A Possible Role of Tetrodotoxin-Sensitive Na+ Channels for Oxidation-Induced Late Na+ Currents in Cardiomyocytes.

An accumulation of reactive oxygen species (ROS) in cardiomyocytes can induce pro-arrhythmogenic late Na+ currents by removing the inactivation of voltage-gated Na+ channels including the tetrodotoxin (TTX)-resistant cardiac α-subunit Nav1.5 as well as TTX-sensitive α-subunits like Nav1.2 and Nav1.3. Here, we explored oxidant-induced late Na+ currents in mouse cardiomyocytes and human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as well as in HEK 293 cells expressing Nav1.2, Nav1.3, or Nav1.5. Na+ currents in mouse cardiomyocytes and hiPSC-CMs treated with the oxidant chloramine T (ChT) developed a moderate reduction in peak current amplitudes accompanied by large late Na+ currents. While ChT induced a strong reduction in peak current amplitudes but only small persistent currents on Nav1.5, both Nav1.2 and Nav1.3 produced increased peak current amplitudes and large persistent currents following oxidation. TTX (300 nM) blocked ChT-induced late Na+ currents significantly stronger as compared to peak Na+ currents in both mouse cardiomyocytes and hiPSC-CMs. Similar differences between Nav1.2, Nav1.3, and Nav1.5 regarding ROS sensitivity were also evident when oxidation was induced with UVA-light (380 nm) or the cysteine-selective oxidant nitroxyl (HNO). To conclude, our data on TTX-sensitive Na+ channels expressed in cardiomyocytes may be relevant for the generation of late Na+ currents following oxidative stress.

SIRT1 mediates the excitability of spinal CaMKIIα-positive neurons and participates in neuropathic pain by controlling Nav1.3.

AIMS: Neuropathic pain is a common chronic pain disorder, which is largely attributed to spinal central sensitization. Calcium/calmodulin-dependent protein kinase II alpha (CaMKIIα) activation in the spinal dorsal horn (SDH) is a major contributor to spinal sensitization. However, the exact way that CaMKIIα-positive (CaMKIIα+) neurons in the SDH induce neuropathic pain is still unclear. This study aimed to explore the role of spinal CaMKIIα+ neurons in neuropathic pain caused by chronic constriction injury (CCI) and investigate the potential epigenetic mechanisms involved in CaMKIIα+ neuron activation. METHODS: CCI-induced neuropathic pain mice model, Sirt1loxP/loxP mice, and chemogenetic virus were used to investigate whether the activation of spinal CaMKIIα+ neurons is involved in neuropathic pain and its involved mechanism. Transcriptome sequence, western blotting, qRT-PCR, and immunofluorescence analysis were performed to assay the expression of related molecules and activation of neurons. Co-immunoprecipitation was used to observe the binding relationship of protein. Chromatin immunoprecipitation (ChIP)-PCR was applied to analyze the acetylation of histone H3 in the Scn3a promoter region. RESULTS: The expression of sodium channel Nav1.3 was increased and the expression of SIRT1 was decreased in the spinal CaMKIIα+ neurons of CCI mice. CaMKIIα neurons became overactive after CCI, and inhibiting their activation relieved CCI-induced pain. Overexpression of SIRT1 reversed the increase of Nav1.3 and alleviated pain, while knockdown of SIRT1 or overexpression of Nav1.3 promoted CaMKIIα+ neuron activation and induced pain. By knocking down spinal SIRT1, the acetylation of histone H3 in the Scn3a (encoding Nav1.3) promoter region was increased, leading to an increased expression of Nav1.3. CONCLUSION: The findings suggest that an aberrant reduction of spinal SIRT1 after nerve injury epigenetically increases Nav1.3, subsequently activating CaMKIIα+ neurons and causing neuropathic pain.

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

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

Targeted blockade of aberrant sodium current in a stem cell-derived neuron model of SCN3A encephalopathy.

Missense variants in SCN3A encoding the voltage-gated sodium (Na+) channel α subunit Nav1.3 are associated with SCN3A-related neurodevelopmental disorder (SCN3A-NDD), a spectrum of disease that includes epilepsy and malformation of cortical development. How genetic variation in SCN3A leads to pathology remains unclear, as prior electrophysiological work on disease-associated variants has been performed exclusively in heterologous cell systems. To further investigate the mechanisms of SCN3A-NDD pathogenesis, we used CRISPR/Cas9 gene editing to modify a control human induced pluripotent stem cell (iPSC) line to express the recurrent de novo missense variant SCN3A c.2624T>C (p.Ile875Thr). With the established Ngn2 rapid induction protocol, we generated glutamatergic forebrain-like neurons (iNeurons), which we showed to express SCN3A mRNA and Nav1.3-mediated Na+ currents. We performed detailed whole-cell patch clamp recordings to determine the effect of the SCN3A-p.Ile875Thr variant on endogenous Na+ currents in, and intrinsic excitability of, human neurons. Compared to control iNeurons, variant-expressing iNeurons exhibit markedly increased slowly-inactivating/persistent Na+ current, abnormal firing patterns with paroxysmal bursting and plateau-like potentials with action potential failure, and a hyperpolarized voltage threshold for action potential generation. We then validated these findings using a separate iPSC line generated from a patient harbouring the SCN3A-p.Ile875Thr variant compared to a corresponding CRISPR-corrected isogenic control line. Finally, we found that application of the Nav1.3-selective blocker ICA-121431 normalizes action potential threshold and aberrant firing patterns in SCN3A-p.Ile1875Thr iNeurons; in contrast, consistent with action as a Na+ channel blocker, ICA-121431 decreases excitability of control iNeurons. Our findings demonstrate that iNeurons can model the effects of genetic variation in SCN3A yet reveal a complex relationship between gain-of-function at the level of the ion channel versus impact on neuronal excitability. Given the transient expression of SCN3A in the developing human nervous system, selective blockade or suppression of Nav1.3-containing Na+ channels could represent a therapeutic approach towards SCN3A-NDD.

Structure and Function of Sodium Channel Nav1.3 in Neurological Disorders.

Nav1.3, encoded by the SCN3A gene, is a voltage-gated sodium channel on the cell membrane. It is expressed abundantly in the fetal brain but little in the normal adult brain. It is involved in the generation and conduction of action potentials in excitable cells. Nav1.3 plays an important role in many neurological diseases. The aim of this review is to summarize new findings about Nav1.3 in the field of neurology. Many mutations of SCN3A can lead to neuronal hyperexcitability and then cause epilepsy. The rapid recovery from inactivation and slow closed-state inactivation kinetics of Nav1.3 leads to a reduced activation threshold of the channel and a high frequency of firing of neurons. Hyperactivity of Nav1.3 also induces increased excitability of sensory neurons, a lower nociceptive threshold, and neuropathic pain. This review summarizes the structure and the function of Nav1.3 and focuses on its relationship with epilepsy and neuropathic pain.

Role of voltage-gated sodium channels in axonal signal propagation of trigeminal ganglion neurons after infraorbital nerve entrapment.

Chronic pain arising from peripheral nerve injuries represents a significant clinical challenge because even the most efficacious anticonvulsant drug treatments are limited by their side effects profile. We investigated pain behavior, changes in axonal signal conduction and excitability of trigeminal neurons, and expression of voltage-gated sodium channels (NaVs) in the infraorbital nerve and trigeminal ganglion (TG) after infraorbital nerve entrapment (IoNE). Compared to Sham, IoNE rats had increased A- and C-fiber compound action potentials (CAPs) and Aδ component of A-CAP area from fibers innervating the vibrissal pad. After IoNE, A- and C-fiber CAPs were more sensitive to blockade by tetrodotoxin (TTX), and those fibers that were TTX-resistant were more sensitive to blockade by the NaV1.8 selective blocker, A-803467. Although NaV1.7 blocker, ICA-121431 alone, did not affect Aδ-fiber signal propagation, cumulative application with A-803467 and 4,9-anhydro-TTX significantly reduced the Aδ-fiber CAP in IoNE rats. In patch clamp recordings from small- and medium-sized TG neurons, IoNE resulted in reduced action potential (AP) depolarizing current threshold, hyperpolarized AP voltage threshold, increased AP duration, and a more depolarized membrane potential. While the transcripts of most NaVs were reduced in the ipsilateral TG after IoNE, NaV1.3, NaV1.7, and NaV1.8 mRNAs, and NaV1.8 protein, were significantly increased in the nerve. Altogether, our data suggest that axonal redistribution of NaV1.8, and to a lesser extent NaV1.3, and NaV1.7 contributes to enhanced nociceptive signal propagation in peripheral nerve after IoNE.

Knockdown of rno_circRNA_009194 Improves Outcomes in Traumatic Brain Injury Rats through Inhibiting Voltage-Gated Sodium Channel Nav1.3.

Excessive activation of voltage-gated sodium channel Nav1.3 has been recently reported in secondary traumatic brain injury (TBI). However, the molecular mechanisms underlying regulating voltage-gated sodium channel (Nav1.3) have not been well understood. The present study used a TBI rat model induced by a fluid percussion device and performed a circular RNA (circRNA) microarray (n = 3) to profile the altered circRNAs in the hippocampus after TBI. After polymerase chain reaction (PCR) validation, certain circRNAs were selected to investigate the function and mechanism in regulating Nav1.3 in the TBI rat model by intracerebroventricular injection with lentivirus. The neurological outcome was evaluated by Morris water maze test, modified Neurological Severity Score (mNSS), brain water content measurement, and hematoxylin and eosin staining. The related molecular mechanisms were explored with PCR, Western blotting, luciferase reporter, chromatin immunoprecipitation assay, and electrophoretic mobility shift assay (EMSA). A total of 347 circRNAs were observed to be differentially expressed (fold change [FC] ≥ 1.2 and p < 0.05) after TBI, including 234 up-regulated and 113 down-regulated circRNAs. Among 10 validated circRNAs, we selected circRNA_009194 with the maximized up-regulated fold change (n = 5, FC = 4.45, p < 0.001) for the in vivo functional experiments. Down-regulation of circRNA_009194 resulted in a 27.5% reduced mNSS in rat brain (n = 6, p < 0.01) after TBI and regulated the expression levels of miR-145-3p, Sp1, and Nav1.3, which was reversed by sh-miR-145-3p or Sp1/Nav1.3 overexpression (n = 5, p < 0.05). Mechanistically, circRNA_009194 might act as a sponge for miR-145-3p to regulate Sp1-mediated Nav1.3. This study demonstrated that circRNA_009194 knockdown could improve neurological outcomes in TBI in vivo by inhibiting Nav1.3, directly or indirectly.

New application of an old drug proparacaine in treating epilepsy via liposomal hydrogel formulation.

Proparacaine (PPC) is a previously discovered topical anesthetic for ophthalmic optometry and surgery by blocking the central Nav1.3. In this study, we found that proparacaine hydrochloride (PPC-HCl) exerted an acute robust antiepileptic effect in pilocarpine-induced epilepsy mice. More importantly, chronic treatment with PPC-HCl totally terminated spontaneous recurrent seizure occurrence without significant toxicity. Chronic treatment with PPC-HCl did not cause obvious cytotoxicity, neuropsychiatric adverse effects, hepatotoxicity, cardiotoxicity, and even genotoxicity that evaluated by whole genome-scale transcriptomic analyses. Only when in a high dose (50 mg/kg), the QRS interval measured by electrocardiography was slightly prolonged, which was similar to the impact of levetiracetam. Nevertheless, to overcome this potential issue, we adopt a liposome encapsulation strategy that could alleviate cardiotoxicity and prepared a type of hydrogel containing PPC-HCl for sustained release. Implantation of thermosensitive chitosan-based hydrogel containing liposomal PPC-HCl into the subcutaneous tissue exerted immediate and long-lasting remission from spontaneous recurrent seizure in epileptic mice without affecting QRS interval. Therefore, this new liposomal hydrogel formulation of proparacaine could be developed as a transdermal patch for treating epilepsy, avoiding the severe toxicity after chronic treatment with current antiepileptic drugs in clinic.

Peripheral Voltage-Gated Cation Channels in Neuropathic Pain and Their Potential as Therapeutic Targets.

The persistence of increased excitability and spontaneous activity in injured peripheral neurons is imperative for the development and persistence of many forms of neuropathic pain. This aberrant activity involves increased activity and/or expression of voltage-gated Na+ and Ca2+ channels and hyperpolarization activated cyclic nucleotide gated (HCN) channels as well as decreased function of K+ channels. Because they display limited central side effects, peripherally restricted Na+ and Ca2+ channel blockers and K+ channel activators offer potential therapeutic approaches to pain management. This review outlines the current status and future therapeutic promise of peripherally acting channel modulators. Selective blockers of Nav1.3, Nav1.7, Nav1.8, Cav3.2, and HCN2 and activators of Kv7.2 abrogate signs of neuropathic pain in animal models. Unfortunately, their performance in the clinic has been disappointing; some substances fail to meet therapeutic end points whereas others produce dose-limiting side effects. Despite this, peripheral voltage-gated cation channels retain their promise as therapeutic targets. The way forward may include (i) further structural refinement of K+ channel activators such as retigabine and ASP0819 to improve selectivity and limit toxicity; use or modification of Na+ channel blockers such as vixotrigine, PF-05089771, A803467, PF-01247324, VX-150 or arachnid toxins such as Tap1a; the use of Ca2+ channel blockers such as TTA-P2, TTA-A2, Z 944, ACT709478, and CNCB-2; (ii) improving methods for assessing "pain" as opposed to nociception in rodent models; (iii) recognizing sex differences in pain etiology; (iv) tailoring of therapeutic approaches to meet the symptoms and etiology of pain in individual patients via quantitative sensory testing and other personalized medicine approaches; (v) targeting genetic and biochemical mechanisms controlling channel expression using anti-NGF antibodies such as tanezumab or re-purposed drugs such as vorinostat, a histone methyltransferase inhibitor used in the management of T-cell lymphoma, or cercosporamide a MNK 1/2 inhibitor used in treatment of rheumatoid arthritis; (vi) combination therapy using drugs that are selective for different channel types or regulatory processes; (vii) directing preclinical validation work toward the use of human or human-derived tissue samples; and (viii) application of molecular biological approaches such as clustered regularly interspaced short palindromic repeats (CRISPR) technology.

Characterization of Synthetic Tf2 as a NaV1.3 Selective Pharmacological Probe.

NaV1.3 is a subtype of the voltage-gated sodium channel family. It has been implicated in the pathogenesis of neuropathic pain, although the contribution of this channel to neuronal excitability is not well understood. Tf2, a ß-scorpion toxin previously identified from the venom of Tityus fasciolatus, has been reported to selectively activate NaV1.3. Here, we describe the activity of synthetic Tf2 and assess its suitability as a pharmacological probe for NaV1.3. As described for the native toxin, synthetic Tf2 (1 µM) caused early channel opening, decreased the peak current, and shifted the voltage dependence of NaV1.3 activation in the hyperpolarizing direction by -11.3 mV, with no activity at NaV1.1, NaV1.2, and NaV1.4-NaV1.8. Additional activity was found at NaV1.9, tested using the hNav1.9_C4 chimera, where Tf2 (1 µM) shifted the voltage dependence of activation by -6.3 mV. In an attempt to convert Tf2 into an NaV1.3 inhibitor, we synthetized the analogue Tf2[S14R], a mutation previously described to remove the excitatory activity of related ß-scorpion toxins. Indeed, Tf2[S14R](10 µM) had reduced excitatory activity at NaV1.3, although it still caused a small -5.8 mV shift in the voltage dependence of activation. Intraplantar injection of Tf2 (1 µM) in mice caused spontaneous flinching and swelling, which was not reduced by the NaV1.1/1.3 inhibitor ICA-121431 nor in NaV1.9-/- mice, suggesting off-target activity. In addition, despite a loss of excitatory activity, intraplantar injection of Tf2[S14R](10 µM) still caused swelling, providing strong evidence that Tf2 has additional off-target activity at one or more non-neuronal targets. Therefore, due to activity at NaV1.9 and other yet to be identified target(s), the use of Tf2 as a selective pharmacological probe may be limited.

IL-6 contributes to Nav1.3 up-regulation in trigeminal nerve following chronic constriction injury.

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 infraorbital nerve chronic constriction injury (CCI) model in rats.Method: The expression of Nav1.3 or IL-6 in the infraorbital nerve (ION) and trigeminal ganglion (TG) were detected by western blot and immunocytochemistry after administration of antisense oligodeoxynucleotide sequence (AS), IL-6 or Anti-IL-6.Results: With intrathecal administration of AS or mismatch oligodeoxynucleotide sequence (MM) in the CCI rats, the Nav1.3-IR in ION and TG accounted for 2.2 ± 0.51% and 8.5 ± 3.1% in AS+CCI group vs. 6.9 ± 1.3% and 38.7 ± 4.8% in MM+CCI group (p < 0.05), respectively. While with local administration of IL-6 in those with sham operation, it accounted for 7.4 ± 2.1% and 45.5 ± 3.4% in IL-6+ sham group vs. 1.9 ± 0.67% and 8.1 ± 1.3% in vehicle+sham group (p < 0.05); with local administration of anti-IL-6 in CCI rats, 4.5 ± 0.78% and 32.1 ± 9.6% in Anti-IL-6+ CCI group vs 8.9 ± 2.1% and 61.4 ± 11.2% in vehicle+CCI group (p < 0.05).Discussion: We believe that the emergence of Nav1.3 from the compressed trigeminal nerve might be an important structural basis for the development of the ectopic excitability on the axon and IL-6 may play a role of necessary precondition.

MicroRNA-212-3p Attenuates Neuropathic Pain via Targeting Sodium Voltage-gated Channel Alpha Subunit 3 (NaV 1.3).

PURPOSE: To explore the role and potential mechanism of miR-212-3p in neuropathic pain regulation. METHODS: Adult male rats were used to establish chronic constriction injury (CCI) model to mimic the neuropathic pain. Then, paw withdrawal threshold (PWT) and paw withdrawal thermal latency (PWL) were determined. The concentrations of interleukin 1 beta (IL-1ß), interleukin 6 (IL-6) and tumor necrosis factor-alpha (TNF-α) were measured with enzyme-linked immune sorbent assay (ELISA) kit and the expression of miR-212-3p was measured by real time quantitative PCR (RTqPCR). Besides, miR-212-3p agomir was intrathecally injected into CCI rats and the expression of key apoptotic proteins was determined by western blot. Furthermore, dual-luciferase reporter assay was used to determine the binding of miR-212-3p and 3' untranslated regions (3'UTR) of NaV1.3 and the expression levels of NaV1.3 were measured by western blot and RT-qPCR. RESULTS: In the CCI group, the PWT and PWL were significantly decreased and IL-1ß, IL-6 and TNF-α were increased. miR-212-3p was decreased in response to CCI. The intrathecal injection of miR-212-3p agomir into CCI rats improved the PWT and PWL, decreased the IL-1ß, IL-6 and TNF-α, decreased the expression levels of BCL2 associated X, apoptosis regulator (Bax), cleaved caspase-3 and increased the expression levels of BCL2 apoptosis regulator (Bcl-2). The results of dual--luciferase reporter assay showed that miR-212-3p could directly bind with 3'UTR of NaV1.3. The expression of NaV1.3 was up-regulated in CCI rats who were intrathecally injected with miRctrl, whereas it decreased in CCI rats intrathecally injected with miR-212-3p agomir. CONCLUSION: The expression of miR-212a-3p attenuates neuropathic pain by targeting NaV1.3.

The expression of voltage-gated sodium channels in trigeminal nerve following chronic constriction injury in rats.

Objectives: Despite the etiology of trigeminal neuralgia has been verified by microvascular decompression as vascular compression of the trigeminal root, very few researches concerning its underlying pathogenesis has been reported in the literature. The present study focused on those voltage-gated sodium channels, which are the structural basis for generation of ectopic action potentials. Methods: The trigeminal neuralgia modeling was obtained with infraorbital nerve chronic constriction injury (ION-CCI) in rats. Two weeks postoperatively, the infraorbital nerve (TN), the trigeminal ganglion (TG), and the brain stem (BS) were removed and analyzed with a series of molecular biological techniques. Results: Western blot depicted a significant up-regulation of Nav1.3 in TN and TG but not in BS, while none of the other isoforms (Nav1.6, Nav1.7, Nav1.8, or Nav1.9) presented a statistical change. The Nav1.3 from ION-CCI group was quantified as 2.5-fold and 1.7-fold than that from sham group in TN and TG, respectively (p < .05). Immunocytochemistry showed the Nav1.3-IR from ION-CCI group accounted for 21.2 ± 2.3% versus 6.1 ± 1.2% from sham group in TN, while the Nav1.3-positive neurons from ION-CCI group accounted for 34.1 ± 3.5% versus 11.2 ± 1.8% from sham group in TG. Immunohistochemical labeling showed the Nav1.3 was co-localized with CGRP and IB4 but not with GFAP or NF-200 in TG. Conclusion: ION-CCI may give rise to an up-regulation of Nav1.3 in trigeminal nerve as well as in C-type neurons at the trigeminal ganglion. It implied that the ectopic action potential may generate from both the compressed site of the trigeminal nerve and the ganglion rather than from the trigeminal nuclei.

Role of Nav1.3 in control of excitability of RIN-14B cells / 上海交通大学学报（医学版）

Objective: To provide electrophysiological evidence for RIN-14B cells as an useful model of enterochromaffin cells (EC) and to study the role of Nav1. 3 channel in the control of its excitability. Methods: Resting membrane potential was recorded and the effects of TTX and ICA-121431 were examined by current-clamp in cultured RIN-14B cells. The effects of TTX and ICA-121431 on Na+ current of RIN-14B cells were examined by voltage-clamp. Results: RIN-14B cells had a resting potential around -60 mV and fired action potentials when stimulated with depolarizing current pulses. The action potential was completely blocked by TTX and inhibited by ICA-121431 in a dose-dependent manner. TTX blocked activation and inactivation of sodium current. In addition ICA-121431 dose-dependently inhibited activation of Na+ current. Conclusion: The action potential of RIN-14B cells is induced by TTX-sensitive sodium channel and the excitability is controlled by Nav1.3. These results suggest RIN-14B cells are similar to EC and it may be a good model of EC.

Effects of acidosis on neuronal voltage-gated sodium channels: Nav1.1 and Nav1.3.

Voltage-gated sodium channels are key contributors to membrane excitability. These channels are expressed in a tissue-specific manner. Mutations and modulation of these channels underlie various physiological and pathophysiological manifestations. The effects of changes in extracellular pH on channel gating have been studied on several sodium channel subtypes. Among these, Nav1.5 is the most pH-sensitive channel, with Nav1.2 and Nav1.4 being mostly pH-resistant channels. However, pH effects have not been characterized on other sodium channel subtypes. In this study, we sought to determine whether Nav1.1 and Nav1.3 display resistance or sensitivity to changes in extracellular pH. These two sodium channel subtypes are predominantly found in inhibitory neurons. The expression of these channels highly depends on age and the developmental stage of neurons, with Nav1.3 being found mostly in neonatal neurons, and Nav1.1 being found in adult neurons. Our present results indicate that, during extracellular acidosis, both channels show a depolarization in the voltage-dependence of activation and moderate reduction in current density. Voltage-dependence of steady-state fast inactivation and recovery from fast inactivation were unchanged. We conclude that Nav1.1 and Nav1.3 have similar pH-sensitivities.

Re-expression of voltage-gated sodium channel subtype Nav1.3 in the substantia nigra after dopamine depletion.

Abnormal synchronized oscillatory bursts occurring in the basal ganglia (BG) are suggested to be correlated with motor symptoms in Parkinson's disease (PD) patients and animal models of PD. Voltage-gated sodium channels (VGSCs) have been demonstrated to play an important role in the abnormal electrical activity of neurons in the BG. Nav1.3, a VGSCs subtype, is predominantly expressed in embryonic and neonatal nervous system but barely detected in the normal adult nervous system in rodents. Here we investigated the expression patterns of Nav1.3 in the BG of 6-OHDA lesioned Sprague Dawley rats. The results showed that Nav1.3 at mRNA and protein levels was abundantly re-expressed in the ipsilateral and contralateral SN at 49 days postlesion, but was rarely detected in the other nuclei of the BG in the 6-OHDA lesioned rats. Furthermore, Nav1.3 was not only expressed in TH-positive dopaminergic neurons of the ipsilateral and contralateral SN, but also in nestin-positive neural progenitor cells surrounding the ipsilateral SN and the midline region adjacent to the ipsilateral SN in the midbrain at 49 days postlesion. These results suggested that the re-expression of Nav1.3 may influence the electrical activity of dopaminergic neurons in the SN in 6-OHDA lesioned rats.

Swimming Exercise Induced Reversed Expression of miR-96 and Its Target Gene NaV1.3 in Diabetic Peripheral Neuropathy in Rats.

Diabetes is a common metabolic disease which leads to diabetic peripheral neuropathy. Recently, the role of microRNA-96 (miR-96) in alleviating neuropathic pain by inhibiting the expression of NaV1.3, an isoform of voltage-gated sodium channels, has been shown. Peripheral nerve injuries result in NaV1.3 elevation. Exercise has beneficial role in diabetes management and peripheral neuropathy. However, the effects of exercise on miR-96 and its target gene NaV1.3 in diabetic rats are unknown. Therefore, the present study investigated the effects of exercise training on the expression of miR-96 and NaV1.3 in diabetic rats. For this purpose, rats were randomly divided into four groups: control, exercise, diabetic and diabetic-exercise groups. Type 2 diabetes was induced by a high-fat diet and the administration of streptozotocin (STZ) (35 mg/kg, i.p.). The exercise groups were subjected to swimming exercise 5 days/week for 10 weeks. At the end of the treatment period, thermal pain threshold, determined through the tail-flick test, and the expression levels of miR-96 and its target gene NaV1.3 were determined by reverse transcription (RT)-PCR in the sciatic nerve tissues of the rats. Data of the present study indicated that diabetes diminished miR-96 expression levels, but significantly upregulated NaV1.3 expression in the sciatic nerve. On exercise training, miR-96 expression was reversed with concurrent down-regulation of the NaV1.3 expression. This study introduced a new and potential miRNA-dependent mechanism for exerciseinduced protective effects against diabetic thermal hyperalgesia.

MiR-30b Attenuates Neuropathic Pain by Regulating Voltage-Gated Sodium Channel Nav1.3 in Rats.

Nav1.3 is a tetrodotoxin-sensitive isoform among voltage-gated sodium channels that are closely associated with neuropathic pain. It can be up-regulated following nerve injury, but its biological function remains uncertain. MicroRNAs (miRNAs) are endogenous non-coding RNAs that can regulate post-transcriptional gene expression by binding with their target mRNAs. Using Target Scan software, we discovered that SCN3A is the major target of miR-30b, and we then determined whether miR-30b regulated the expression of Nav1.3 by transfecting miR-30b agomir through the stimulation of TNF-α or by transfecting miR-30b antagomir in primary dorsal root ganglion (DRG) neurons. The spinal nerve ligation (SNL) model was used to determine the contribution of miR-30b to neuropathic pain, to evaluate changes in Nav1.3 mRNA and protein expression, and to understand the sensitivity of rats to mechanical and thermal stimuli. Our results showed that miR-30b agomir transfection down-regulated Nav1.3 mRNA stimulated with TNF-α in primary DRG neurons. Moreover, miR-30b overexpression significantly attenuated neuropathic pain induced by SNL, with decreases in the expression of Nav1.3 mRNA and protein both in DRG neurons and spinal cord. Activation of Nav1.3 caused by miR-30b antagomir was identified. These data suggest that miR-30b is involved in the development of neuropathic pain, probably by regulating the expression of Nav1.3, and might be a novel therapeutic target for neuropathic pain. Perspective: This study is the first to explore the important role of miR-30b and Nav1.3 in spinal nerve ligation-induced neuropathic pain, and our evidence may provide new insight for improving therapeutic approaches to pain.

Electro-acupuncture-modulated miR-214 prevents neuronal apoptosis by targeting Bax and inhibits sodium channel Nav1.3 expression in rats after spinal cord injury.

Electro-acupuncture (EA) has been proven to contribute towards neurologic and functional recoveries in spinal cord injury (SCI), but the underlying mechanism remains largely unknown especially regarding the effects of preventing neuronal apoptosis and alleviating neuropathic pain involved in the development of EA. In this study, we evaluated the effect of EA treatment in an animal model of SCI using the Basso, Beattie, and Bresnahan (BBB) score method, lesion volume by cresyl violet staining and neuronal apoptosis by TUNEL staining. Our results showed that EA therapy improved functional recovery, and reduced tissue loss and neuronal apoptosis after SCI. Meanwhile, we found that proapoptotic proteins (cleaved-caspase-3, 9 and cleaved-PARP) were downregulated and antiapoptotic protein Bcl-2 was upregulated following EA. To further explore the antiapoptotic effect of EA treatment, we verified that a large set of microRNAs (miRNAs) expression were altered following EA treatment and the miR-214 was one of the miRNAs being most significantly upregulated. Importantly, we validated both apoptosis related protein Bax and pain related protein Nav1.3 as two functional targets of miR-214 in vitro and vivo. Furthermore, our data showed that EA attenuates SCI-induced Nav1.3 and Bax upregulation in injured spinal cord via upregulating miR-214. These results suggest that miR-214 played an important role after SCI in the process of EA therapy, and the miR-214 could become an attractive novel therapeutic target for the treatment of SCI.