Actions of veratridine on tetrodotoxin-sensitive voltage-gated Na+ currents,NaV1.6, in murine vas deferens myocytes

Background and purpose:

The effects of veratridine, an alkaloid found in Liliaceae plants, on tetrodotoxin (TTX)-sensitive voltage-gated Na⁺ channels were investigated in mouse vas deferens.

Experimental approach:

Effects of veratridine on TTX-sensitive Na⁺ currents (I_Na) in vas deferens myocytes dispersed from BALB/c mice, homozygous mice with a null allele of Na_V1.6 (Na_V1.6^−/−) and wild-type mice (Na_V1.6^+/+) were studied using patch-clamp techniques. Tension measurements were also performed to compare the effects of veratridine on phasic contractions in intact tissues.

Key results:

In whole-cell configuration, veratridine had a concentration-dependent dual action on the peak amplitude of I_Na: I_Na was enhanced by veratridine (1–10 µM), while higher concentrations (≥30 µM) inhibited I_Na. Additionally, two membrane current components were evoked by veratridine, namely a sustained inward current during the duration of the depolarizing rectangular pulse and a tail current at the repolarization. Although veratridine caused little shift of the voltage dependence of the steady-state inactivation curve and the activation curve for I_Na, veratridine enhanced a non-inactivating component of I_Na. Veratridine caused no detectable contractions in vas deferens from Na_V1.6^−/− mice, although in tissues from Na_V1.6^+/+ mice, veratridine (≥3 µM) induced TTX-sensitive contractions. Similarly, no detectable inward currents were evoked by veratridine in Na_V1.6^−/− vas deferens myocytes, while veratridine elicited both the sustained and tail currents in cells taken from Na_V1.6^+/+ mice.

Conclusions and implications:

These results suggest that veratridine possesses a dual action on I_Na and that the veratridine-induced activation of contraction is induced by the activation of Na_V1.6 channels.     

The facilitatory actions of snake venom phospholipase A2 neurotoxins at the neuromuscular junction are not mediated through voltage-gated K channels

Electrophysiological investigations have previously suggested that phospholipase A₂ (PLA₂) neurotoxins from snake venoms increase the release of acetylcholine (Ach) at the neuromuscular junction by blocking voltage-gated K⁺ channels in motor nerve terminals.We have tested some of the most potent presynaptically-acting neurotoxins from snake venoms, namely β-bungarotoxin (BuTx), taipoxin, notexin, crotoxin, ammodytoxin C and A (Amotx C & A), for effects on several types of cloned voltage-gated K⁺ channels (mKv1.1, rKv1.2, mKv1.3, hKv1.5 and mKv3.1) stably expressed in mammalian cell lines. By use of the whole-cell configuration of the patch clamp recording technique and concentrations of toxins greater than those required to affect acetylcholine release, these neurotoxins have been shown not to block any of these voltage-gated K⁺ channels. In addition, internal perfusion of the neurotoxins (100 μg/ml) into mouse B82 fibroblast cells that expressed rKv1.2 channels also did not substantially depress K⁺ currents. The results of this study suggest that the mechanism by which these neurotoxins increase the release of acetylcholine at the neuromuscular junction is not related to the direct blockage of voltage-activated Kv1.1, Kv1.2, Kv1.3, Kv1.5 and Kv3.1 K⁺ channels.     

The calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalene sulphonamide directly blocks human ether ��-go-go-related gene potassium channels stably expressed in human embryonic kidney 293 cells

Background and purpose:

Results from several studies point to voltage-gated Na⁺ channels as potential mediators of the immobility produced by inhaled anaesthetics. We hypothesized that the intrathecal administration of tetrodotoxin, a drug that blocks Na⁺ channels, should enhance anaesthetic potency, and that concurrent administration of veratridine, a drug that augments Na⁺ channel opening, should reverse the increase in potency.

Experimental approach:

We measured the change in isoflurane potency for reducing movement in response to a painful stimulus as defined by MAC (minimum alveolar concentration of anaesthetic required to abolish movement in 50% of subjects) caused by intrathecal infusion of various concentrations of tetrodotoxin into the lumbothoracic subarachnoid space of rats, and the change in MAC caused by the administration of a fixed dose of tetrodotoxin plus various doses of intrathecal veratridine.

Key results:

Intrathecal infusion of tetrodotoxin (0.078–0.63 µM) produced a reversible dose-related decrease in MAC, of more than 50% at the highest concentration. Intrathecal co-administration of veratridine (1.6–6.4 µM) reversed this decrease in a dose-related manner, with nearly complete reversal at the highest veratridine dose tested.

Conclusions and implications:

Intrathecal administration of tetrodotoxin increases isoflurane potency (decreases isoflurane MAC), and intrathecal administration of veratridine counteracts this effect in vivo. These findings are consistent with a role for voltage-gated Na⁺ channel blockade in the immobility produced by inhaled anaesthetics.     

Inhibition of mechanical activation of guinea-pig airway afferent neurons by amiloride analogues

1. The aim of this study was to investigate a role for Epithelial Sodium Channels (ENaCs) in the mechanical activation of low-threshold vagal afferent nerve terminals in the guinea-pig trachea/bronchus. 2. Using extracellular single-unit recording techniques, we found that the ENaC blocker amiloride, and its analogues dimethylamiloride and benzamil caused a reduction in the mechanical activation of guinea-pig airway afferent fibres. 3. Amiloride and it analogues also reduced the sensitivity of afferent fibres to electrical stimulation such that greater stimulation voltages were required to induce action potentials from their peripheral terminals within the trachea/bronchus. 4. The relative potencies of these compounds for inhibiting electrical excitability of afferent nerves were similar to that observed for inhibition of mechanical stimulation (dimethylamiloride approximately benzamil > amiloride). This rank order of potency is incompatible with the known rank order of potency for blockade of ENaCs (benzamil > amiloride > dimethylamiloride). 5. As voltage-gated sodium channels play an important role in determining the electrical excitability of neurons, we used whole-cell patch recordings of nodose neuron cell bodies to investigate the possibility that amiloride analogues caused blockade of these channels. At the concentration required to inhibit mechanical activation of vagal nodose afferent fibres (100 microM), benzamil caused significant inhibition of voltage-gated sodium currents in neuronal cell bodies acutely isolated from guinea-pig nodose ganglia. 6. Combined, our findings suggest that amiloride and its analogues did not selectively block mechanotransduction in airway afferent neurons, but rather they reduced neuronal excitability, possibly by inhibiting voltage-gated sodium currents.     

Antinociceptive effect of ambroxol in rats with neuropathic spinal cord injury pain

Symptoms of neuropathic spinal cord injury (SCI) pain include evoked cutaneous hypersensitivity and spontaneous pain, which can be present below the level of the injury. Adverse side-effects obtained with currently available analgesics complicate effective pain management in SCI patients. Voltage-gated Na⁺ channels expressed in primary afferent nociceptors have been identified to mediate persistent hyperexcitability in dorsal root ganglia (DRG) neurons, which in part underlies the symptoms of nerve injury-induced pain. Ambroxol has previously demonstrated antinociceptive effects in rat chronic pain models and has also shown to potently block Na⁺ channel current in DRG neurons. Ambroxol was tested in rats that underwent a mid-thoracic spinal cord compression injury. Injured rats demonstrated robust hind paw (below-level) heat and mechanical hypersensitivity. Orally administered ambroxol significantly attenuated below-level hypersensitivity at doses that did not affect performance on the rotarod test. Intrathecal injection of ambroxol did not ameliorate below-level hypersensitivity. The current data suggest that ambroxol could be effective for clinical neuropathic SCI pain. Furthermore, the data suggest that peripherally expressed Na⁺ channels could lend themselves as targets for the development of pharmacotherapies for SCI pain.     

Effects of beta-adrenoceptor antagonists in the neural nitric oxide release induced by electrical field stimulation and sodium channel activators in the rabbit corpus cavernosum

Beta-Adrenoceptor antagonists may present receptor-independent mechanisms, such as blockade of voltage-gated sodium channels. This study aimed to investigate the effects of non-selective (propranolol), and selective beta1- (atenolol, metoprolol and betaxolol) and beta2-adrenoceptor (ICI 118,551) antagonists in the nitric oxide (NO)-mediated rabbit corpus cavernosum relaxations induced by either electrical field stimulation (EFS) or activators of voltage-gated sodium channels. The sodium channel blockers tetrodotoxin and saxitoxin abolished the relaxations induced by EFS or sodium channel activators of binding site-2 (aconitine and veratridine), site-3 (Ts3 toxin), site-4 (Ts1 toxin) and site-5 (brevetoxin-3). The beta-adrenoceptor antagonists failed to affect the relaxations induced by EFS, aconitine and veratridine. Relaxations induced by Ts3 and Ts1 toxins, as well as brevetoxin-3, were markedly reduced by prior addition of propranolol, betaxolol and ICI 118,551. During the established relaxation induced by Ts3 toxin, propranolol failed to restore the basal tone. In conclusion, beta-adrenoceptor antagonists may cause an allosteric inhibition at the binding site-3, -4 and -5 of voltage-gated sodium channels, leading to blockade of neural NO release.     

Dual action of a dinoflagellate-derived precursor of Pacific ciguatoxins (P-CTX-4B) on voltage-dependent K and Na channels of single myelinated axons

The effects of Pacific ciguatoxin-4B (P-CTX-4B, also named gambiertoxin), extracted from toxic Gambierdiscus dinoflagellates, were assessed on nodal K⁺ and Na⁺ currents of frog myelinated axons, using a conventional voltage-clamp technique. P-CTX-4B decreased, within a few minutes, both K⁺ and Na⁺ currents in a dose-dependent manner, without inducing any marked change in current kinetics. The toxin was more effective in blocking K⁺ than Na⁺ channels. P-CTX-4B shifted the voltage-dependence of Na⁺ conductance by about 14 mV towards more negative membrane potentials. This effect was reversed by increasing Ca²⁺ in the external solution. A negative shift of about 16 mV in the steady-state Na⁺ inactivation-voltage curve was also observed in the presence of the toxin. Unmodified and P-CTX-4B-modified Na⁺ currents were similarly affected by the local anaesthetic lidocaine. The decrease of the two currents by lidocaine was dependent on both the concentration and the membrane potential during pre-pulses. In conclusion, P-CTX-4B appears about four times more effective than P-CTX-1B to affect K⁺ channels, whereas it is about 50 times less efficient to affect Na⁺ channels of axonal membranes. These actions may be related to subtle differences between the two chemical structures of molecules.     

Characterization of Ionic Currents in Human Neural Stem Cells

The profile of membrane currents was investigated in differentiated neuronal cells derived from human neural stem cells (hNSCs) that were obtained from aborted fetal cortex. Whole-cell voltage clamp recording revealed at least 4 different currents: a tetrodotoxin (TTX)-sensitive Na⁺ current, a hyperpolarization-activated inward current, and A-type and delayed rectifier-type K⁺ outward currents. Both types of K⁺ outward currents were blocked by either 5 mM tetraethylammonium (TEA) or 5 mM 4-aminopyridine (4-AP). The hyperpolarization-activated current resembled the classical K⁺ inward current in that it exhibited a voltage-dependent block in the presence of external Ba²⁺ (30µM) or Cs⁺ (3µM). However, the reversal potentials did not match well with the predicted K⁺ equilibrium potentials, suggesting that it was not a classical K⁺ inward rectifier current. The other Na⁺ inward current resembled the classical Na⁺ current observed in pharmacological studies. The expression of these channels may contribute to generation and repolarization of action potential and might be regarded as functional markers for hNSCs-derived neurons.     

Effects of carbamazepine and amitriptyline on tetrodotoxin-resistant Na<Superscript>+</Superscript> channels in immature rat trigeminal ganglion neurons

Although anticonvulsant drugs that block voltage-dependent Na⁺ channels have been widely used for neuropathic pain, including peripheral nerve injury-induced pain, much less is known about the actions of these drugs on immature trigeminal ganglion (TG) neurons. Here we report the effects of carbamazepine (CBZ) and amitriptyline (ATL) on tetrodotoxin-resistant (TTX-R) Na⁺ channels expressed on immature rat TG neurons. TTX-R Na⁺ currents (I_Na) were recorded in the presence of 300 nM TTX by use of a conventional whole-cell patch clamp method. Both CBZ and ATL inhibited TTX-R I_Na in a concentration-dependent manner, but ATL was more potent. While CBZ and ATL did not affect the overall voltage-activation relationship of TTX-R Na⁺ channels, both drugs shifted the voltage-activation relationship to the left, indicating that they inhibited TTX-R Na⁺ channels more efficiently at depolarized membrane potentials. ATL showed a profound use-dependent blockade of TTX-R I_Na, but CBZ had little effect. The present results suggest that both CBZ and ATL, common drugs used for treating neuropathic pain, efficiently inhibit TTX-R Na⁺ channels expressed on immature TG neurons, and that these drugs might be useful for the treatment of trigeminal nerve injury-induced neuropathic pain, as well as the inhibition of ongoing central sensitization, even during immature periods.     

Modulation of ion channels in clinical psychopharmacology: adults and younger people

This review focuses on the use of Na⁺, Ca²⁺ and Cl^- channel modulators in psychiatric disease. Drugs that modulate ion channels have been used in psychiatry for more than a century, and in this review we critically evaluate clinical research that reports the therapeutic effects of drugs acting on GABA_A, voltage-gated Na⁺ and voltage-gated Ca²⁺ channels in pediatric and adult patients. As in other fields, the evidence underpinning the use of medicines in younger people is far less robust than for adults. In addition, we discuss some current developments and highlight clinical disorders in which current molecules could be further tested. Notable success stories, such as benzodiazepines (in sleep and anxiety disorders) and antiepileptics (in bipolar disorder), have been the result of serendipitous discoveries or refinements of serendipitous discoveries, as in all other major treatments in psychiatry. Genomic, high-throughput screening and molecular pharmacology discoveries may, however, guide further developments in the future. This could include increased research in promising targets that have been perceived as commercially risky, such as selective α-subunit GABA_A receptors.     

Bepridil up-regulates cardiac Na+ channels as a long-term effect by blunting proteasome signals through inhibition of calmodulin activity

Background and purpose:

Bepridil is an anti-arrhythmic agent with anti-electrical remodelling effects that target many cardiac ion channels, including the voltage-gated Na⁺ channel. However, long-term effects of bepridil on the Na⁺ channel remain unclear. We explored the long-term effect of bepridil on the Na⁺ channel in isolated neonatal rat cardiomyocytes and in a heterologous expression system of human Na_v1.5 channel.

Experimental approach:

Na⁺ currents were recorded by whole-cell voltage-clamp technique. Na⁺ channel message and protein were evaluated by real-time RT-PCR and Western blot analysis.

Key results:

Treatment of cardiomyocytes with 10 µmol·L⁻¹ bepridil for 24 h augmented Na⁺ channel current (I_Na) in a dose- and time-dependent manner. This long-term effect of bepridil was mimicked or masked by application of W-7, a calmodulin inhibitor, but not KN93 [2-[N-(2-hydroxyethyl)-N-(4-methoxy benzenesulphonyl)]-amino-N-(4-chlorocinnamyl)-N-methylbenzylamine], a Ca²⁺/calmodulin-dependent kinase inhibitor. During inhibition of protein synthesis by cycloheximide, the I_Na increase due to bepridil was larger than the increase without cycloheximide. Bepridil and W-7 significantly slowed the time course of Na_v1.5 protein degradation in neonatal cardiomyocytes, although the mRNA levels of Na_v1.5 were not modified. Bepridil and W-7 did not increase I_Na any further in the presence of the proteasome inhibitor MG132 [N-[(phenylmethoxy)carbonyl]-L-leucyl-N-[(1S)-1-formyl-3-methylbutyl]-L-leucinamide]. Bepridil, W-7 and MG132 but not KN93 significantly decreased 20S proteasome activity in a concentration-dependent manner.

Conclusions and implications:

We conclude that long-term exposure of cardiomyocytes to bepridil at therapeutic concentrations inhibits calmodulin action, which decreased degradation of the Na_v1.5 α-subunit, which in turn increased Na⁺ current.     

Drosotoxin, a selective inhibitor of tetrodotoxin-resistant sodium channels

The design of animal toxins with high target selectivity has long been a goal in protein engineering. Based on evolutionary relationship between the Drosophila antifungal defensin (drosomycin) and scorpion depressant Na⁺ channel toxins, we exploited a strategy to create a novel chimeric molecule (named drosotoxin) with high selectivity for channel subtypes, which was achieved by using drosomycin to substitute the structural core of BmKITc, a depressant toxin acting on both insect and mammalian Na⁺ channels. Recombinant drosotoxin selectively inhibited tetrodotoxin-resistant (TTX-R) Na⁺ channels in rat dorsal root ganglion (DRG) neurons with a 50% inhibitory concentration (IC₅₀) of 2.6 ± 0.5 μM. This chimeric peptide showed no activity on K⁺, Ca²⁺ and TTX-sensitive (TTX-S) Na⁺ channels in rat DRG neurons and Drosophila para/tipE channels at micromolar concentrations. Drosotoxin represents the first chimeric toxin and example of a non-toxic core scaffold with high selectivity on mammalian TTX-R Na⁺ channels.     

Biophysical properties of Na(v) 1.8/Na(v) 1.2 chimeras and inhibition by µO-conotoxin MrVIB

BACKGROUND AND PURPOSE

Voltage-gated sodium channels are expressed primarily in excitable cells and play a pivotal role in the initiation and propagation of action potentials. Nine subtypes of the pore-forming α-subunit have been identified, each with a distinct tissue distribution, biophysical properties and sensitivity to tetrodotoxin (TTX). Na_v1.8, a TTX-resistant (TTX-R) subtype, is selectively expressed in sensory neurons and plays a pathophysiological role in neuropathic pain. In comparison with TTX-sensitive (TTX-S) Na_vα-subtypes in neurons, Na_v1.8 is most strongly inhibited by the µO-conotoxin MrVIB from Conus marmoreus. To determine which domain confers Na_v1.8 α-subunit its biophysical properties and MrVIB binding, we constructed various chimeric channels incorporating sequence from Na_v1.8 and the TTX-S Na_v1.2 using a domain exchange strategy.

EXPERIMENTAL APPROACH

Wild-type and chimeric Na_v channels were expressed in Xenopus oocytes, and depolarization-activated Na⁺ currents were recorded using the two-electrode voltage clamp technique.

KEY RESULTS

MrVIB (1 µM) reduced Na_v1.2 current amplitude to 69 ± 12%, whereas Na_v1.8 current was reduced to 31 ± 3%, confirming that MrVIB has a binding preference for Na_v1.8. A similar reduction in Na⁺ current amplitude was observed when MrVIB was applied to chimeras containing the region extending from S6 segment of domain I through the S5-S6 linker of domain II of Na_v1.8. In contrast, MrVIB had only a small effect on Na⁺ current for chimeras containing the corresponding region of Na_v1.2.

CONCLUSIONS AND IMPLICATIONS

Taken together, these results suggest that domain II of Na_v1.8 is an important determinant of MrVIB affinity, highlighting a region of the α-subunit that may allow further nociceptor-specific ligand targeting.     

Selective alteration of sodium channel gating by Australian funnel-web spider toxins

The actions of potent mammalian neurotoxins isolated from the venom of two Australian funnel-web spiders were investigated using both electrophysiological and neurochemical techniques. Whole-cell patch clamp recording of sodium currents in rat dorsal root ganglion neurons revealed that versutoxin (VTX), isolated from the venom of Hadronyche versuta, produced a concentration-dependent slowing or removal of tetrodotoxin-sensitive (TTX-S) sodium current inactivation and a reduction in peak TTX-S sodium current. In contrast, VTX had no effect on tetrodotoxin-resistant (TTX-R) sodium currents or potassium currents. VTX also shifted the voltage dependence of sodium channel activation in the hyperpolarizing direction and increased the rate of recovery from inactivation. Ion flux studies performed in rat brain synaptosomes also revealed that robustoxin (RTX), from the venom of Atrax robustus, and VTX both produced a partial activation of ²²Na ⁺ flux and an inhibition of batrachotoxin-activated ²²Na⁺ flux. This inhibition of flux through batrachotoxin-activated channels was not due to an interaction with neurotoxin receptor site 1 since [³H]saxitoxin binding was unaffected. In addition, the partial activation of ²²Na⁺ flux was not enhanced in the presence of α-scorpion toxin and further experiments suggest that VTX also enhances [³H]batrachotoxin binding. These selective actions of funnel-web spider toxins on sodium channel function are comparable to those of α-scorpion and sea anemone toxins which bind to neurotoxin receptor site 3 on the channel to slow channel inactivation profoundly. Also, these modifications of sodium channel gating and kinetics are consistent with actions of the spider toxins to produce repetitive firing of action potentials.     

Inhibition of Na+-pump enhances carbachol-induced influx of 45Ca2+ and secretion of catecholamines by elevation of cellular accumulation of 22Na+ in cultured bovine adrenal medullary cells

Summary In bovine adrenal medullary cells, we reported that ²²Na⁺ influx via nicotinic receptor-associated Na⁺ channels is involved in ⁴⁵Ca²⁺ influx, a requisite for initiating the secretion of catecholamines (Wada et al. 1984, 1985b).In the present study, we investigated whether the inhibition of Na⁺-pump modulates carbachol-induced ²²Na⁺ influx, ⁴⁵Ca²⁺ influx and catecholamine secretion in cultured bovine adrenal medullary cells. We also measured ⁸⁶Rb⁺ uptake by the cells to estimate the activity of Na⁺, K⁺-ATPase. (1) Ouabain and extracellular K⁺ deprivation remarkably potentiated carbachol-induced ²²Na⁺ influx, ⁴⁵Ca²⁺ influx and catecholamine secretion; this potentiation of carbachol-induced ⁴⁵Ca²⁺ influx and catecholamine secretion was not observed in Na⁺ free medium. (2) Carbachol increased the uptake of ⁸⁶Rb⁺; this increase was inhibited by hexamethonium and d-tubocurarine. In Na⁺ free medium, carbachol failed to increase ⁸⁶Rb⁺ uptake. (3) Ouabain inhibited carbachol-induced ⁸⁶Rb⁺ uptake in a concentration-dependent manner, as it increased the accumulation of cellular ²²Na⁺. These results suggest that Na⁺ influx via nicotinic receptor-associated Na⁺ channels increases the activity of Na⁺, K⁺-ATPase and the inhibition of Na⁺, K⁺-ATPase augmented carbachol-induced Ca²⁺ influx and catecholamine secretion by potentiating cellular accumulation of Na⁺. It seems that nicotinic receptor-associated Na⁺ channels and Na⁺, K⁺-ATPase, both modulate the influx of Ca²⁺ and secretion of catecholamines by accomodating cellular concentration of Na⁺.     

Actions of sea anemone type 1 neurotoxins on voltage-gated sodium channel isoforms

As voltage-gated Na⁺ channels are responsible for the conduction of electrical impulses in most excitable tissues in the majority of animals (except nematodes), they have become important targets for the toxins of venomous animals, from sea anemones to molluscs, scorpions, spiders and even fishes. During their evolution, different animals have developed a set of cysteine-rich peptides capable of binding different extracellular sites of this channel protein. A fundamental question concerning the mechanism of action of these toxins is whether they act at a common receptor site in Na⁺ channels when exerting their different pharmacological effects, or at distinct receptor sites in different Na_v channels subtypes whose particular properties lead to these pharmacological differences. The α-subunits of voltage-gated Na⁺ channels (Na_v1.x) have been divided into at least nine subtypes on the basis of amino acid sequences. Sea anemones have been extensively studied from the toxinological point of view for more than 40 years. There are about 40 sea anemone type 1 peptides known to be active on Na_v1.x channels and all are 46-49 amino acid residues long, with three disulfide bonds and their molecular weights range between 3000 and 5000 Da. About 12 years ago a general model of Na_v1.2-toxin interaction, developed for the α-scorpion toxins, was shown to fit also to action of sea anemone toxin such as ATX-II. According to this model these peptides are specifically acting on the type 3 site known to be between segments 3 and 4 in domain IV of the Na⁺ channel protein. This region is indeed responsible for the normal Na⁺ currents fast inactivation that is potently slowed by these toxins. This fundamental “gain-of-function” mechanism is responsible for the strong increase in the action potential duration. They constitute a class of tools by means of which physiologists and pharmacologists can study the structure/function relationships of channel proteins. As most of the structural and electrophysiological studies were performed on type 1 sea anemone sodium channel toxins, we will present a comprehensive and updated review on the current understanding of the physiological actions of these Na channel modifiers.     

Concomitant facilitation of GABAA receptors and KV7 channels by the non-opioid analgesic flupirtine

BACKGROUND AND PURPOSE

Flupirtine is a non-opioid analgesic that has been in clinical use for more than 20 years. It is characterized as a selective neuronal potassium channel opener (SNEPCO). Nevertheless, its mechanisms of action remain controversial and are the purpose of this study.

EXPERIMENTAL APPROACH

Effects of flupirtine on native and recombinant voltage- and ligand-gated ion channels were explored in patch-clamp experiments using the following experimental systems: recombinant K_IR3 and K_V7 channels and α3β4 nicotinic acetylcholine receptors expressed in tsA 201 cells; native voltage-gated Na⁺, Ca²⁺, inward rectifier K⁺, K_V7 K⁺, and TRPV1 channels, as well as GABA_A, glycine, and ionotropic glutamate receptors expressed in rat dorsal root ganglion, dorsal horn and hippocampal neurons.

KEY RESULTS

Therapeutic flupirtine concentrations (≤10 µM) did not affect voltage-gated Na⁺ or Ca²⁺ channels, inward rectifier K⁺ channels, nicotinic acetylcholine receptors, glycine or ionotropic glutamate receptors. Flupirtine shifted the gating of K_V7 K⁺ channels to more negative potentials and the gating of GABA_A receptors to lower GABA concentrations. These latter effects were more pronounced in dorsal root ganglion and dorsal horn neurons than in hippocampal neurons. In dorsal root ganglion and dorsal horn neurons, the facilitatory effect of therapeutic flupirtine concentrations on K_V7 channels and GABA_A receptors was comparable, whereas in hippocampal neurons the effects on K_V7 channels were more pronounced.

CONCLUSIONS AND IMPLICATIONS

These results indicate that flupirtine exerts its analgesic action by acting on both GABA_A receptors and K_V7 channels.     

Underlying mechanism of actions of tefluthrin,a pyrethroid insecticide,on voltage-gated ion currents and on action currents in pituitary tumor (GH3) cells and GnRH-secreting (GT1-7) neurons

Tefluthrin is a synthetic pyrethroid and involved in acute neurotoxic effects. How this compound affects ion currents in endocrine or neuroendocrine cells remains unclear. Its effects on membrane ion currents in pituitary tumor (GH₃) cells and in hypothalamic (GT1-7) neurons were investigated. Application of Tef (10 μM) increased the amplitude of voltage-gated Na⁺ current (I_Na), along with a slowing in current inactivation and deactivation in GH₃ cells. The current–voltage relationship of I_Na was shifted to more negative potentials in the presence of this compound. Tef increased I_Na with an EC₅₀ value of 3.2 ± 0.8 μM. It also increased the amplitude of persistent I_Na. Tef reduced the amplitude of L-type Ca²⁺ current. This agent slightly inhibited K⁺ outward current; however, it had no effect on the activity of large-conductance Ca²⁺-activated K⁺ channels. Under cell-attached voltage-clamp recordings, Tef (10 μM) increased amplitude and frequency of spontaneous action currents, along with appearance of oscillatory inward currents. Tef-induced inward currents were suppressed after further application of tetrodotoxin, riluzole or ranolazine. In GT1-7 cells, Tef also increased the amplitude and frequency of action currents. Taken together, the effects of Tef and its structural related pyrethroids on ion currents can contribute to the underlying mechanisms through which they affect endocrine or neuroendocrine function in vivo.     

Blood-Pressure Lowering,Positive Chronotropy and Inotropy by the Veratrum Alkaloids Germidine and Germerine but Negative Chronotropy by Veratridine in Mice

Abstract

Germidine and germerine, the Veratrum alkaloids lowered blood pressure accompanied with positive chronotropy and inotropy in mice. Germerine was more potent than germidine in both blood-pressure lowering and positive inotropy, whereas veratridine produced negative chronotropy and positive inotropy. An acyl group (an acetyl or a 2-methylbutyroyl group) at 3-O-R¹ position and a 2-methylbutyroyl group at 15-O-R² position in germine were important to produce the positive inotropy and chronotropy. The presence of a veratroyl group at 3-O-R¹ position and a free hydroxyl group at 15-O-R² position may be essential to produce the negative chronotropy by veratridine. The positive inotropy by germidine and veratridine may be due to TTX-resistant Na⁺ channel activation.