrophage proliferation in atherosclerotic plaques. Blockage of VGSCs may play an important role in cardiovascular protection., (Copyright © 2019 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Medication-overuse headaches (MOH) occur with both over-the-counter and pain-relief medicines, including paracetamol, opioids and combination analgesics. The mechanisms that lead to MOH are still uncertain. Here, we show that abnormal activation of Nav1.9 channels by Nitric Oxide (NO) is responsible for MOH induced by triptan migraine medicine. Deletion of the Scn11a gene in MOH mice abrogates NO-mediated symptoms, including cephalic and extracephalic allodynia, photophobia and phonophobia. NO strongly activates Nav1.9 in dural afferent neurons from MOH but not normal mice. Abnormal activation of Nav1.9 triggers CGRP secretion, causing artery dilatation and degranulation of mast cells. In turn, released mast cell mediators potentiates Nav1.9 in meningeal nociceptors, exacerbating inflammation and pain signal. Analysis of signaling networks indicates that PKA is downregulated in trigeminal neurons from MOH mice, relieving its inhibitory action on NO-Nav1.9 coupling. Thus, anomalous activation of Nav1.9 channels by NO, as a result of chronic medication, promotes MOH.

Itch (pruritis) and pain represent two distinct sensory modalities; yet both have evolved to alert us to potentially harmful external stimuli. Compared with pain, our understanding of itch is still nascent. Here, we report a new clinical case of debilitating itch and altered pain perception resulting from the heterozygous de novo p.L811P gain-of-function mutation in NaV1.9, a voltage-gated sodium (NaV) channel subtype that relays sensory information from the periphery to the spine. To investigate the role of NaV1.9 in itch, we developed a mouse line in which the channel is N-terminally tagged with a fluorescent protein, thereby enabling the reliable identification and biophysical characterization of NaV1.9-expressing neurons. We also assessed NaV1.9 involvement in itch by using a newly created NaV1.9-/- and NaV1.9L799P/WT mouse model. We found that NaV1.9 is expressed in a subset of nonmyelinated, nonpeptidergic small-diameter dorsal root ganglia (DRGs). In WT DRGs, but not those of NaV1.9-/- mice, pruritogens altered action potential parameters and NaV channel gating properties. Additionally, NaV1.9-/- mice exhibited a strong reduction in acute scratching behavior in response to pruritogens, whereas NaV1.9L799P/WT mice displayed increased spontaneous scratching. Altogether, our data suggest an important contribution of NaV1.9 to itch signaling.

Background: Oxaliplatin-induced cold allodynia is a frequent complication appearing in patients treated with this anti-tumor drug. Since, there are no clear algorithms to overcome this painful condition effectively, it is important to establish novel strategies for its treatment., Aim: In this study, the ability of pregabalin and ambroxol, used as single drugs or in combinations administered in a time-shifted manner to attenuate cold allodynia was assessed in the mouse cold plate test. The hot plate test was additionally used to assess antinociceptive properties of ambroxol in the acute, thermally-induced pain model. Locomotor activity and motor coordination of mice were also evaluated. In silico studies were undertaken to predict potential binding of ambroxol to sodium channel (Na v ) subtypes whose overexpression is implicated in the development of oxaliplatin-induced neuropathic pain., Key Findings: A hyperadditive antiallodynic effect of combined sub-analgesic ambroxol and pregabalin was demonstrated in oxaliplatin-treated mice. This effect was particularly strong when these drugs were given 4 h apart. Both drugs used in combination reduced animals' locomotor activity, but they did not impair motor coordination in the rotarod test. Ambroxol did not show antinociceptive properties in the hot plate test. The molecular docking studies predicted that in mice ambroxol might bind to Na v 1.6 and Na v 1.9 rather than Na v 1.7 and Na v 1.8., Significance: Time-shifted co-administration of sub-analgesic doses of ambroxol and pregabalin effectively attenuates oxaliplatin-induced cold allodynia. Molecular docking model predicts preferential binding of ambroxol to mouse Na v 1.6, Na v 1.9 channels. This mechanism, if confirmed in vitro, might explain pharmacological activities observed in vivo., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

The exercise pressor reflex (EPR) is activated by muscle contractions to increase heart rate and blood pressure during exercise. While this reflex is beneficial in healthy individuals, the reflex activity is exaggerated in patients with cardiovascular disease, which is associated with increased mortality. Group III and IV afferents mediate the EPR and have been shown to express both tetrodotoxin-sensitive (TTX-S, Na V 1.6, and Na V 1.7) and -resistant (TTX-R, Na V 1.8, and Na V 1.9) voltage-gated sodium (Na V ) channels, but Na V 1.9 current has not yet been demonstrated. Using a F - -containing internal solution, we found a Na V current in muscle afferent neurons that activates at around -70 mV with slow activation and inactivation kinetics, as expected from Na V 1.9 current. However, this current ran down with time, which resulted, at least in part, from increased steady-state inactivation since it was slowed by both holding potential hyperpolarization and a depolarized shift of the gating properties. We further show that, following Na V 1.9 current rundown (internal F - ), application of the Na V 1.8 channel blocker A803467 inhibited significantly more TTX-R current than we had previously observed (internal Cl - ), which suggests that Na V 1.9 current did not rundown with that internal solution. Using immunohistochemistry, we found that the majority of group IV somata and axons were Na V 1.9 positive. The majority of small diameter myelinated afferent somata (putative group III) were also Na V 1.9 positive, but myelinated muscle afferent axons were rarely labeled. The presence of Na V 1.9 channels in muscle afferents supports a role for these channels in activation and maintenance of the EPR. NEW & NOTEWORTHY Small diameter muscle afferents signal pain and muscle activity levels. The muscle activity signals drive the cardiovascular system to increase muscle blood flow, but these signals can become exaggerated in cardiovascular disease to exacerbate cardiac damage. The voltage-dependent sodium channel Na V 1.9 plays a unique role in controlling afferent excitability. We show that Na V 1.9 channels are expressed in muscle afferents, which supports these channels as a target for drug development to control hyperactivity of these neurons.

Damage-sensing nociceptors in the skin provide an indispensable protective function thanks to their specialized ability to detect and transmit hot temperatures that would block or inflict irreversible damage in other mammalian neurons. Here we show that the exceptional capacity of skin C-fiber nociceptors to encode noxiously hot temperatures depends on two tetrodotoxin (TTX)-resistant sodium channel α-subunits: Na V 1.8 and Na V 1.9. We demonstrate that Na V 1.9, which is commonly considered an amplifier of subthreshold depolarizations at 20°C, undergoes a large gain of function when temperatures rise to the pain threshold. We also show that this gain of function renders Na V 1.9 capable of generating action potentials with a clear inflection point and positive overshoot. In the skin, heat-resistant nociceptors appear as two distinct types with unique and possibly specialized features: one is blocked by TTX and relies on Na V 1.9, and the second type is insensitive to TTX and composed of both Na V 1.8 and Na V 1.9. Independent of rapidly gated TTX-sensitive Na V channels that form the action potential at pain threshold, Na V 1.8 is required in all heat-resistant nociceptors to encode temperatures higher than ∼46°C, whereas Na V 1.9 is crucial for shaping the action potential upstroke and keeping the Na V 1.8 voltage threshold within reach., (© 2018 Touska et al.)

Many veterans of Operation Desert Storm (ODS) struggle with the chronic pain of Gulf War Illness (GWI). Exposure to insecticides and pyridostigmine bromide (PB) have been implicated in the etiology of this multisymptom disease. We examined the influence of 3 (DEET (N,N-diethyl-meta-toluamide), permethrin, chlorpyrifos) or 4 GW agents (DEET, permethrin, chlorpyrifos, pyridostigmine bromide (PB)) on the post-exposure ambulatory and resting behaviors of rats. In three independent studies, rats that were exposed to all 4 agents consistently developed both immediate and delayed ambulatory deficits that persisted at least 16 weeks after exposures had ceased. Rats exposed to a 3 agent protocol (PB excluded) did not develop any ambulatory deficits. Cellular and molecular studies on nociceptors harvested from 16WP (weeks post-exposure) rats indicated that vascular nociceptor Na v 1.9 mediated currents were chronically potentiated following the 4 agent protocol but not following the 3 agent protocol. Muscarinic linkages to muscle nociceptor TRPA1 were also potentiated in the 4 agent but not the 3 agent, PB excluded, protocol. Although K v 7 activity changes diverged from the behavioral data, a K v 7 opener, retigabine, transiently reversed ambulation deficits. We concluded that PB played a critical role in the development of pain-like signs in a GWI rat model and that shifts in Na v 1.9 and TRPA1 activity were critical to the expression of these pain behaviors., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The aim of the present study was to identify whether the sodium voltage-gated channel alpha subunit 9 (SCN9A) gene modification is a potential treatment for diarrhea‑predominant irritable bowel syndrome (D‑IBS), via regulating the Na+ channel and the expression of nerve growth factor (NGF). The recombinant adenovirus vector of the SCN9A gene was established, and rat colon cells were isolated for SCN9A gene modification. All subjects were divided into four groups: i) The SCN9A‑modified (D‑IBS rat model implanted with SCN9A‑modified colon cells), ii) negative control (NC; D‑IBS rat model implanted with colon cells without SCN9A gene modification), iii) blank (D‑IBS rat model without any treatment) and iv) normal (normal rats without any treatment). Western blotting and reverse transcription‑quantitative polymerase chain reaction were used to detect the protein and mRNA expression levels of SCN9A, NGF and voltage gated sodium channels (Nav)1.8 and Nav1.9 in rat colon tissues. Compared with the normal group, the rats in the SCN9A, NC and blank groups had significantly elevated mRNA and protein expression levels of NGF, SCN9A, Nav1.8 and Nav1.9. The rats in the SCN9A group demonstrated significantly increased mRNA and protein expression levels of NGF, SCN9A, Nav1.8 and Nav1.9 compared with the NC group and the blank group (all P<0.05). SCN9A gene modification can promote the expression of Nav1.8 and Nav1.9 channels, in addition to NGF which may provide a novel therapeutic basis for treating of D‑IBS.

Developmental changes that occur in the prefrontal cortex during adolescence alter behavior. These behavioral alterations likely stem from changes in prefrontal cortex neuronal activity, which may depend on the properties and expression of ion channels. Nav1.9 sodium channels conduct a Na + current that is TTX resistant with a low threshold and noninactivating over time. The purpose of this study was to assess the presence of Nav1.9 channels in medial prefrontal cortex (mPFC) layer II and V pyramidal neurons in young (20-day old), late adolescent (60-day old), and adult (6- to 7-month old) rats. First, we demonstrated that layer II and V mPFC pyramidal neurons in slices obtained from young rats exhibited a TTX-resistant, low-threshold, noninactivating, and voltage-dependent Na + current. The mRNA expression of the SCN11a gene (which encodes the Nav1.9 channel) in mPFC tissue was significantly higher in young rats than in late adolescent and adult rats. Nav1.9 protein was immunofluorescently labeled in mPFC cells in slices and analyzed via confocal microscopy. Nav1.9 immunolabeling was present in layer II and V mPFC pyramidal neurons and was more prominent in the neurons of young rats than in the neurons of late adolescent and adult rats. We conclude that Nav1.9 channels are expressed in layer II and V mPFC pyramidal neurons and that Nav1.9 protein expression in the mPFC pyramidal neurons of late adolescent and adult rats is lower than that in the neurons of young rats. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1371-1384, 2017., (© 2017 Wiley Periodicals, Inc.)

The goal of these studies was to investigate the links between chronic exposure to the pro-inflammatory cytokine tumor necrosis factor (TNF), hyperalgesia and the excitability of dorsal root ganglion (DRG) sensory neurons. We employed transgenic mice that constitutively express TNF (TNFtg mice), a well-established model of chronic systemic inflammation. At 6 months of age, TNFtg mice demonstrated increased sensitivity to both mechanical and thermal heat stimulation relative to aged-matched wild-type controls. These increases in stimulus-evoked behaviors are consistent with nociceptor sensitization to normal physiological stimulation. The mechanisms underlying nociceptor sensitization were investigated using single-cell analysis to quantitatively compare gene expression in small-diameter (<30μm) DRG neurons. This analysis revealed the upregulation of mRNA encoding for tetrodotoxin-resistant (TTX-R) sodium (Na + ) channels (Nav1.8, Nav1.9), Na + channel β subunits (β 1 -β 3 ), TNF receptor 1 (TNFR1) and p38α mitogen-activated protein kinase in neurons of TNFtg mice. Whole-cell electrophysiology demonstrated a corresponding increase in TTX-R Na + current density, hyperpolarizing shifts in activation and steady-state inactivation, and slower recovery from inactivation in the TNFtg neurons. Increased overlap of activation and inactivation in the TNFtg neurons produces inward Na + currents at voltages near the resting membrane potential of sensory neurons (i.e. window currents). The combination of increased Na + current amplitude, hyperpolarized shifts in Na + channel activation and increased window current predicts a reduction in the action potential threshold and increased firing of small-diameter DRG neurons. Together, these data suggest that increases in the expression of Nav1.8 channels, regulatory β 1 subunits and TNFR1 contribute to increased nociceptor excitability and hyperalgesia in the TNFtg mice., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Voltage-gated sodium channel (NaV) mutations cause genetic pain disorders that range from severe paroxysmal pain to a congenital inability to sense pain. Previous studies on NaV1.7 and NaV1.8 established clear relationships between perturbations in channel function and divergent clinical phenotypes. By contrast, studies of NaV1.9 mutations have not revealed a clear relationship of channel dysfunction with the associated and contrasting clinical phenotypes. Here, we have elucidated the functional consequences of a NaV1.9 mutation (L1302F) that is associated with insensitivity to pain. We investigated the effects of L1302F and a previously reported mutation (L811P) on neuronal excitability. In transfected heterologous cells, the L1302F mutation caused a large hyperpolarizing shift in the voltage-dependence of activation, leading to substantially enhanced overlap between activation and steady-state inactivation relationships. In transfected small rat dorsal root ganglion neurons, expression of L1302F and L811P evoked large depolarizations of the resting membrane potential and impaired action potential generation. Therefore, our findings implicate a cellular loss of function as the basis for impaired pain sensation. We further demonstrated that a U-shaped relationship between the resting potential and the neuronal action potential threshold explains why NaV1.9 mutations that evoke small degrees of membrane depolarization cause hyperexcitability and familial episodic pain disorder or painful neuropathy, while mutations evoking larger membrane depolarizations cause hypoexcitability and insensitivity to pain.

Sequestration of nerve growth factor has been used successfully in the management of pain in animal models of bone disease and in human osteoarthritis. However, the mechanisms of nerve growth factor-induced bone pain and its role in modulating inflammatory bone pain remain to be determined. In this study, we show that nerve growth factor receptors (TrkA and p75) and some other nerve growth factor-signaling molecules (TRPV1 and Nav1.8, but not Nav1.9) are expressed in substantial proportions of rat bone nociceptors. We demonstrate that nerve growth factor injected directly into rat tibia rapidly activates and sensitizes bone nociceptors and produces acute behavioral responses with a similar time course. The nerve growth factor-induced changes in the activity and sensitivity of bone nociceptors we report are dependent on signaling through the TrkA receptor, but are not affected by mast cell stabilization. We failed to show evidence for longer term changes in expression of TrkA, TRPV1, Nav1.8 or Nav1.9 in the soma of bone nociceptors in a rat model of inflammatory bone pain. Thus, retrograde transport of NGF/TrkA and increased expression of some of the common nerve growth factor signaling molecules do not appear to be important for the maintenance of inflammatory bone pain. The findings are relevant to understand the basis of nerve growth factor sequestration and other therapies directed at nerve growth factor signaling, in managing pain in bone disease.

Aim: Voltage-gated sodium channel subtype 9 (Nav1.9) are expressed in dorsal root ganglion neurons and are known to be involved in pain during inflammation. Animal studies have reported Nav1.9 channel expression in myenteric intrinsic primary afferent neurons (IPANs). More recently, a study involving Nav1.9 knockout mice showed clear evidence of colonic dysmotility. However, there are no data regarding the expression of these channels in the human intestine, thus, the aim of our study was to determine Nav1.9 channel expression within the human colon and to elucidate if Nav1.9 channel expression is altered in Hirschsprung's disease (HD)., Methods: HD tissue specimens (n=10) were collected at the time of pull-through surgery, while normal controls were obtained at the time of colostomy closure in patients with imperforate anus (n=10). Nav1.9 immunofluorescence was visualized using confocal microscopy to assess the distribution of the protein. Western blot analysis was undertaken to determine Nav1.9 protein quantification., Results: Confocal microscopy revealed Nav1.9-immunoreactive neurons within the submucosal and myenteric plexus in normal controls, with a reduction in the HD specimens. Calbindin double-labeling showed that Nav1.9-immunoreactive neurons were IPANs. Nav1.9 channels were also seen to be co-localized on smooth muscle cells in all tissues. Western blotting revealed high levels of Nav1.9 protein expression in normal controls, while there was a marked decrease in Nav1.9 protein expression in the HD tissue., Conclusion: Our results show the expression of Nav1.9 channels within the human colon for the first time. Furthermore, Nav1.9 channel expression is decreased in HD versus normal controls., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The voltage dependent sodium channel Nav1.9, is expressed preferentially in peripheral sensory neurons and has been linked to human genetic pain disorders, which makes it target of interest for the development of new pain therapeutics. However, characterization of Nav1.9 pharmacology has been limited due in part to the historical difficulty of functionally expressing recombinant channels. Here we report the successful generation and characterization of human, mouse and rat Nav1.9 stably expressed in human HEK-293 cells. These cells exhibit slowly activating and inactivating inward sodium channel currents that have characteristics of native Nav1.9. Optimal functional expression was achieved by coexpression of Nav1.9 with β1/β2 subunits. While recombinantly expressed Nav1.9 was found to be sensitive to sodium channel inhibitors TC-N 1752 and tetracaine, potency was up to 100-fold less than reported for other Nav channel subtypes despite evidence to support an interaction with the canonical local anesthetic (LA) binding region on Domain 4 S6. Nav1.9 Domain 2 S6 pore domain contains a unique lysine residue (K799) which is predicted to be spatially near the local anesthetic interaction site. Mutation of this residue to the consensus asparagine (K799N) resulted in an increase in potency for tetracaine, but a decrease for TC-N 1752, suggesting that this residue can influence interaction of inhibitors with the Nav1.9 pore. In summary, we have shown that stable functional expression of Nav1.9 in the widely used HEK-293 cells is possible, which opens up opportunities to better understand channel properties and may potentially aid identification of novel Nav1.9 based pharmacotherapies., Competing Interests: The authors have declared that no competing interests exist.

Voltage-gated sodium channels (VGSCs), especially the tetrodotoxin-sensitive Nav1.3 and Nav1.7, and the tetrodotoxin-resistant Nav1.8 and Nav1.9, have been implicated in acute and chronic neuropathic pain. The aim of this study was to investigate the expression of VGSC Nav1.3, Nav1.7, Nav1.8, and Nav1.9 after nerve injury and their roles in the development of trigeminal neuralgia (TN). We used the infraorbital nerve-chronic constriction injury model of TN in the rat. The time course of changes in the mechanical pain threshold was examined. In addition, real-time PCR and double immunofluorescence staining of VGSC α subunits were used to evaluate messenger RNA and protein expression, respectively, in the trigeminal ganglion. Behavioral tests showed that the mechanical pain threshold decreased significantly 4-42 days after surgery and reached the lowest observed value by day 12. Compared with sham-operated controls, we found that trigeminal ganglion in rats subjected to an infraorbital nerve-chronic constriction injury showed upregulation of Nav1.3 and downregulation of Nav1.7, Nav1.8, and Nav1.9 messenger RNA and protein levels. Our findings suggest that VGSC may participate in the regulation of TN.

Painful peripheral neuropathy has been correlated with various voltage-gated sodium channel mutations in sensory neurons. Recently Nav1.9, a voltage-gated sodium channel subtype, has been established as a genetic influence for certain peripheral pain syndromes. In this study, we performed a genetic study in six unrelated multigenerational Japanese families with episodic pain syndrome. Affected participants (n = 23) were characterized by infantile recurrent pain episodes with spontaneous mitigation around adolescence. This unique phenotype was inherited in an autosomal-dominant mode. Linkage analysis was performed for two families with 12 affected and nine unaffected members, and a single locus was identified on 3p22 (LOD score 4.32). Exome analysis (n = 14) was performed for affected and unaffected members in these two families and an additional family. Two missense variants were identified: R222H and R222S in SCN11A. Next, we generated a knock-in mouse model harboring one of the mutations (R222S). Behavioral tests (Hargreaves test and cold plate test) using R222S and wild-type C57BL/6 (WT) mice, young (8-9 weeks old; n = 10-12 for each group) and mature (36-38 weeks old; n = 5-6 for each group), showed that R222S mice were significantly (p < 0.05) more hypersensitive to hot and cold stimuli than WT mice. Electrophysiological studies using dorsal root ganglion neurons from 8-9-week-old mice showed no significant difference in resting membrane potential, but input impedance and firing frequency of evoked action potentials were significantly increased in R222S mice compared with WT mice. However, there was no significant difference among Nav1.9 (WT, R222S, and R222H)-overexpressing ND7/23 cell lines. These results suggest that our novel mutation is a gain-of-function mutation that causes infantile familial episodic pain. The mouse model developed here will be useful for drug screening for familial episodic pain syndrome associated with SCN11A mutations.

Gain-of-function mutations in the human SCN11A-encoded voltage-gated Na(+) channel NaV1.9 cause severe pain disorders ranging from neuropathic pain to congenital pain insensitivity. However, the entire spectrum of the NaV1.9 diseases has yet to be defined. Applying whole-exome sequencing we here identify a missense change (p.V1184A) in NaV1.9, which leads to cold-aggravated peripheral pain in humans. Electrophysiological analysis reveals that p.V1184A shifts the voltage dependence of channel opening to hyperpolarized potentials thereby conferring gain-of-function characteristics to NaV1.9. Mutated channels diminish the resting membrane potential of mouse primary sensory neurons and cause cold-resistant hyperexcitability of nociceptors, suggesting a mechanistic basis for the temperature dependence of the pain phenotype. On the basis of direct comparison of the mutations linked to either cold-aggravated pain or pain insensitivity, we propose a model in which the physiological consequence of a mutation, that is, augmented versus absent pain, is critically dependent on the type of NaV1.9 hyperactivity.

SCN11A encodes the voltage-gated sodium channel NaV1.9, which deviates most strongly from the other eight NaV channels expressed in mammals. It is characterized by resistance to the prototypic NaV channel blocker tetrodotoxin and exhibits slow activation and inactivation gating. Its expression in dorsal root ganglia neurons suggests a role in motor or pain signaling functions as also recently demonstrated by the occurrence of various mutations in human SCN11A leading to altered pain sensation syndromes. The systematic investigation of human NaV1.9, however, is severely hampered because of very poor heterologous expression in host cells. Using patch-clamp and two-electrode voltage-clamp methods, we show that this limitation is caused by the C-terminal structure of NaV1.9. A chimera of NaV1.9 harboring the C terminus of NaV1.4 yields functional expression not only in neuronal cells but also in non-excitable cells, such as HEK 293T or Xenopus oocytes. The major functional difference of the chimeric channel with respect to NaV1.9 is an accelerated activation and inactivation. Since the entire transmembrane domain is preserved, it is suited for studying pharmacological properties of the channel and the functional impact of disease-causing mutations. Moreover, we demonstrate how mutation S360Y makes NaV1.9 channels sensitive to tetrodotoxin and saxitoxin and that the unusual slow open-state inactivation of NaV1.9 is also mediated by the IFM (isoleucine-phenylalanine-methionine) inactivation motif located in the linker connecting domains III and IV.

Damage to the cholinergic input to the prefrontal cortex has been implicated in neuropsychiatric disorders. Cholinergic endings release acetylcholine, which activates nicotinic and/or G-protein-coupled muscarinic receptors. Muscarinic receptors activate transduction systems, which control cellular effectors that regulate the membrane potential in medial prefrontal cortex (mPFC) neurons. The mechanisms responsible for the cholinergic-dependent depolarization of mPFC layer V pyramidal neurons in slices obtained from young rats were elucidated in this study. Glutamatergic and GABAergic transmission as well as tetrodotoxin (TTX)-sensitive Na(+) and voltage-dependent Ca(++) currents were eliminated. Cholinergic receptor stimulation by carbamoylcholine chloride (CCh; 100 μM) evoked depolarization (10.0 ± 1.3 mV), which was blocked by M1/M4 (pirenzepine dihydrochloride, 2 μM) and M1 (VU 0255035, 5 μM) muscarinic receptor antagonists and was not affected by a nicotinic receptor antagonist (mecamylamine hydrochloride, 10 μM). CCh-dependent depolarization was attenuated by extra- (20 μM) or intracellular (50 μM) application of an inhibitor of the βγ-subunit-dependent transduction system (gallein). It was also inhibited by intracellular application of a βγ-subunit-binding peptide (GRK2i, 10μM). mPFC pyramidal neurons express Nav1.9 channels. CCh-dependent depolarization was abolished in the presence of antibodies against Nav1.9 channels in the intracellular solution and augmented by the presence of ProTx-I toxin (100 nM) in the extracellular solution. CCh-induced depolarization was not affected by the following reagents: intracellular transduction system blockers, including U-73122 (10 μM), chelerythrine chloride (5 μM), SQ 22536 (100 μM) and H-89 (2 μM); channel blockers, including Ba(++) ions (200 μM), apamin (100 nM), flufenamic acid (200 μM), 2-APB (200 μM), SKF 96365 (50 μM), and ZD 7288 (50 μM); and a Na(+)/Ca(++) exchanger blocker, benzamil (20 μM). We conclude that muscarinic M1 receptor-dependent depolarization in mPFC pyramidal neurons is evoked by the activation of Nav1.9 channels and that the signal transduction pathway involves G-protein βγ subunits., (Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.)

Neuropathic pain is a global medical concern, characterized by spontaneous pain, heat hyperalgesia and mechanical allodynia. The condition has been associated with alterations in the voltage‑gated sodium channels, Nav1.8 and Nav1.9, in nociceptive neurons termed nociceptors. However, an explanation for the contribution of these channels to the phenotype observed in neuropathic pain remains to be elucidated. The changes induced by chronic constriction injury (CCI) to Nav1.8 and Nav1.9 mRNA and protein levels, as well as electrical currents in injured and contralateral non‑injured dorsal root ganglion (DRG) neurons are described in the present study. A marked downregulation was observed for each Nav isoform transcript and protein expressed in injured neurons with the exception of the Nav1.9 protein, which exhibited no change, while in contralateral non‑injured neurons, the levels of protein and mRNA remained unchanged. Nav isoform functional analysis was then performed in L(4‑6) DRG neurons 14 days after CCI. The Nav1.8 current density was markedly decreased in injured DRG neurons following CCI. The voltage‑dependent activation of the Nav1.8 channel in these neurons was shifted to depolarized potentials by 5.3 mV, while it was shifted to hyperpolarized potentials by 10 mV for inactivation. The electrophysiological function of Nav1.9 was not affected by CCI. The present study demonstrated that ectopic discharge following CCI, which was likely induced by a positive shift in the Nav1.8 current inactivation curve in injured neurons, enhanced the excitability of the neurons by facilitating tetrodotoxin‑resistant sodium channels into the fast inactivation state and did not occur as a result of a compensatory redistribution in the contralateral uninjured neurons.

Cold-triggered pain is essential to avoid prolonged exposure to harmfully low temperatures. However, the molecular basis of noxious cold sensing in mammals is still not completely understood. Here, we show that the voltage-gated Nav1.9 sodium channel is important for the perception of pain in response to noxious cold. Nav1.9 activity is upregulated in a subpopulation of damage-sensing sensory neurons responding to cooling, which allows the channel to amplify subthreshold depolarizations generated by the activation of cold transducers. Consequently, cold-triggered firing is impaired in Nav1.9(-/-) neurons, and Nav1.9 null mice and knockdown rats show increased cold pain thresholds. Disrupting Nav1.9 expression in rodents also alleviates cold pain hypersensitivity induced by the antineoplastic agent oxaliplatin. We conclude that Nav1.9 acts as a subthreshold amplifier in cold-sensitive nociceptive neurons and is required for the perception of cold pain under normal and pathological conditions., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

In the central auditory system, cartwheel cells (CWCs) are a group of interneurons in the dorsal cochlear nucleus (DCN). While other DCN neurons respond to stimuli with a simple discharge pattern of single action potentials (SAPs), CWCs respond with complex action potentials (CAPs), consisting of SAPs superimposed on a slow depolarization. The CAPs in CWCs may participate in various auditory or non‑auditory signaling processing but its intrinsic mechanisms are largely unknown. In the present study, in vitro whole‑cell current clamp recordings on neonatal mice brain slices were used to demonstrate that CWCs respond to brief voltage stimulation with CAPs. Western blotting and immunohistochemistry were also utilized to demonstrate that Nav1.9 was expressed in the CWCs. Finally, when Nav1.9 was genetically silenced, CWCs responded to voltage stimulation with SAPs, not CAPs. The results strongly suggested that Nav1.9 was expressed and functionally contributed to the signaling processing in the central auditory pathway.

Chronic visceral pain affects millions of individuals worldwide and remains poorly understood, with current therapeutic options constrained by gastrointestinal adverse effects. Visceral pain is strongly associated with inflammation and distension of the gut. Here we report that the voltage-gated sodium channel subtype NaV1.9 is expressed in half of gut-projecting rodent dorsal root ganglia sensory neurons. We show that NaV1.9 is required for normal mechanosensation, for direct excitation and for sensitization of mouse colonic afferents by mediators from inflammatory bowel disease tissues, and by noxious inflammatory mediators individually. Excitatory responses to ATP or PGE2 were substantially reduced in NaV1.9(-/-) mice. Deletion of NaV1.9 substantially attenuates excitation and subsequent mechanical hypersensitivity after application of inflammatory soup (IS) (bradykinin, ATP, histamine, PGE2, and 5HT) to visceral nociceptors located in the serosa and mesentery. Responses to mechanical stimulation of mesenteric afferents were also reduced by loss of NaV1.9, and there was a rightward shift in stimulus-response function to ramp colonic distension. By contrast, responses to rapid, high-intensity phasic distension of the colon are initially unaffected; however, run-down of responses to repeat phasic distension were exacerbated in NaV1.9(-/-) afferents. Finally colonic afferent activation by supernatants derived from inflamed human tissue was greatly reduced in NaV1.9(-/-) mice. These results demonstrate that NaV1.9 is required for persistence of responses to intense mechanical stimulation, contributes to inflammatory mechanical hypersensitivity, and is essential for activation by noxious inflammatory mediators, including those from diseased human bowel. These observations indicate that NaV1.9 represents a high-value target for development of visceral analgesics., (Crown Copyright © 2014. Published by Elsevier B.V. All rights reserved.)

Many veterans of the 1991 Gulf War (GW) returned from that conflict with a widespread chronic pain affecting deep tissues. Recently, we have shown that a 60day exposure to the insecticides permethrin, chlorpyrifos, and pyridostigmine bromide (NTPB) had little influence on nociceptor action potential forming Nav1.8, but increased Kv7 mediated inhibitory currents 8weeks after treatment. Using the same exposure regimen, we used whole cell patch methods to examine whether the influences of NTPB could be observed on Nav1.9 expressed in muscle and vascular nociceptors. During a 60day exposure to NTPB, rats exhibited lowered muscle pain thresholds and increased rest periods, but these measures subsequently returned to normal levels. Eight and 12weeks after treatments ceased, DRG neurons were excised from the sensory ganglia. Whole cell patch studies revealed little change in voltage dependent activation and deactivation of Nav1.9, but significant increases in the amplitude of Nav1.9 were observed 8weeks after exposure. Cellular studies, at the 8week delay, revealed that NTPB also significantly prolonged action potential duration and afterhyperpolarization (22°C). Acute application of permethrin (10μM) also increased the amplitude of Nav1.9 in skin, muscle and vascular nociceptors. In conclusion, chronic exposure to Gulf War agents produced long term changes in the amplitude of Nav1.9 expressed in muscle and vascular nociceptors. The reported increases in Kv7 amplitude may have been an adaptive response to increased Nav1.9, and effectively suppressed behavioral pain measures in the post treatment period. Factors that alter the balance between Nav1.9 and Kv7 could release spontaneous discharge and produce chronic deep tissue pain., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Voltage-gated sodium (Nav) channels play a central role in gastrointestinal physiology because they transmit depolarizing impulses in enteric neurons, thereby enabling the coordination of intestinal motility. However, little is known about the ion channel machinery that specifies firing pattern of enteric neurons. Here, we used in situ patch-clamp recording of myenteric neurons from mice to define functionally the Nav channel subtypes responsible for the electrical signature of myenteric neurons. We found that mouse myenteric neurons exhibit two types of tetrodotoxin-resistant Na(+) currents: an early inactivating Na(+) current (INaT) and a persistent Na(+) current (INaP). INaT was encountered in all myenteric neurons, whereas INaP was preferentially found in Dogiel type II sensory neurons. Knock-out mouse studies, in combination with pharmacological assays, indicate that INaT is carried by the Scn5a-encoded "cardiac" Nav1.5, whereas INaP is attributed to the Scn11a-encoded Nav1.9. Current-clamp experiments show that Nav1.9 flows at subthreshold voltages, generating tonic firing. In addition, action potential (AP) clamp reveals that Nav1.5 contributes to the upstroke velocity of APs, whereas Nav1.9, which remains active during the falling phase, opposes AP repolarization. We developed a computational model of a Dogiel type II myenteric neuron that successfully reproduces all experimentally observed phenomena and highlights the differential roles of Nav1.5 and Nav1.9 in the control of excitability. Our data illustrate how excitability can be finely tuned to provide specific firing templates by the selective deployment of Nav1.5 and Nav1.9 isoforms. We propose that Nav-dependent ENS disorders of excitability may play important roles in the pathogenesis of digestive diseases.

Osmoregulation in mammals is tightly controlled by the release of vasopressin and oxytocin from magnocellular neurosecretory cells (MSC) of the supraoptic nucleus (SON). The release of vasopressin and oxytocin in the neurohypophysis by axons of MSC is regulated by bursting activity of these neurons, which is influenced by multiple sources, including intrinsic membrane properties, paracrine contributions of glial cells, and extrinsic synaptic inputs. Previous work has shown that bursting activity of MSC is tetrodotoxin (TTX)-sensitive, and that TTX-S sodium channels Nav1.2, Nav1.6 and Nav1.7 are expressed by MSC and upregulated in response to osmotic challenge in rats. The TTX-resistant sodium channels, NaV1.8 and Nav1.9, are preferentially expressed, at relatively high levels, in peripheral neurons, where their properties are linked to repetitive firing and subthreshold electrogenesis, respectively, and are often referred to as "peripheral" sodium channels. Both sodium channels have been implicated in pain pathways, and are under study as potential therapeutic targets for pain medications which might be expected to have minimal CNS side effects. We show here, however, that Nav1.9 is expressed by vasopressin- and oxytocin-producing MSC of the rat supraoptic nucleus (SON). We also show that cultured MSC exhibit sodium currents that have characteristics of Nav1.9 channels. In contrast, Nav1.8 is not detectable in the SON. These results suggest that Nav1.9 may contribute to the firing pattern of MSC of the SON, and that careful assessment of hypothalamic function be performed as NaV1.9 blocking agents are studied as potential pain therapies., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Objective: To investigate the relationship between pulpitis pain and voltage-gated sodium channel (Nav1.9) by detecting the expression of Nav1.9 at different time points of the rat pulpal lesion model., Methods: Thirty-six SD pulpal lesions rat models were divided into three experimental groups, 1 d (n = 12), 3 d (n = 12) and 5 d group(n = 12).Normal SD rats served as control(n = 12). Tumor necrosis factor-α (TNF-α) and Nav1.9 mRNA expressions were evaluated by reverse transcription PCR (RT-PCR) .Nav1.9 protein expressions were analyzed by enzyme-linked immunosorbent assay (ELISA) and Western blotting., Results: The expression of TNF-α in the experimental group (1 d:0.514 ± 0.098, 3 d:0.739 ± 0.104, 5 d:1.238 ± 0.082) was higher than those in the control group (0.147 ± 0.016) (P < 0.01). Nav1.9 mRNA was up-regulated markedly in experimental groups (1 d: 0.296 ± 0.038, 3 d:0.409 ± 0.013, 5 d: 0.487 ± 0.028) , compare with control group (0.223 ± 0.020) (P < 0.05). The ELISA results revealed that the level of Nav1.9 in control pulp tissue was (4.013 ± 0.292) µg/L, in painful pulp tissue of 1 d group was (5.143 ± 0.101) µg/L, in painful pulp tissue of 3 d group was (5.835 ± 0.088) µg/L and in painful pulp tissue of 5 d group was (6.307 ± 0.137) µg/L (P < 0.05). Western blotting showed the expression of Nav1.9 in experimental groups (1 d: 0.106 ± 0.007, 3 d:0.170 ± 0.013, 5 d:0.238 ± 0.004) was up-regulated significantly compared with control group (0.073 ± 0.004)(P < 0.05)., Conclusions: The level of Nav1.9 had a significant increase in painful pulp tissue. Moreover, with the degree of pain aggravation, the expression of Nav1.9 increased in pulp tissue.It suggests that Nav1.9 may play an important role in the development of pulpitis pain.

Recent advances in our understanding of voltage-gated sodium channels (NaVs) lead to the rational hypothesis that drugs capable of selective blockade of NaV subtypes may be a safe and effective strategy for the treatment of unwanted cough. Among the nine NaV subtypes (NaV1.1-NaV1.9), the afferent nerves involved in initiating cough, in common with nociceptive neurons in the somatosensory system, express mainly NaV1.7, NaV1.8, and NaV1.9. Although knowledge about the effect of selectively blocking these channels on the cough reflex is limited, their biophysical properties indicate that each may contribute to the hypertussive and allotussive state that typifies subacute and chronic nonproductive cough.

Background: To determine effects on calcium and sodium channels of Ca(2+) and Na(+) channel blockers in the present study, expression levels of TRPM1, TRPM2, TRPM3, TRPM4, TRPM5, TRPM6, TRPM7, TRPM8, and NaV1.9 genes were evaluated in kidney tissues after induced ischemia-reperfusion., Material and Methods: Forty albino Wistar male rats were equally divided into 4 groups as follows: group I: control group (n = 10), group II: ischemia group (60 minutes of ischemia + 48 hours of reperfusion; n = 10), group III: ischemia (60 minutes of ischemia + 48 hours of reperfusion) + calcium channel blocker (n = 8), group IV: ischemia (60 minutes of ischemia + 48 hours of reperfusion) + sodium channel blocker (n = 8)., Results: When compared to ischemia group expression levels of TRPM2, TRPM4, TRPM6, and NaV1.9 in Ca(2+) and Na(+) channel blocker groups were increased, whereas that of TRPM7 was decreased. However, expression levels of TRPM1, TRPM3, TRPM5, and TRPM8 were not determined in kidney tissue. Histologically, the Ca(2+) channel blocker verapamil and the Na(+) channel blocker lidocaine inhibited the cell death in kidney tissue compared to control., Conclusion: Our study suggested that verapamil and lidocaine significantly reduce the degree of ischemia-reperfusion injury due to effects to TRPM and Nav1.9 genes., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Enteric neurons located in the gastro-intestinal tract are of particular importance to control digestive functions such as motility and secretion. In our recent publication, we showed that mouse myenteric neurons exhibit 2 types of tetrodotoxin-resistant Na(+) currents: a fast inactivating Na(+) current produced by Nav1.5 channels, present in nearly all myenteric neurons, and a persistent Na(+) current attributed to Nav1.9 channels, restricted to the intrinsic primary afferent neurons (IPANs). By combination of experimental recording and computer simulation we found that Nav1.5 contributed to the upstroke velocity of action potentials (APs), whereas Nav1.9 opposed AP repolarization. Here, we detailed the Na(+), Ca(2+) and K(+) currents used in our computational model of IPAN. We refined the prototype cell to reproduce the sustained firing pattern recorded in situ. As shown in experimental conditions we demonstrated that Nav1.9 channels critically determine the up-state life-time and thus, are essential to sustain tonic firing.

Background: Voltage-gated sodium channels Nav1.8 and Nav1.9 are expressed preferentially in small diameter sensory neurons, and are thought to play a role in the generation of ectopic activity in neuronal cell bodies and/or their axons following peripheral nerve injury. The expression of Nav1.8 and Nav1.9 has been quantified in human lingual nerves that have been previously injured inadvertently during lower third molar removal, and any correlation between the expression of these ion channels and the presence or absence of dysaesthesia investigated., Results: Immunohistochemical processing and quantitative image analysis revealed that Nav1.8 and Nav1.9 were expressed in human lingual nerve neuromas from patients with or without symptoms of dysaesthesia. The level of Nav1.8 expression was significantly higher in patients reporting pain compared with no pain, and a significant positive correlation was observed between levels of Nav1.8 expression and VAS scores for the symptom of tingling. No significant differences were recorded in the level of expression of Nav1.9 between patients with or without pain., Conclusions: These results demonstrate that Nav1.8 and Nav1.9 are present in human lingual nerve neuromas, with significant correlations between the level of expression of Nav1.8 and symptoms of pain. These data provide further evidence that changes in expression of Nav1.8 are important in the development and/or maintenance of nerve injury-induced pain, and suggest that Nav1.8 may be a potential therapeutic target.

The classical role ascribed to voltage-gated Na channels is the conduction of action potentials. Some excitable tissues such as cardiac muscle and skeletal muscle predominantly express a single voltage-gated Na channels isoform. Of the nine voltage-gated Na channels, seven are expressed in neurons, of these Nav 1.7, 1.8 and 1.9 are expressed in sensory neurons including vagal sensory neurons that innervate the airways and initiate cough. Nav 1.7 and Nav 1.9 are of particular interest as they represent two extremes in the functional diversity of voltage-gated Na channels. Voltage-gated Na channel isoforms expressed in airway sensory neurons produce multiple distinct Na currents that underlie distinct aspects of sensory neuron function. The interaction between voltage-gated Na currents underlies the characteristic ability of airway sensory nerves to encode encounters with irritant stimuli into action potential discharge and evoke the cough reflex., (© 2013 Elsevier Ltd. All rights reserved.)

Cold allodynia, pain in response to cooling, occurs during or within hours of oxaliplatin infusion and is thought to arise from a direct effect of oxaliplatin on peripheral sensory neurons. To characterize the pathophysiological mechanisms underlying acute oxaliplatin-induced cold allodynia, we established a new intraplantar oxaliplatin mouse model that rapidly developed long-lasting cold allodynia mediated entirely through tetrodotoxin-sensitive Nav pathways. Using selective inhibitors and knockout animals, we found that Nav1.6 was the key isoform involved, while thermosensitive transient receptor potential channels were not involved. Consistent with a crucial role for delayed-rectifier potassium channels in excitability in response to cold, intraplantar administration of the K(+)-channel blocker 4-aminopyridine mimicked oxaliplatin-induced cold allodynia and was also inhibited by Nav1.6 blockers. Intraplantar injection of the Nav1.6 activator Cn2 elicited spontaneous pain, mechanical allodynia, and enhanced 4-aminopyridine-induced cold allodynia. These findings provide behavioural evidence for a crucial role of Nav1.6 in multiple peripheral pain pathways including cold allodynia., (Copyright © 2013 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.)

Background: Amitriptyline (AMI) is tricyclic antidepressant that has been widely used to manage various chronic pains such as migraines. Its efficacy is attributed to its blockade of voltage-gated sodium channels (VGSCs). However, the effects of AMI on the tetrodotoxin-resistant (TTX-r) sodium channel Nav1.9 currents have been unclear to present., Results: Using a whole-cell patch clamp technique, this study showed that AMI efficiently inhibited Nav1.9 currents in a concentration-dependent manner and had an IC50 of 15.16 μM in acute isolated trigeminal ganglion (TG) neurons of the rats. 10 μM AMI significantly shifted the steady-state inactivation of Nav1.9 channels in the hyperpolarizing direction without affecting voltage-dependent activation. Surprisingly, neither 10 nor 50 μM AMI caused a use-dependent blockade of Nav1.9 currents elicited by 60 pulses at 1 Hz., Conclusion: These data suggest that AMI is a state-selective blocker of Nav1.9 channels in rat nociceptive trigeminal neurons, which likely contributes to the efficacy of AMI in treating various pains, including migraines.

Objective: To establish the cell line with stable voltage-gated sodium channels (VGSCs/NaVs) α subunit NaV1.9 gene silencing through RNA interference (RNAi) in murine RAW264.7 macrophages, and to investigate proliferation, phagocytosis and migration in this cell line., Methods: The stable NaV1.9-deficient cell line was generated by selection in G418 after the transfection of short hairpin (shRNA) plasmid with Lipofectamine TM2000. RNAi efficiency was qualified by RT-PCR; proliferation ability was measured by CCK-8 assay; cell cycle and phagocytic ability were analyzed by flow cytometry; and migrating ability was detected by Transwell migration assay., Results: Stable NaV1.9-deficient cell line was established and the expression of NaV1.9 was reduced by 80%. CCK-8 assay and flow cytometry showed that the proliferation of the NaV1.9-deficient cell line was inhibited (P<0.05). Flow cytometry revealed that phagocytic ability was reduced in the cell line (P<0.05). Transwell migration assay demonstrated that migrating ability was depressed in the cell line (P<0.05)., Conclusion: In the stable NaV1.9-deficient cells we successfully constructed, proliferation, phagocytosis and migration were obviously inhibited.

Tetrodotoxin (TTX)-resistant voltage-gated Na (Na(V)) channels have been implicated in nociception. In particular, Na(V)1.9 contributes to expression of persistent Na current in small diameter, nociceptive sensory neurons in dorsal root ganglia and is required for inflammatory pain sensation. Using ND7/23 cells stably expressing human Na(V)1.9, we elucidated the biophysical mechanisms responsible for potentiation of channel activity by G-protein signaling to better understand the response to inflammatory mediators. Heterologous Na(V)1.9 expression evoked TTX-resistant Na current with peak activation at -40 mV with extensive overlap in voltage dependence of activation and inactivation. Inactivation kinetics were slow and incomplete, giving rise to large persistent Na currents. Single-channel recording demonstrated long openings and correspondingly high open probability (P(o)) accounting for the large persistent current amplitude. Channels exposed to intracellular GTPγS, a proxy for G-protein signaling, exhibited twofold greater current density, slowing of inactivation, and a depolarizing shift in voltage dependence of inactivation but no change in activation voltage dependence. At the single-channel level, intracellular GTPγS had no effect on single-channel amplitude but caused an increased mean open time and greater P(o) compared with recordings made in the absence of GTPγS. We conclude that G-protein activation potentiates human Na(V)1.9 activity by increasing channel open probability and mean open time, causing the larger peak and persistent current, respectively. Our results advance our understanding about the mechanism of Na(V)1.9 potentiation by G-protein signaling during inflammation and provide a cellular platform useful for the discovery of Na(V)1.9 modulators with potential utility in treating inflammatory pain.

Spontaneous electrical activity preceding synapse formation contributes to the precise regulation of neuronal development. Examining the origins of spontaneous activity revealed roles for neurotransmitters that depolarize neurons and activate ion channels. Recently, we identified a new molecular mechanism underlying fluctuations in spontaneous neuronal excitability. We found that embryonic motoneurons with a genetic loss of the low-threshold sodium channel NaV1.9 show fewer fluctuations in intracellular calcium in axonal compartments and growth cones than wild-type littermates. As a consequence, axon growth of NaV1.9-deficient motoneurons in cell culture is drastically reduced while dendritic growth and cell survival are not affected. Interestingly, NaV1.9 function is observed under conditions that would hardly allow a ligand- or neurotransmitter-dependent depolarization. Thus, NaV1.9 may serve as a cell-autonomous trigger for neuronal excitation. In this addendum, we discuss a model for the interplay between cell-autonomous local neuronal activity and local cytoskeleton dynamics in growth cone function.