Activation Of Voltage-gated Sodium Channels Research Articles

Structural Foundation for Insect-Selective Activity of Acylpolyamine Toxins from Spider Araneus ventricosus.

Spider venoms are insecticidal mixtures with diverse biological activities, and acylpolyamines are their small molecular active components. However, the mechanism for the insecticidal activity of acylpolyamines remains to be elucidated. Here, the structure and function of two acylpolyamine toxins, AVTX-622 and AVTX-636, from Araneus ventricosus were investigated. Nuclear magnetic resonance (NMR) analysis illustrated that the structure of two toxins was very similar, and compared to AVTX-636, AVTX-622 only missed a methylene group in the linker region between the polyamine head and tail. Both the two toxins did not inhibit on voltage-gated sodium channels in mammalian neuronal cells. Intriguingly, AVTX-622, but not AVTX-636, inhibited voltage-gated sodium channels in DUM neuronal cells of Periplaneta americana. Further animal test displayed that the paralyzing potency of AVTX-622 on insect was over ten-times stronger than that of AVTX-636. These findings indicate that a single methylene deletion from AVTX-636 offered AVTX-622 the insect-selective voltage-gated sodium channel activity, which not only elucidated structure-function of the toxins, but also provided new clues for insect-selective insecticide design.

Type II toxins from sea anemone Heteractis crispa with various effects on activation and inactivation of voltage-gated sodium channels

Type II toxins from sea anemone Heteractis crispa with various effects on activation and inactivation of voltage-gated sodium channels

BmK AEP, an Anti-Epileptic Peptide Distinctly Affects the Gating of Brain Subtypes of Voltage-Gated Sodium Channels.

BmK AEP, a scorpion peptide purified form the venom of Buthus martensii Karsch, has been reported to display anti-epileptic activity. Voltage-gated sodium channels (VGSCs) are responsible for the rising phase of action potentials (APs) in neurons and, therefore, controlling neuronal excitability. To elucidate the potential molecular mechanisms responsible for its anti-epileptic activity, we examined the influence of BmK AEP on AP firing in cortical neurons and how BmK AEP influences brain subtypes of VGSCs (Nav1.1–1.3 and Nav1.6). BmK AEP concentration-dependently suppresses neuronal excitability (AP firing) in primary cultured cortical neurons. Consistent with its inhibitory effect on AP generation, BmK AEP inhibits Na+ peak current in cortical neurons with an IC50 value of 2.12 µM by shifting the half-maximal voltage of activation of VGSC to hyperpolarized direction by ~7.83 mV without affecting the steady-state inactivation. Similar to its action on Na+ currents in cortical neurons, BmK AEP concentration-dependently suppresses the Na+ currents of Nav1.1, Nav1.3, and Nav1.6, which were heterologously expressed in HEK-293 cells, with IC50 values of 3.20, 1.46, and 0.39 µM with maximum inhibition of 82%, 56%, and 93%, respectively. BmK AEP shifts the voltage-dependent activation in the hyperpolarized direction by ~15.60 mV, ~9.97 mV, and ~6.73 mV in Nav1.1, Nav1.3, and Nav1.6, respectively, with minimal effect on steady-state inactivation. In contrast, BmK AEP minimally suppresses Nav1.2 currents (~15%) but delays the inactivation of the channel with an IC50 value of 1.69 µM. Considered together, these data demonstrate that BmK AEP is a relatively selective Nav1.6 gating modifier which distinctly affects the gating of brain subtypes of VGSCs.

Jaburetox, a natural insecticide derived from Jack Bean Urease, activates voltage-gated sodium channels to modulate insect behavior

Jaburetox (Jbtx) is an insecticidal peptide derived from Canavalia ensiformis urease, whose mechanism of action is not completely elucidated. We employed behavioral, electromyographical and electrophysiological protocols to identify the cellular and molecular targets involved in the Jbtx entomotoxicity in cockroaches and locusts. In Nauphoeta cinerea, Jbtx (32 μg/g) altered the locomotory behaviour inducing a significative decrease in the distance travelled followed by a significant increase in stopped time (52 ± 85 cm and 2573 ± 89 s, p < .05, n = 40). Jbtx (8 to 32 μg/g body weight, respectively) also increased the leg and antennae grooming activities (p < .05, n = 40, respectively). Jbtx (8 to 16 μg/g) induced a maximum neuromuscular blockade of 80.72% (n = 6, p < .05) and was cardiotoxic, decreasing the cockroach heart rate. The electrophysiological profiles of both muscle and nerve of L. migratoria showed that Jbtx (2.5 × 10−7 and 2.5 × 10−3 μg/ body weight) induced a significant increase in the amplitude of nerve action potentials (n = 5, p < .05). Voltage clamp analysis of Jbtx (200 nM) applied in Xenopus laevis oocytes heterologously expressed with Nav 1.1 channels showed a significant increase in the sodium currents. In conclusion, this work revealed that the entomotoxic activity of Jbtx involves complex behavioral alterations that begins with an initial activation of voltage-gated sodium channels.

µ-TRTX-Ca1a: a novel neurotoxin from Cyriopagopus albostriatus with analgesic effects.

Human genetic and pharmacological studies have demonstrated that voltage-gated sodium channels (VGSCs) are promising therapeutic targets for the treatment of pain. Spider venom contains many toxins that modulate the activity of VGSCs. To date, only 0.01% of such spider toxins has been explored, and thus there is a great potential for discovery of novel VGSC modulators as useful pharmacological tools or potential therapeutics. In the current study, we identified a novel peptide, µ-TRTX-Ca1a (Ca1a), in the venom of the tarantula Cyriopagopus albostriatus. This peptide consisted of 38 residues, including 6 cysteines, i.e. IFECSISCEIEKEGNGKKCKPKKCKGGWKCKFNICVKV. In HEK293T or ND7/23 cells expressing mammalian VGSCs, this peptide exhibited the strongest inhibitory activity on Nav1.7 (IC50 378 nM), followed by Nav1.6 (IC50 547 nM), Nav1.2 (IC50 728 nM), Nav1.3 (IC50 2.2 µM) and Nav1.4 (IC50 3.2 µM), and produced negligible inhibitory effect on Nav1.5, Nav1.8, and Nav1.9, even at high concentrations of up to 10 µM. Furthermore, this peptide did not significantly affect the activation and inactivation of Nav1.7. Using site-directed mutagenesis of Nav1.7 and Nav1.4, we revealed that its binding site was localized to the DIIS3-S4 linker region involving the D816 and E818 residues. In three different mouse models of pain, pretreatment with Cala (100, 200, 500 µg/kg) dose-dependently suppressed the nociceptive responses induced by formalin, acetic acid or heat. These results suggest that Ca1a is a novel neurotoxin against VGSCs and has a potential to be developed as a novel analgesic.

Activation of human macrophage sodium channels regulates RNA processing to increase expression of the DNA repair protein PPP1R10

Prior work demonstrated that a splice variant of SCN5A, a voltage-gated sodium channel gene, acts as a cytoplasmic sensor for viral dsRNA in human macrophages. Expression of this channel also polarizes macrophages to an anti-inflammatory phenotype in vitro and in vivo. Here we utilized global expression analysis of splice variants to identify novel channel-dependent signaling mechanisms. Pharmacological activation of voltage-gated sodium channels in human macrophages, but not treatment with cytoplasmic poly I:C, was associated with splicing of a retained intron in transcripts of PPP1R10, a regulator of phosphatase activity and DNA repair. Microarray analysis also demonstrated expression of a novel sodium channel splice variant, human macrophage SCN10A, that contains a similar exon deletion as SCN5A. SCN10A localizes to cytoplasmic and nuclear vesicles in human macrophages. Simultaneous expression of human macrophage SCN5A and SCN10A was required to decrease expression of the retained intron and increase protein expression of PPP1R10. Channel activation also increased protein expression of the splicing factor EFTUD2, and knockdown of EFTUD2 prevented channel dependent splicing of the retained PPP1R10 intron. Knockdown of the SCN5A and SCN10A variants in human macrophages reduced the severity of dsDNA breaks induced by treatment with bleomycin and type 1 interferon. These results suggested that human macrophage SCN5A and SCN10A variants mediate an innate immune signaling pathway that limits DNA damage through increased expression of PPP1R10. The functional significance of this pathway is that it may prevent cytotoxicity during inflammatory responses.

In-vitro effects of the FS50 protein from salivary glands of Xenopsylla cheopis on voltage-gated sodium channel activity and motility of MDA-MB-231 human breast cancer cells.

Voltage-gated sodium channel activity enhances the motility and oncogene expression of metastasic cancer cells that express a neonatal alternatively spliced form of the NaV1.5 isoform. We reported previously that FS50, a salivary protein from Xenopsylla cheopis, showed inhibitory activity against the NaV1.5 channel when assayed in HEK 293T cells and antiarrhythmia effects on rats and monkeys after induction of arrhythmia by BaCl2. This study aims to identify the effect of FS50 on voltage-gated sodium channel activity and the motility of MDA-MB-231 human breast cancer cells in vitro. NaV1.5 was abnormally expressed in the highly metastatic breast cancer cell line MDA-MB-231, but not in the MCF-7 cell line. FS50 significantly inhibited sodium current, migration, and invasion in a dose-dependent manner, but had no effect on the proliferation of MDA-MB-231 cells at the working concentrations (1.5-12 μmol/l) after a long-term treatment for 48 h. Meanwhile, FS50 decreased NaV1.5 mRNA expression without altering the total protein level in MDA-MB-231 cells. Correspondingly, the results also showed that MMP-9 activity and the ratio of MMP-9 mRNA to TIMP-1 mRNA were markedly decreased by FS50. Taken together, our findings highlighted for the first time an inhibitory effect of a salivary protein from a blood-feeding arthropod on breast cancer cells through the NaV1.5 channel. Furthermore, this study provided a new candidate leading molecule against antitumor cells expressing NaV1.5.

Somatosensory Neurons Enter a State of Altered Excitability during Hibernation

Hibernation in mammals involves prolonged periods of inactivity, hypothermia, hypometabolism, and decreased somatosensation. Peripheral somatosensory neurons play an essential role in the detection and transmission of sensory information to CNS and in the generation of adaptive responses. During hibernation, when body temperature drops to as low as 2°C, animals dramatically reduce their sensitivity to physical cues [1, 2]. It is well established that, in non-hibernators, cold exposure suppresses energy production, leading to dissipation of the ionic and electrical gradients across the plasma membrane and, in the case of neurons, inhibiting the generation of action potentials [3]. Conceivably, such cold-induced elimination of electrogenesis could be part of a general mechanism that inhibits sensory abilities in hibernators. However, when hibernators become active, the bodily functions-including the ability to sense environmental cues-return to normal within hours, suggesting the existence of mechanisms supporting basal functionality of cells during torpor and rapid restoration of activity upon arousal. We tested this by comparing properties of somatosensory neurons from active and torpid thirteen-lined ground squirrels (Ictidomys tridecemlineatus). We found that torpid neurons can compensate for cold-induced functional deficits, resulting in unaltered resting potential, input resistance, and rheobase. Torpid neurons can generate action potentials but manifest markedly altered firing patterns, partially due to decreased activity of voltage-gated sodium channels. Our results provide insights into the mechanism that preserves somatosensory neurons in a semi-active state, enabling fast restoration of sensory function upon arousal. These findings contribute to the development of strategies enabling therapeutic hypothermia and hypometabolism.

Abstract 1950: Targeted silencing of NaV1.5 channel suppresses cell proliferation and invasion in ovarian cancer cells through inhibition of PI3K/AKT and Integrinβ-1/FAK/Src axis

Abstract Ovarian cancer (OCa) is the leading cause of gynecological cancer related deaths. OCa is one of the most aggressive cancer and associated with poor prognosis and survival rates (5-year survival rates about 50%). OCa cells have highly invasive metastatic phenotype due to mutations, altered signaling pathways and deregulated of control mechanisms and patients with metastatic disease have poorer prognosis (5-year survival is 20%). The major reasons for patient death include significant intratumoral heterogeneity, early metastasis and development of resistance to currently used chemotherapeutics. Therefore, identification of novel molecular targets and therapeutics strategies are urgently needed to enhance the efficacy of current therapies and prolong patient survival. Ion channels are important signaling molecules expressed in a wide range of tissues where they have significant involvement in determining a variety of cellular functions: solute transport, volume control, enzyme activity, secretion, invasion, gene expression, excitation-contraction coupling and intercellular communication. Studies indicated that there are significant differences in the regulation and the function of ion channels between normal and cancer cells. Voltage gated sodium channels (VGSC) is a group of ion channels that has been correlated with OCa because of their higher expression in highly metastatic ovarian cancer cells. Importantly, VGSC activity contributes to many cellular behaviors integral to metastasis in breast and other cancers including OCA. In this study, we investigated the NaV1.5 as integral component of the metastatic process in human OCa. The aim of the current study was to reveal molecular mechanisms underlying the effects of NaV1.5 down-regulation and investigate the effects on OCa in vitro. In this study, the ovarian cancer cell lines (HEYA8, SKOV3-IP1, SKOV3-TR (taxol resistant)) were used. NaV1.5 and Control non-silencing small interfering RNAs (siRNA) were employed for therapy. As in vitro experiments, cell proliferation, colony formation, invasion, western blot analysis were performed. Our results showed that specific NaV1.5 siRNA treatments caused a significant reduction in cell proliferation, colony formation, and invasion capacity in OCa cells (p&amp;lt;0.0001). To reveal molecular mechanisms underlying the effects of NaV1.5 down-regulation, we evaluated signaling pathways regulating cell proliferation and invasion/metastasis by western blot analysis and found that NaV1.5 expression promotes expression of PI3K/AKT, Integrinβ1/FAK/Src and P70S6K. In conclusion, our results suggest that Nav1.5 channels may contribute OCa tumorigenesis and metastasis through the upregulation of oncogenic pathways and serve as a potential therapeutic target in OCa. Citation Format: Mumin Alper Erdogan, Bulent Ozpolat. Targeted silencing of NaV1.5 channel suppresses cell proliferation and invasion in ovarian cancer cells through inhibition of PI3K/AKT and Integrinβ-1/FAK/Src axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1950.

VOLTAGE‐GATED SODIUM CHANNEL ACTIVATION TRIGGERS CALCIUM INFLUX AND HIPPOCAMPAL NEURON COMPLEXITY

Background and significanceAfter stroke, the molecular mechanisms underlying recovery are similar to the development of the nervous system. During development, neuronal activity regulates both the morphology and connectivity of neurons. Neuronal activity primarily involves voltage‐gated sodium channel (VGNaC) activation. Calcium signaling through the N‐methyl‐D‐aspartate receptor (NMDAR) drives morphological changes such as, dendritic arborization, synaptogenesis, and spinogenesis. VGNaC activation influences NMDAR function making this pathway an attractive therapeutic target for stroke recovery.HypothesisActivation of VGNaCs by PbTx‐2 augments NMDAR signaling resulting in and increased Ca2+ influx leading to dendritic arborization and spinogenesis in hippocampal neurons.Experimental design and MethodsWe used the VGNaC gating modifier, brevetoxin (PbTx‐2), to assess the effects of VGNaC activation on neuronal morphology. We defined the calcium‐signaling pathway triggered by PbTx‐2 in a population of hippocampal neurons (HNs), and evaluated PbTx‐2‐induced effects on dendritic arborization and spinogenesis in organotypic hippocampal slice culture (OHSC).HNscells were prepared from E18 Swiss Webster mouse embryos and plated in 96‐well plates. All experiments were run on (10 Days in vitro). A FlexStation2 was used to monitor PbTx‐2‐induced Ca2+ influx.OHSCSlices were prepared from P5, Thy‐1 YFP transgenic, mouse pups. Slices were treated with Pbtx‐2 18H after plating. A Leica SP8 confocal microscope was used to generate Z‐stack images of neurons or dendrites. Imaris‐XT software was used to reconstruct 3D‐images of the dendritic arbor, run Sholl analysis, and measure spine density.Results and ConclusionIn HNs, the EC50 for PbTx‐2‐induced Ca2+ influx was 376 nM (95% CI, 281–504 nM). The PbTx‐2 triggered Ca2+ influx in HNs was through the reverse mode of operation of Na+/Ca2+ exchanger, the NMDAR and the L‐type Ca2+ channel pathways. In OHSC, four‐day treatment with PbTx‐2 (100 nM) produced significant increases in dendritic arbor complexity as determined by Sholl analysis (p&lt;0.005, ANOVA). PbTx‐2 treatment (300 nM) produced significant increases in spine density (p&lt;0.001, ANOVA). Modifying the gating properties of VGNaC using PbTx‐2 enhanced the morphology of hippocampal neurons. Overall, these studies are consistent with the hypothesis that VGNaC activation may represent a novel pharmacological strategy to regulate NMDAR function and aid in recovery post‐stroke.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Spatiotemporal magnetic fields enhance cytosolic Ca2+ levels and induce actin polymerization via activation of voltage-gated sodium channels in skeletal muscle cells

Cellular function is modulated by the electric membrane potential controlling intracellular physiology and signal propagation from a motor neuron to a muscle fiber resulting in muscle contraction. Unlike electric fields, magnetic fields are not attenuated by biological materials and penetrate deep into the tissue. We used complex spatiotemporal magnetic fields (17–70 mT) to control intracellular signaling in skeletal muscle cells. By changing different parameters of the alternating magnetic field (amplitude, inversion time, rotation frequency), we induced transient depolarization of cellular membranes leading to i) Na+ influx through voltage-gated sodium channels (VGSC), ii) cytosolic calcium increase, and iii) VGSC- and ryanodine receptor-dependent increase of actin polymerization. The ion fluxes occurred only, when the field was applied and returned to baseline after the field was turned off. The 30-s-activation-cycle could be repeated without any loss of signal intensity. By contrast, static magnetic fields of the same strength exhibited no effect on myotube Ca2+ levels. Mathematical modeling suggested a role for the alternating magnetic field-induced eddy current, which mediates a local change in the membrane potential triggering the activation of VGSC. These findings might pave the way for the use of complex magnetic fields to improve function of skeletal muscles in myopathies.

Reduced cooperativity of voltage-gated sodium channels in the hippocampal interneurons of an aged mouse model of Alzheimer's disease.

Beta amyloid (A[Formula: see text] ) associated with Alzheimer's disease (AD) leads to abnormal behavior in inhibitory neurons, resulting in hyperactive neuronal networks, epileptiform behavior, disrupted gamma rhythms, and aberrant synaptic plasticity. Previously, we used a dual modeling-experimental approach to explain several observations, including failure to reliably produce action potentials (APs), smaller AP amplitudes, higher resting membrane potential, and higher membrane depolarization in response to a range of stimuli in hippocampal inhibitory neurons from 12- to 16-month-old female AP Pswe/PSEN1DeltaE9 (APdE9) AD mice as compared to age-matched non-transgenic (NTG) mice. Our experimental results also showed that AP initiation in interneurons from APdE9 mice are significantly different from that of NTG mice. APs in interneurons from NTG mice are characterized by abrupt onset and an upstroke that is much steeper and occurs with larger variability as compared to cells from APdE9 mice. The phase plot (the rate of change of membrane potential versus the instantaneous membrane potential) of APs produced by interneurons from APdE9 mice shows a biphasic behavior, whereas that from NTG mice shows a monophasic behavior. Here we show that using the classic Hodgkin-Huxley (HH) formalism for the gating of voltage-gated sodium channels (VGSCs) in a single-compartment neuron, we cannot reproduce these features, and a model that takes into account a cooperative activation of VGSCs is needed. We also argue that considering a realistic multi-compartment neuron where the kinetics of VGSC is modeled by HH formalism, as done in the past, would not explain our observations when APs from both NTG and APdE9 mice are considered simultaneously. Wefurther show that VGSCs in interneurons from APdE9 mice exhibit significantly lower cooperativity in their activation as compared to those from NTG mice.

Immune effects of the neurotoxins ciguatoxins and brevetoxins

Ciguatoxins (CTXs) and brevetoxins (PbTxs) are phycotoxins that can accumulate along the marine food chain and thus cause seafood poisoning in humans, namely “ciguatera fish poisoning” (CFP) and “neurotoxic shellfish poisoning” (NSP), respectively. CFP is characterized by early gastrointestinal symptoms and typical sensory disorders (paraesthesia, pain, pruritus and cold dysaesthesia), which can persist several weeks and, in some cases, several months or years. NSP is considered a mild form of CFP with similar but less severe symptoms. After inhaled exposure, PbTxs can also cause respiratory tract irritation in healthy subjects and asthma exacerbations in predisposed subjects, whose respiratory functions may be disrupted for several days following PbTx inhalation. Mechanistically, it is well established that CTX- or PbTx-induced disturbances are primarily mainly due to voltage-gated sodium channel activation in sensory and motor peripheral nervous system. However, little is known about the pathophysiology or a potential individual susceptibility to long lasting effects of CFP/NSP. In addition to their action on the nervous system, PbTxs and CTXs were also shown to exert effects on the immune system. However, their role in the pathophysiology of syndromes induced by CTX or PbTx exposure is poorly documented. The aim of this review is to inventory the literature thus far on the inflammatory and immune effects of PbTxs and CTXs.

Activation of Veratridine Sensitive Sodium Channels, But not Electrical Field Stimulation, Dilates Porcine Retinal Arterioles with Preserved Perivascular Tissue

ABSTRACTPurpose: Disturbances in retinal blood flow are a prominent feature of vision threatening retinal diseases. The regulation of tone in retinal resistance vessels involves the perivascular retinal tissue, but it is unknown to what extent neurons or glial cells contribute to the effect. Therefore, the purpose of the present study was to study the contribution of neurons in the perivascular retina to vascular tone during activation of voltage-gated sodium channels with veratridine and electrical field stimulation (EFS).Methods: Porcine retinal arterioles with and without perivascular tissue were mounted in an isometric myograph system for studying the effects of the voltage-gated sodium channel opener veratridine and EFS on retinal vascular tone.Results: Veratridine induced concentration-dependent relaxation of retinal arterioles which was more pronounced in arterioles with preserved perivascular retinal tissue than in isolated vessels. In the presence of this tissue, veratridine-induced relaxation was inhibited by the voltage-gated sodium channel blocker tetrodotoxin and the nitric oxide synthase inhibitor, Nω-Nitro-L-arginine methyl ester (L-NAME), but was unaffected by the inhibition of the cyclo-oxygenase inbitior ibuprofen and by blocking of adenosine receptors with 8-(p-Sulfophenyl)theophylline hydrate (8-PSPT). Electrical field stimulation induced no changes in retinal vascular tone.Conclusions: Sodium channels of neuronal origin are likely to be involved in the regulation of retinal vascular tone. The lack of effect of EFS on retinal vascular tone may be due to the lack of autonomic nerves in the retina.

Damage-free peripheral nerve stimulation by 12-ns pulsed electric field

Modern technologies enable deep tissue focusing of nanosecond pulsed electric field (nsPEF) for non-invasive nerve and muscle stimulation. However, it is not known if PEF orders of magnitude shorter than the activation time of voltage-gated sodium channels (VGSC) would evoke action potentials (APs). One plausible scenario requires the loss of membrane integrity (electroporation) and resulting depolarization as an intermediate step. We report, for the first time, that the excitation of a peripheral nerve can be accomplished by 12-ns PEF without electroporation. 12-ns stimuli at 4.1–11 kV (3.3–8.8 kV/cm) evoked APs similarly to conventional stimuli (100–250 μs, 1–5 V, 103–515 V/m), except for having higher selectivity for the faster nerve fibers. Nerves sustained repeated tetanic stimulations (50 Hz or 100 Hz for 1 min) alternately by 12-ns PEF and by conventional pulses. Such tetani caused a modest AP decrease, to a similar extent for both types of stimuli. Nerve refractory properties were not affected. The lack of cumulative damages even from tens of thousands of 12-ns stimuli and the similarities with the conventional stimulation prove VGSC activation by nsPEF without nerve membrane damage.

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.

Neuronal excitation and permeabilization by 200-ns pulsed electric field: An optical membrane potential study with FluoVolt dye

Electric field pulses of nano- and picosecond duration are a novel modality for neurostimulation, activation of Ca2+ signaling, and tissue ablation. However it is not known how such brief pulses activate voltage-gated ion channels. We studied excitation and electroporation of hippocampal neurons by 200-ns pulsed electric field (nsPEF), by means of time-lapse imaging of the optical membrane potential (OMP) with FluoVolt dye. Electroporation abruptly shifted OMP to a more depolarized level, which was reached within <1ms. The OMP recovery started rapidly (τ=8–12ms) but gradually slowed down (to τ>10s), so cells remained above the resting OMP level for at least 20–30s. Activation of voltage-gated sodium channels (VGSC) enhanced the depolarizing effect of electroporation, resulting in an additional tetrodotoxin-sensitive OMP peak in 4–5ms after nsPEF. Omitting Ca2+ in the extracellular solution did not reduce the depolarization, suggesting no contribution of voltage-gated calcium channels (VGCC). In 40% of neurons, nsPEF triggered a single action potential (AP), with the median threshold of 3kV/cm (range: 1.9–4kV/cm); no APs could be evoked by stimuli below the electroporation threshold (1.5–1.9kV/cm). VGSC opening could already be detected in 0.5ms after nsPEF, which is too fast to be mediated by the depolarizing effect of electroporation. The overlap of electroporation and AP thresholds does not necessarily reflect the causal relation, but suggests a low potency of nsPEF, as compared to conventional electrostimulation, for VGSC activation and AP induction.

Cytokine cascades induced by mechanical trauma injury alter voltage-gated sodium channel activity in intact cortical neurons

BackgroundTraumatic brain injury (TBI) triggers both immediate (primary) and long-term (secondary) tissue damages. Secondary damages can last from hours to days or even a lifetime. Secondary damages implicate several mechanisms, including influence of inflammatory mediators, mainly cytokines, on excitability of ion channels. However, studies should further explore the effects of inflammatory cytokines on voltage-gated sodium channels (VGSCs) and excitability in distal intact neurons.MethodsMixed cultures of mouse cortical astrocytes and neurons were subjected to mechanical injury (trauma) to mimic TBI in vitro. Expression of various cytokines in these cultures were measured by real-time polymerase chain reaction and enzyme-linked immunosorbent assay. A trauma-conditioned medium with or without brain-derived neurotrophic factor (BDNF) was added to mouse primary cortical neurons for 6 and 24 h to mimic combined effects of multiple inflammatory cytokines on VGSCs. Spike behaviors of distal intact neurons were examined by whole-cell patch-clamp recordings.ResultsMechanical injury in mixed cortical neuron–astrocyte cultures significantly increased expression levels of multiple cytokines, including interleukin (IL)-1β, IL-6, tumor necrosis factor-α, monocyte chemoattractant protein-1, chemokine (C-C motif) ligand-5, IL-10, and transforming growth factor-β1, at 6 and 24 h after injury. Incubation in trauma-conditioned medium increased functional VGSCs in neuronal membranes and Na+ currents. Enhanced VGSCs were almost completely abolished by BDNF, and reinforcement of Na+ currents was also reduced in a dose-dependent manner. BDNF (30 ng/mL) also significantly reversed reduced neuronal cell viability, which was induced by medium conditioned at 6 h. At 6 and 24 h, trauma-conditioned medium significantly increased spike frequency but not spike threshold.ConclusionsIn TBI, the combined effect of inflammatory cytokines is directly involved in VGSC, Na+ current, and excitability dysfunction in distal intact neurons. BDNF may partly exert neuroprotective effects by maintaining balance of VGSC function in distal intact neurons.

Differential effect of brief electrical stimulation on voltage-gated potassium channels.

Electrical stimulation of neuronal tissue is a promising strategy to treat a variety of neurological disorders. The mechanism of neuronal activation by external electrical stimulation is governed by voltage-gated ion channels. This stimulus, typically brief in nature, leads to membrane potential depolarization, which increases ion flow across the membrane by increasing the open probability of these voltage-gated channels. In spiking neurons, it is activation of voltage-gated sodium channels (NaV channels) that leads to action potential generation. However, several other types of voltage-gated channels are expressed that also respond to electrical stimulation. In this study, we examine the response of voltage-gated potassium channels (KV channels) to brief electrical stimulation by whole cell patch-clamp electrophysiology and computational modeling. We show that nonspiking amacrine neurons of the retina exhibit a large variety of responses to stimulation, driven by different KV-channel subtypes. Computational modeling reveals substantial differences in the response of specific KV-channel subtypes that is dependent on channel kinetics. This suggests that the expression levels of different KV-channel subtypes in retinal neurons are a crucial predictor of the response that can be obtained. These data expand our knowledge of the mechanisms of neuronal activation and suggest that KV-channel expression is an important determinant of the sensitivity of neurons to electrical stimulation.NEW & NOTEWORTHY This paper describes the response of various voltage-gated potassium channels (KV channels) to brief electrical stimulation, such as is applied during prosthetic electrical stimulation. We show that the pattern of response greatly varies between KV channel subtypes depending on activation and inactivation kinetics of each channel. Our data suggest that problems encountered when artificially stimulating neurons such as cessation in firing at high frequencies, or "fading," may be attributed to KV-channel activation.

Modification of distinct ion channels differentially modulates Ca2+ dynamics in primary cultured rat ventricular cardiomyocytes

Primary cultured cardiomyocytes show spontaneous Ca2+ oscillations (SCOs) which not only govern contractile events, but undergo derangements that promote arrhythmogenesis through Ca2+ -dependent mechanism. We systematically examined influence on SCOs of an array of ion channel modifiers by recording intracellular Ca2+ dynamics in rat ventricular cardiomyocytes using Ca2+ specific fluorescence dye, Fluo-8/AM. Voltage-gated sodium channels (VGSCs) activation elongates SCO duration and reduces SCO frequency while inhibition of VGSCs decreases SCO frequency without affecting amplitude and duration. Inhibition of voltage-gated potassium channel increases SCO duration. Direct activation of L-type Ca2+ channels (LTCCs) induces SCO bursts while suppressing LTCCs decreases SCO amplitude and slightly increases SCO frequency. Activation of ryanodine receptors (RyRs) increases SCO duration and decreases both SCO amplitude and frequency while inhibiting RyRs decreases SCO frequency without affecting amplitude and duration. The potencies of these ion channel modifiers on SCO responses are generally consistent with their affinities in respective targets demonstrating that modification of distinct targets produces different SCO profiles. We further demonstrate that clinically-used drugs that produce Long-QT syndrome including cisapride, dofetilide, sotalol, and quinidine all induce SCO bursts while verapamil has no effect. Therefore, occurrence of SCO bursts may have a translational value to predict cardiotoxicants causing Long-QT syndrome.