Function Of Voltage-gated Sodium Channels Research Articles

Dual receptor-sites reveal the structural basis for hyperactivation of sodium channels by poison-dart toxin batrachotoxin

The poison dart toxin batrachotoxin is exceptional for its high potency and toxicity, and for its multifaceted modification of the function of voltage-gated sodium channels. By using cryogenic electron microscopy, we identify two homologous, but nonidentical receptor sites that simultaneously bind two molecules of toxin, one at the interface between Domains I and IV, and the other at the interface between Domains III and IV of the cardiac sodium channel. Together, these two bound toxin molecules stabilize α/π helical conformation in the S6 segments that gate the pore, and one of the bound BTX-B molecules interacts with the crucial Lys1421 residue that is essential for sodium conductance and selectivity via an apparent water-bridged hydrogen bond. Overall, our structure provides insight into batrachotoxin’s potency, efficacy, and multifaceted functional effects on voltage-gated sodium channels via a dual receptor site mechanism.

Transforming growth factor-beta 1 enhances discharge activity of cortical neurons

Abstract Transforming growth factor-beta 1 (TGF-β1) has been extensively studied for its pleiotropic effects on central nervous system diseases. The neuroprotective or neurotoxic effects of TGF-β1 in specific brain areas may depend on the pathological process and cell types involved. Voltage-gated sodium channels (VGSCs) are essential ion channels for the generation of action potentials in neurons, and are involved in various neuroexcitation-related diseases. However, the effects of TGF-β1 on the functional properties of VGSCs and firing properties in cortical neurons remain unclear. In this study, we investigated the effects of TGF-β1 on VGSC function and firing properties in primary cortical neurons from mice. We found that TGF-β1 increased VGSC current density in a dose- and time-dependent manner, which was attributable to the upregulation of Nav1.3 expression. Increased VGSC current density and Nav1.3 expression were significantly abolished by preincubation with inhibitors of mitogen-activated protein kinase kinase (PD98059), p38 mitogen-activated protein kinase (SB203580), and Jun NH2-terminal kinase 1/2 inhibitor (SP600125). Interestingly, TGF-β1 significantly increased the firing threshold of action potentials but did not change their firing rate in cortical neurons. These findings suggest that TGF-β1 can increase Nav1.3 expression through activation of the ERK1/2–JNK– MAPK pathway, which leads to a decrease in the firing threshold of action potentials in cortical neurons under pathological conditions. Thus, this contributes to the occurrence and progression of neuroexcitatory-related diseases of the central nervous system.

The Role of MAPRE2 and Microtubules in Maintaining Normal Ventricular Conduction.

Brugada syndrome is associated with loss-of-function SCN5A variants, yet these account for only ≈20% of cases. A recent genome-wide association study identified a novel locus within MAPRE2, which encodes EB2 (microtubule end-binding protein 2), implicating microtubule involvement in Brugada syndrome. A mapre2 knockout zebrafish model was generated using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated protein 9) and validated by Western blot. Larval hearts at 5 days post-fertilization were isolated for voltage mapping and immunocytochemistry. Adult fish hearts were used for ECG, patch clamping, and immunocytochemistry. Morpholinos were injected into embryos at 1-cell stage for knockdown experiments. A transgenic zebrafish line with cdh2 tandem fluorescent timer was used to study adherens junctions. Microtubule plus-end tracking and patch clamping were performed in human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) with MAPRE2 knockdown and knockout, respectively. Voltage mapping of mapre2 knockout hearts showed a decrease in ventricular maximum upstroke velocity of the action potential and conduction velocity, suggesting loss of cardiac voltage-gated sodium channel function. ECG showed QRS prolongation in adult knockout fish, and patch clamping showed decreased sodium current density in knockout ventricular myocytes and arrhythmias in knockout iPSC-CMs. Confocal imaging showed disorganized adherens junctions and mislocalization of mature Ncad (N-cadherin) with mapre2 loss of function, associated with a decrease of detyrosinated tubulin. MAPRE2 knockdown in iPSC-CMs led to an increase in microtubule growth velocity and distance, indicating changes in microtubule dynamics. Finally, knockdown of ttl encoding tubulin tyrosine ligase in mapre2 knockout larvae rescued tubulin detyrosination and ventricular maximum upstroke velocity of the action potential. Genetic ablation of mapre2 led to a decrease in voltage-gated sodium channel function, a hallmark of Brugada syndrome, associated with disruption of adherens junctions, decrease of detyrosinated tubulin as a marker of microtubule stability, and changes in microtubule dynamics. Restoration of the detyrosinated tubulin fraction with ttl knockdown led to rescue of voltage-gated sodium channel-related functional parameters in mapre2 knockout hearts. Taken together, our study implicates microtubule dynamics in the modulation of ventricular conduction.

Beyond CBD: Inhibitory effects of lesser studied phytocannabinoids on human voltage-gated sodium channels.

Introduction: Cannabis contains cannabidiol (CBD), the main non-psychoactive phytocannabinoid, but also many other phytocannabinoids that have therapeutic potential in the treatment of epilepsy. Indeed, the phytocannabinoids cannabigerolic acid (CBGA), cannabidivarinic acid (CBDVA), cannabichromenic acid (CBCA) and cannabichromene (CBC) have recently been shown to have anti-convulsant effects in a mouse model of Dravet syndrome (DS), an intractable form of epilepsy. Recent studies demonstrate that CBD inhibits voltage-gated sodium channel function, however, whether these other anti-convulsant phytocannabinoids affect these classic epilepsy drug-targets is unknown. Voltage-gated sodium (NaV) channels play a pivotal role in initiation and propagation of the neuronal action potential and NaV1.1, NaV1.2, NaV1.6 and NaV1.7 are associated with the intractable epilepsies and pain conditions. Methods: In this study, using automated-planar patch-clamp technology, we assessed the profile of the phytocannabinoids CBGA, CBDVA, cannabigerol (CBG), CBCA and CBC against these human voltage-gated sodium channels subtypes expressed in mammalian cells and compared the effects to CBD. Results: CBD and CBGA inhibited peak current amplitude in the low micromolar range in a concentration-dependent manner, while CBG, CBCA and CBC revealed only modest inhibition for this subset of sodium channels. CBDVA inhibited NaV1.6 peak currents in the low micromolar range in a concentration-dependent fashion, while only exhibiting modest inhibitory effects on NaV1.1, NaV1.2, and NaV1.7 channels. CBD and CBGA non-selectively inhibited all channel subtypes examined, whereas CBDVA was selective for NaV1.6. In addition, to better understand the mechanism of this inhibition, we examined the biophysical properties of these channels in the presence of each cannabinoid. CBD reduced NaV1.1 and NaV1.7 channel availability by modulating the voltage-dependence of steady-state fast inactivation (SSFI, V0.5 inact), and for NaV1.7 channel conductance was reduced. CBGA also reduced NaV1.1 and NaV1.7 channel availability by shifting the voltage-dependence of activation (V0.5 act) to a more depolarized potential, and for NaV1.7 SSFI was shifted to a more hyperpolarized potential. CBDVA reduced channel availability by modifying conductance, SSFI and recovery from SSFI for all four channels, except for NaV1.2, where V0.5 inact was unaffected. Discussion: Collectively, these data advance our understanding of the molecular actions of lesser studied phytocannabinoids on voltage-gated sodium channel proteins.

Abstract 276: Inflammatory co-regulation of voltage-gated sodium channels and Na,K-ATPases in metastatic breast cancer

Abstract Changes in membrane potential dramatically impact the progression of cancer, yet little is known as to how ion transport contributes to disease progression. Voltage-gated sodium channels (VGSCs) and Na,K-ATPases are particularly attractive targets as both are upregulated in many cancers and have been correlated specifically with invasiveness in vitro and metastasis and in vivo. However, their precise upstream regulators and downstream effectors remain unclear. Additionally, it has not been determined whether these transporters are co-regulated in cancer, although they display overlap in their downstream effectors and have complementary activity on sodium ions. Such a system has, however, been described in neuronal models of pain, wherein inflammatory injury leads to an upregulation in VGSC expression and a subsequent proportional increase in Na,K-ATPase. We sought to describe a similar phenomenon in cancer cells, which could indicate that this neuronal response to injury may be hijacked as a driver of metastatic development. To elucidate the impact of inflammatory mediators on these ion transporters in cancer cells, we assessed the effects of TNFα and PGE2 on VGSCs and Na,K-ATPases in the human breast epithelial or cancer cells MCF-10a, MCF-7, and MDA-MB-231. RT-qPCR revealed increases in RNA expression for the clinically relevant VGSC subunit Nav1.5. Similar RNA increases were observed in the α1 and α3 subunits of Na,K-ATPase, although in all cases the magnitude and time point of maximal effect differed by cell line. Then a 1hr challenge of TNFα on MDA-MB-231 cells was used to examine whether the targets were independently regulated by inflammatory mediators, co-regulated via the transport of sodium ions, or co-regulated by the expression of one protein influencing the expression of the other protein. Transfection with siRNA against either the α1 or α3 subunit of Na,K-ATPase did not inhibit the inflammatory-mediated increase in RNA for Nav1.5. However, transfection with anti-Nav1.5 siRNA inhibited the upregulation of both Na,K-ATPase subunits. TNFα challenge following pre-incubation of MDA-MB-231 cells in a sodium-free medium completely inhibited the inflammatory-mediated upregulation of Nav1.5, Na,K-ATPase α1 or α3 RNA. Co-incubation of TNFα with the VGSC blocking drug protoxin I inhibited the inflammatory-mediated increase in Na,K-ATPase α1 or α3 RNA, but co-incubation with the Na,K-ATPase blocking drug ouabain did not inhibit the increase in Nav1.5 RNA and indeed may have potentiated the increase. Together these results are evidence that VGSCs and Na,K-ATPase are co-regulated by inflammatory mediators in metastatic breast cancer, that sodium is essential to this mechanism, and that the presence and ion-transporting function of VGSCs is necessary for the inflammatory-mediated effect on Na,K-ATPase but not vice versa. Citation Format: Steven D. Scahill, Harry J. Gould, Dennis Paul. Inflammatory co-regulation of voltage-gated sodium channels and Na,K-ATPases in metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 276.

A nutraceutical product, extracted from Cannabis sativa, modulates voltage-gated sodium channel function

BackgroundPurified cannabidiol (CBD), a non-psychoactive phytocannabinoid, has gained regulatory approval to treat intractable childhood epilepsies. Despite this, artisanal and commercial CBD-dominant hemp-based products continue to be used by epilepsy patients. Notably, the CBD doses used in these latter products are much lower than that found to be effective in reducing seizures in clinical trials with purified CBD. This might be because these CBD-dominant hemp products contain other bioactive compounds, including phytocannabinoids and terpenes, which may exert unique effects on epilepsy-relevant drug targets. Voltage-gated sodium (NaV) channels are vital for initiation of neuronal action potential propagation and genetic mutations in these channels result in epilepsy phenotypes. Recent studies suggest that NaV channels are inhibited by purified CBD. However, the effect of cannabis-based products on the function of NaV channels is unknown.MethodsUsing automated-planar patch-clamp technology, we profile a hemp-derived nutraceutical product (NP) against human NaV1.1–NaV1.8 expressed in mammalian cells to examine effects on the biophysical properties of channel conductance, steady-state fast inactivation and recovery from fast inactivation.ResultsNP modifies peak current amplitude of the NaV1.1–NaV1.7 subtypes and has variable effects on the biophysical properties for all channel subtypes tested. NP potently inhibits NaV channels revealing half-maximal inhibitory concentration (IC50) values of between 1.6 and 4.2 μg NP/mL. Purified CBD inhibits NaV1.1, NaV1.2, NaV1.6 and NaV1.7 to reveal IC50 values in the micromolar range. The CBD content of the product equates to IC50 values (93–245 nM), which are at least an order of magnitude lower than purified CBD. Unlike NP, hemp seed oil vehicle alone did not inhibit NaV channels, suggesting that the inhibitory effects of NP are independent of hemp seed oil.ConclusionsThis CBD-dominant NP potently inhibits NaV channels. Future study of the individual elements of NP, including phytocannabinoids and terpenes, may reveal a potent individual component or that its components interact to modulate NaV channels.

Functional Voltage-Gated Sodium Channels Are Present in the Human B Cell Membrane.

B cells express various ion channels, but the presence of voltage-gated sodium (NaV) channels has not been confirmed in the plasma membrane yet. In this study, we have identified several NaV channels, which are expressed in the human B cell membrane, by electrophysiological and molecular biology methods. The sensitivity of the detected sodium current to tetrodotoxin was between the values published for TTX-sensitive and TTX-insensitive channels, which suggests the co-existence of multiple NaV1 subtypes in the B cell membrane. This was confirmed by RT-qPCR results, which showed high expression of TTX-sensitive channels along with the lower expression of TTX-insensitive NaV1 channels. The biophysical characteristics of the currents also supported the expression of multiple NaV channels. In addition, we investigated the potential functional role of NaV channels by membrane potential measurements. Removal of Na+ from the extracellular solution caused a reversible hyperpolarization, supporting the role of NaV channels in shaping and maintaining the resting membrane potential. As this study was mainly limited to electrophysiological properties, we cannot exclude the possible non-canonical functions of these channels. This work concludes that the presence of voltage-gated sodium channels in the plasma membrane of human B cells should be recognized and accounted for in the future.

Scorpion venom heat-resistant synthesized peptide ameliorates 6-OHDA-induced neurotoxicity and neuroinflammation: likely role of Nav 1.6 inhibition in microglia.

Microglia-related inflammation is associated with the pathology of Parkinson's disease. Functional voltage-gated sodium channels (VGSCs) are involved in regulating microglial function. Here, we aim to investigate the effects of scorpion venom heat-resistant synthesized peptide (SVHRSP) on 6-hydroxydopamine (6-OHDA)-induced Parkinson's disease-like mouse model and reveal its underlying mechanism. Unilateral brain injection of 6-OHDA was performed to establish Parkinson's disease mouse model. After behaviour test, brain tissues were collected for morphological analysis and protein/gene expression examination. Primary microglia culture was used to investigate the role of sodium channel Nav 1.6 in the regulation of microglia inflammation by SVHRSP. SVHRSP treatment attenuated motor deficits, dopamine neuron degeneration, activation of glial cells and expression of pro-inflammatory cytokines induced by 6-OHDA lesion. Primary microglia activation and the production of pro-inflammatory cytokines induced by lipopolysaccharide (LPS) were also suppressed by SVHRSP treatment. In addition, SVHRSP could inhibit mitogen-activated protein kinases (MAPKs) pathway, which plays pivotal roles in the pro-inflammatory response. Notably, SVHRSP treatment suppressed the overexpression of microglial Nav 1.6 induced by 6-OHDA and LPS. Finally, it was shown that the anti-inflammatory effect of SVHRSP in microglia was Nav 1.6 dependent and was related to suppression of sodium current and probably the consequent Na+ /Ca2+ exchange. SVHRSP might inhibit neuroinflammation and protect dopamine neurons via down-regulating microglial Nav 1.6 and subsequently suppressing intracellular Ca2+ accumulation to attenuate the activation of MAPKs signalling pathway in microglia.

Hyperexcitability of Nucleus Tractus Solitarii Neurons in the Streptozotocin‐Induced Model of Alzheimer's Disease

Besides the stereotypical problems of memory and cognition in patients with Alzheimer's Disease (AD), another highly prevalent symptom is of respiratory origin. We have repeatedly shown blunted respiratory responses to hypoxia in the streptozotocin (STZ) -induced AD model, which is similar to sleep disordered breathing found in nearly 80% of AD patients. Our current study focuses on the electrophysiological changes within the nucleus tractus solitarii (nTS), a brainstem area known to control respiratory function. Using male Sprague-Dawley rats (n = 15 rats, 240±8 g), we induced AD symptoms with microinjections of 2 mg/kg Streptozotocin (STZ) into the lateral cerebral ventricles. Control animals received 0.9 mM citrate buffer as vehicle control (CTL). The disease model was verified using the Morris water maze to test for memory dysfunction. We recorded nTS neuronal responses via whole cell patch clamp. Action potential (AP) discharge upon current-stimulation was higher in STZ-AD when compared to control. Also, spontaneous spike activity (no stimulation) was elevated. These data corroborate earlier results using a higher (2x1.5 mg) STZ dosage. Spontaneous post-synaptic currents in the absence of stimulation resemble nTS network activity. The number of spontaneous events in nTS neurons of the STZ-AD group was surprisingly similar to CTL in the current study. On the other hand, network activity mirrors the hyperexcitability seen from our current injection when using the higher STZ dosage. While there was no appreciable change in resting membrane potential, AP threshold of STZ-AD nTS cells shifted towards a more negative membrane potential, overall contributing to increased excitability in these cells. AP phase plane plots in STZ-AD had a significantly smaller overshoot with a reduced velocity of up and down stroke, possibly indicating altered function of voltage-gated sodium channels. Analysis of transient K+ currents (A-type) revealed a slight shift to higher activation voltages (from -20 mV [CTL] to 0 mV [STZ-AD]) in STZ-AD. This shift favors AP generation and may contribute to a lower spike threshold in STZ-AD. There were no changes in the steady state K+ currents between groups. In summary, nTS cells of the STZ-AD group showed increased excitability that may involve gating properties of voltage-gated sodium channels and A-type potassium channels. The observed neurophysiological changes in the nTS may be part of the neuronal correlate for increased breathing frequency and blunted (ceiling effect) respiratory responses to hypoxia in the STZ-AD model.

Pyrethrins elicit olfactory response and spatial repellency in Aedes albopictus.

Pyrethrum from dry flowers of Chrysanthemum is a well-known botanical insecticide and repellent. Its insecticidal activity attributes to its six insecticidal esters, collectively known as pyrethrins. Pyrethrins and its synthetic analogs pyrethroids exert their toxic action by modifying the function of voltage-gated sodium channels. Aside from insecticidal activity, pyrethrum has also been used to repel mosquitoes for centuries. Today, pyrethrum continues to be used as an active ingredient in mosquito coils and other mosquito-repellent devices globally. However, the mechanism of pyrethrum repellency remains largely unknown. Here we report that pyrethrum vapor induced spatial (non-contact) repellency in Aedes albopictus, a major vector of dengue and West Nile viruses. Using electroantennogram (EAG) recordings from adult antennae, we found that pyrethrum elicited EAG response in a dose-dependent manner. We then isolated the six insecticidal esters, pyrethrins I and II, cinerins I and II, jasmolins I and II from pyrethrum extract and discovered that five of the six esters, except jasmolin I, all elicited EAG responses. Furthermore, pyrethrins I and II, cinerin II and jasmolin II induced repellency, whereas cinerin I and jasmolin I did not. Of the six pyrethrins, four of them, pyrethrins I and II, cinerin II and jasmolin II, activate olfactory-receptor neurons and elicit spatial repellency in Ae. albopictus. Our study provided a foundation for future structure-function studies of pyrethrins, their cognate olfactory receptors and efficacies of repellency and for the development of new and more effective mosquito repellents for controlling vector-borne human diseases. © 2021 Society of Chemical Industry.

Structural Studies of the Interaction Between the Paddle Motif from NaV1.5Div and the Sea Anemone Toxin Anthopleurin-A

Voltage gated sodium channels (VGSC) are membrane proteins that are important in the central and peripheral nervous systems and cardiac muscles among others. The main function of VGSC is in the propagation of electrical signals by depolarizing excitable cells. Numerous diseases have been linked to defects in VGSC including epilepsy, mental retardation, and several ailments that could lead to sudden cardiac death. Furthermore, these channels are one of the primary targets of toxins from venomous animals. These toxins have been used as an excellent probe to study the function of VGSC due to their specific and potent effect on VGSC. The sea anemone toxin anthopleurin-A (ApA) inhibits the fast inactivation of the cardiac channel NaV1.5 by binding to the S3b-S4a motif (paddle motif) on the voltage sensing domain (VSD) of DIV. Here we used NMR spectroscopy to determine the solution structures of NaV1.5 DIV paddle motif and of ApA toxin, both in DPC micelles. The structure of the paddle motif takes a helix-turn-helix motif that aligns well with the cryo-EM structure of the full channel. Through paramagnetic relaxation enhancement, we determined that the paddle motif is mainly interacting with the interface region of the micelle. Based on 15N NMR relaxation studies, the paddle motif is mostly rigid with some residues at and around the loop in addition to the C-terminus undergoing ps-ns motion. NMR titration experiments between a 15N labeled paddle motif with unlabeled ApA toxin uncovered specific residues on the paddle that likely interact with the toxin. Further electrophysiology studies are needed to confirm the importance of these residues in toxin binding. We are in the process of determining the three-dimensional structure of the toxin-paddle complex in micelle.

N-alkylisatin-based microtubule destabilizers bind to the colchicine site on tubulin and retain efficacy in drug resistant acute lymphoblastic leukemia cell lines with less in vitro neurotoxicity

BackgroundDrug resistance and chemotherapy-induced peripheral neuropathy continue to be significant problems in the successful treatment of acute lymphoblastic leukemia (ALL). 5,7-Dibromo-N-alkylisatins, a class of potent microtubule destabilizers, are a promising alternative to traditionally used antimitotics with previous demonstrated efficacy against solid tumours in vivo and ability to overcome P-glycoprotein (P-gp) mediated drug resistance in lymphoma and sarcoma cell lines in vitro. In this study, three di-brominated N-alkylisatins were assessed for their ability to retain potency in vincristine (VCR) and 2-methoxyestradiol (2ME2) resistant ALL cell lines. For the first time, in vitro neurotoxicity was also investigated in order to establish their suitability as candidate drugs for future use in ALL treatment.MethodsVincristine resistant (CEM-VCR R) and 2-methoxyestradiol resistant (CEM/2ME2-28.8R) ALL cell lines were used to investigate the ability of N-alkylisatins to overcome chemoresistance. Interaction of N-alkylisatins with tubulin at the the colchicine-binding site was studied by competitive assay using the fluorescent colchicine analogue MTC. Human neuroblastoma SH-SY5Y cells differentiated into a morphological and functional dopaminergic-like neurotransmitter phenotype were used for neurotoxicity and neurofunctional assays. Two-way ANOVA followed by a Tukey’s post hoc test or a two-tailed paired t test was used to determine statistical significance.ResultsCEM-VCR R and CEM/2ME2-28.8R cells displayed resistance indices of > 100 to VCR and 2-ME2, respectively. CEM-VCR R cells additionally displayed a multi-drug resistant phenotype with significant cross resistance to vinblastine, 2ME2, colchicine and paclitaxel consistent with P-gp overexpression. Despite differences in resistance mechanisms observed between the two cell lines, the N-alkylisatins displayed bioequivalent dose-dependent cytotoxicity to that of the parental control cell line. The N-alkylisatins proved to be significantly less neurotoxic towards differentiated SH-SY5Y cells than VCR and vinblastine, evidenced by increased neurite length and number of neurite branch points. Neuronal cells treated with 5,7-dibromo-N-(p-hydroxymethylbenzyl)isatin showed significantly higher voltage-gated sodium channel function than those treated with Vinca alkaloids, strongly supportive of continued action potential firing.ConclusionsThe N-alkylisatins are able to retain cytotoxicity towards ALL cell lines with functionally distinct drug resistance mechanisms and show potential for reduced neurotoxicity. As such they pose as promising candidates for future implementation into anticancer regimes for ALL. Further in vivo studies are therefore warranted.

Neuroprotective effects of ranolazine versus pioglitazone in experimental diabetic neuropathy: Targeting Nav1.7 channels and PPAR-γ

Diabetic neuropathy (DN) is a common complication of diabetes mellitus (DM). Pathophysiology of DN includes inflammation and changes in expression and function of voltage-gated sodium channels (Nav) in peripheral nerves; and central reduction of Peroxisome Proliferator Activated Receptor-Gamma (PPAR-γ) expression. AimThis study explored the effect of ranolazine (RN) versus pioglitazone (PIO) in DN induced in rats. The role of sciatic interleukin (IL)-1β, tumor necrosis factor-alpha (TNF)-α, Nav1.7, and spinal PPAR-γ expressions were determined. Materials and methodsFor induction of Type-2 DM, 40 high fat diet-fed rats were challenged by a single dose of intraperitoneal streptozotocin (30 mg/kg). One week later, oral PIO (10 mg/kg; once daily) or RN (20, 50 and 100 mg/kg; twice daily) were administered for six weeks. Weekly body weight and fasting blood sugar (FBS) were measured. Rats were tested for thermal hyperalgesia and mechanical allodynia. At the end of the experiment, sciatic nerves homogenates were examined for TNF-α and IL-1B levels, and Nav1.7 channel expression. Segments of spinal cords were investigated for the PPAR-γ gene expression. Evaluation of histopathology of sciatic nerves and spinal cords were done. Key findingsIn diabetic rats, PIO and RN individually improved evoked-pain behaviors, reduced sciatic TNF-α and 1L-1B levels; downregulated expressional levels of Nav1.7 channels; and increased the spinal PPAR-γ gene expression. RN in the dose of 100 mg/kg/day showed the most advantageous effects. SignificanceRN has neuroprotective effects in Type-2 diabetes-induced DN. Further studies of combined RN-PIO treatment are recommended, especially in diabetic patients with cardiovascular co-morbidity.

The General Anesthetic Isoflurane Bilaterally Modulates Neuronal Excitability

SummaryVolatile anesthetics induce hyperactivity during induction while producing anesthesia at higher concentrations. They also bidirectionally modulate many neuronal functions. However, the neuronal mechanism is unclear. The effects of isoflurane on sodium channel currents were analyzed in acute mouse brain slices, including sodium leak (NALCN) currents and voltage-gated sodium channels (Nav) currents. Isoflurane at sub-anesthetic concentrations increased the spontaneous firing rate of CA3 pyramidal neurons, whereas anesthetic concentrations of isoflurane decreased the firing rate. Isoflurane at sub-anesthetic concentrations enhanced NALCN conductance but minimally inhibited Nav currents. Isoflurane at anesthetic concentrations depressed Nav currents and action potential amplitudes. Isoflurane at sub-anesthetic concentrations depolarized resting membrane potential (RMP) of neurons, whereas hyperpolarized the RMP at anesthetic concentrations. Isoflurane at low concentrations induced hyperactivity in vivo, which was diminished in NALCN knockdown mice. In conclusion, enhancement of NALCN by isoflurane contributes to its bidirectional modulation of neuronal excitability and the hyperactivity during induction.

Ca2+-dependent recruitment of voltage-gated sodium channels underlies bilirubin-induced overexcitation and neurotoxicity

Neonatal jaundice is prevalent among newborns and can lead to severe neurological deficits, particularly sensorimotor dysfunction. Previous studies have shown that bilirubin (BIL) enhances the intrinsic excitability of central neurons and this can potentially contribute to their overexcitation, Ca2+ overload, and neurotoxicity. However, the cellular mechanisms underlying elevated neuronal excitability remain unknown. By performing patch-clamp recordings from neonatal neurons in the rat medial vestibular nucleus (MVN), a crucial relay station for locomotor and balance control, we found that BIL (3 μM) drastically increases the spontaneous firing rates by upregulating the current-mediated voltage-gated sodium channels (VGSCs), while shifting their voltage-dependent activation toward more hyperpolarized potentials. Immunofluorescence labeling and western immunoblotting with an anti-NaV1.1 antibody, revealed that BIL elevates the expression of VGSCs by promoting their recruitment to the membrane. Furthermore, we found that this VGSC-trafficking process is Ca2+ dependent because preloading MVN neurons with the Ca2+ buffer BAPTA-AM, or exocytosis inhibitor TAT-NSF700, prevents the effects of BIL, indicating the upregulated activity and density of functional VGSCs as the core mechanism accountable for the BIL-induced overexcitation of neonatal neurons. Most importantly, rectification of such overexcitation with a low dose of VGSC blocker lidocaine significantly attenuates BIL-induced cell death. We suggest that this enhancement of VGSC currents directly contributes to the vulnerability of neonatal brain to hyperbilirubinemia, implicating the activity and trafficking of NaV1.1 channels as a potential target for neuroprotection in cases of severe jaundice.

The Emerging Role of Voltage-Gated Sodium Channels in Tumor Biology.

Voltage-gated sodium channels (VGSCs) are transmembrane proteins which function as gates that control the flux of ions across the cell membrane. They are key ion channels for action potentials in excitable tissues and have important physiological functions. Abnormal function of VGSCs will lead to dysfunction of the body and trigger a variety of diseases. Various studies have demonstrated the participation of VGSCs in the progression of different tumors, such as prostate cancer, cervical cancer, breast cancer, and others, linking VGSC to the invasive capacity of tumor cells. However, it is still unclear whether the VGSC regulate the malignant biological behavior of tumors. Therefore, this paper systematically addresses the latest research progress on VGSCs subunits and tumors and the underlying mechanisms, and it summarizes the potential of VGSCs subunits to serve as potential targets for tumor diagnosis and treatment.

Change in hippocampal voltage-gated I sodium channel of Lithium chloride-Pilocarpine epileptic rat model

Objective To research the changes in hippocampal voltage-gated sodium channel of Lithium chloride-Pilocarpine epileptic rat models, includingⅠsodium channel α subunit protein (Nav1.1), mRNA of Ⅰso-dium channel alpha subunit protein gene and function of sodium channel. Methods Epileptic rat models of Lithium chloride-Pilocarpine were established.Nav1.1 expression in the hippocampus of experimental rats was detected by immunohistochemical staining method, and the changes in voltage-gated sodium channel function (the current-voltage curves, activation and inactivation curves and the recovery curve) of hippocampus nerve cells were detected by whole-cell patch-clamp technique. Results (1)The Lithium chloride-Pilocarpine rat models were successfully reproduced.Three stages of behavior (acute, latent and chronic) of rat models were observed.The blank control group was free of seizure.(2)Immunohistochemistry results: neurons in CA1 and DG regions of hippocampal of epileptic rats were normal, and there was no obvious change in the expression of Nav1.1.In CA3 area, the degeneration and necrosis of neurons were obvious.Staining of Nav1.1 became superficial and even disappeared in these areas, but the normal tissues were enhanced around degenerative and necrotic neurons.Compared with the blank control group, the expression of Nav1.1 in the model group was higher(0.235±0.008 vs.0.210±0.002), and there was statistically significant diffe-rence (t′=-7.426, P<0.05). (3) The whole-cell patch-clamp technique showed that the sodium current density of the model group increased significantly compared with that of the blank group [(-319.70±28.24) pA/pF vs. (-229.06±26.01) pA/pF, t=8.178, P<0.05], the threshold value of activation curve decreased (4.15±0.80 vs. 4.50±0.85, t=11.020, P<0.05), the threshold value of inactivation curve increased (7.47±0.53 vs. 6.24±0.31, t=6.940, P<0.05), and the recovery time after inactivation shortened [(1.36±0.15) ms vs. (1.86±0.21) ms, t=6.712, P<0.05], and there were all statistically significant differences. Conclusion Repeated seizures can lead to increase Nav1.1 compensatory expression of, and significantly increase sodium channel current density, while the threshold value of activation curve decreases, the threshold value of inactivation curve rises, and the recovery time after inactivation is shortened, which eventually leads to increased neuron excitability and is more likely to cause seizures. Key words: Voltage-gated Ⅰ sodium channel, α subunit protein; Epilepsy; Pilocarpine; Whole-cell patch-clamp technique

The influence of voltage-gated sodium channels on human gastrointestinal nociception.

Abdominal pain is a frequent and persistent problem in the most common gastrointestinal disorders, including irritable bowel syndrome and inflammatory bowel disease. Pain adversely impacts quality of life, incurs significant healthcare expenditures, and remains a challenging issue to manage with few safe therapeutic options currently available. It is imperative that new methods are developed for identifying and treating this symptom. A variety of peripherally active neuroendocrine signaling elements have the capability to influence gastrointestinal pain perception. A large and growing body of evidence suggests that voltage-gated sodium channels (VGSCs) play a critical role in the development and modulation of nociceptive signaling associated with the gut. Several VGSC isoforms demonstrate significant promise as potential targets for improved diagnosis and treatment of gut-based disorders associated with hyper- and hyposensitivity to abdominal pain. In this article, we critically review key investigations that have evaluated the potential role that VGSCs play in visceral nociception and discuss recent advances related to this topic. Specifically, we discuss the following: (a) what is known about the structure and basic function of VGSCs, (b) the role that each VGSC plays in gut nociception, particularly as it relates to human physiology, and (c) potential diagnostic and therapeutic uses of VGSCs to manage disorders associated with chronic abdominal pain.

The peptide toxin δ-hexatoxin-MrIX inhibits fast inactivation of NaVs in mouse cerebellar granule cells

Spider venom is rich in peptide toxins that could be used to explore the structure and function of voltage-gated sodium channels (NaVs). This study has characterized a 44-amino acid peptide toxin, δ-hexatoxin-MrIX (δ-HXTX-MrIX), from the venom of the spider Macrothele raveni. δ-hexatoxin-MrIX potently inhibited the fast inactivation of NaVs in mouse cerebellar granule cells (CGCs) with an EC50 of 35.3 ± 5.9 nM. The toxin shifted both the steady-state activation and the steady-state inactivation curves of CGC NaVs to the hyperpolarized direction. δ-hexatoxin-MrIX also acted on NaV1.3 and NaV1.4 channels heterologously expressed in HEK293T cells, as well as on NaVs in acutely isolated cockroach DUM neurons. However, the NaV1.5, NaV1.7 and NaV1.8 channels were resistant to δ-hexatoxin-MrIX. The toxin inhibited the fast inactivation of NaV1.3 and NaV1.4 with high affinity (EC50 values of 82.0 ± 3.0 nM and 24.0 ± 4.7 nM, respectively), but the saturating dose of toxin showed distinct efficacy on these two types of channels. δ-hexatoxin-MrIX is a peptide toxin acting on CGC NaVs and could be used as a pharmacological tool to explore the role of NaVs in granule cell maturation during cerebellum development.

Activation of sodium channel by a novel α-scorpion toxin, BmK NT2, stimulates ERK1/2 and CERB phosphorylation through a Ca2+ dependent pathway in neocortical neurons

Neuronal excitability controls the expression of a variety of genes and proteins and therefore regulates neurite outgrowth and synapse formation, fundamental physiological processes controlling learning and memory. Scorpion venom contains many neurotoxins which alter ion channel activities that influence neuronal excitability. In this study, a novel scorpion peptide termed BmK NT2 was purified from venom of Chinese scorpion Buthus martensii Karsch by combining mass spectrum mapping and intracellular Ca2+ concentration measurement in primary cultured neocortical neurons. Electrophysiological experiments demonstrated that BmK NT2 concentration-dependently delayed inactivation of voltage-gated sodium channels (VGSCs) with an EC50 value of 0.91μM, and shifted the steady-state activation and inactivation of VGSCs to hyperpolarized direction. The effects of BmK NT2 on electrophysiological characteristics of VGSCs were similar to that of α-scorpion toxins. BmK NT2 altered Ca2+ dynamics and increased phosphorylation of extracellular-regulated protein kinases (ERK) 1/2 and cAMP-response element binding (CREB) proteins, which were eliminated by the VGSC blocker, tetrodotoxin. These data demonstrate that BmK NT2 is a novel VGSC α-scorpion toxin which is sufficient to increase the phosphorylation of ERK1/2 and CREB proteins, suggesting that modulation of VGSC function by α-scorpion toxin exerts neurotrophic effect in primary cultured neocortical neurons.