Inactivation Of Sodium Channels Research Articles

NaV1.4 DI-S4 periodic paralysis mutation R222W enhances inactivation and promotes leak current to attenuate action potentials and depolarize muscle fibers

Hypokalemic periodic paralysis is a skeletal muscle disease characterized by episodic weakness associated with low serum potassium. We compared clinical and biophysical effects of R222W, the first hNaV1.4 domain I mutation linked to this disease. R222W patients exhibited a higher density of fibers with depolarized resting membrane potentials and produced action potentials that were attenuated compared to controls. Functional characterization of the R222W mutation in heterologous expression included the inactivation deficient IFM/QQQ background to isolate activation. R222W decreased sodium current and slowed activation without affecting probability. Consistent with the phenotype of muscle weakness, R222W shifted fast inactivation to hyperpolarized potentials, promoted more rapid entry, and slowed recovery. R222W increased the extent of slow inactivation and slowed its recovery. A two-compartment skeletal muscle fiber model revealed that defects in fast inactivation sufficiently explain action potential attenuation in patients. Molecular dynamics simulations showed that R222W disrupted electrostatic interactions within the gating pore, supporting the observation that R222W promotes omega current at hyperpolarized potentials. Sodium channel inactivation defects produced by R222W are the primary driver of skeletal muscle fiber action potential attenuation, while hyperpolarization-induced omega current produced by that mutation promotes muscle fiber depolarization.

β1 subunit stabilises sodium channel Nav1.7 against mechanical stress.

The voltage-gated sodium channel Nav1.7 is a key player in neuronal excitability and pain signalling. In addition to voltage sensing, the channel is also modulated by mechanical stress. Using whole-cell patch-clamp experiments, we discovered that the sodium channel subunit β1 is able to prevent the impact of mechanical stress on Nav1.7. An intramolecular disulfide bond of β1 was identified to be essential for stabilisation of inactivation, but not activation, against mechanical stress using molecular dynamics simulations, homology modelling and site-directed mutagenesis. Our results highlight the role of segment 6 of domain IV in fast inactivation. We present a candidate mechanism for sodium channel stabilisation against mechanical stress, ensuring reliable channel functionality in living systems. Voltage-gated sodium channels are key players in neuronal excitability and pain signalling. Precise gating of these channels is crucial as even small functional alterations can lead to pathological phenotypes such as pain or heart failure. Mechanical stress has been shown to affect sodium channel activation and inactivation. This suggests that stabilising components are necessary to ensure precise channel gating in living organisms. Here, we show that mechanical shear stress affects voltage dependence of activation and fast inactivation of the Nav1.7 channel. Co-expression of the β1 subunit, however, protects both gating modes of Nav1.7 against mechanical shear stress. Using molecular dynamics simulation, homology modelling and site-directed mutagenesis, we identify an intramolecular disulfide bond of β1 (Cys21-Cys43) which is partially involved in this process: the β1-C43A mutant prevents mechanical modulation of voltage dependence of activation, but not of fast inactivation. Our data emphasise the unique role of segment 6 of domain IV for sodium channel fast inactivation and confirm previous reports that the intracellular process of fast inactivation can be modified by interfering with the extracellular end of segment 6 of domain IV. Thus, our data suggest that physiological gating of Nav1.7 may be protected against mechanical stress in a living organism by assembly with the β1 subunit.

Action of six pyrethrins purified from the botanical insecticide pyrethrum on cockroach sodium channels expressed in Xenopus oocytes

Pyrethrin I, pyrethrin II, cinerin I, cinerin II, jasmolin I and jasmolin II are six closely related insecticidal active esters, known as pyrethrins, found in the pyrethrum extract from the dry flowers of Tanacetum cinerariifolium. The chemical structures of the six compounds differ only in the terminal moieties at the acid and alcohol ends, but the compounds' in vivo toxicities are substantially different. Pyrethrins are lead compounds for pyrethroids, a large family of synthetic insecticides that alter nerve functions by prolonging the opening of voltage-gated sodium channels. However, data on the mechanism of action of natural pyrethrins are very limited. In this study, we examined the actions of all six pyrethrins on cockroach sodium channels expressed in Xenopus oocytes. Although the six compounds showed comparable potencies in inhibiting the inactivation of sodium channels, they had greatly variable potencies in inhibiting channel deactivation. Furthermore, unlike pyrethroids, the action of pyrethrins neither depend on nor were enhanced by repeated channel activation. We created a NavMs-based model of the cockroach sodium channel, in which pyrethrin II was docked at the pyrethroid receptor site 1 (PyR1), and proposed a rationale for the observed structure-activity relationship of the six pyrethrins. Our study sheds light on the molecular mechanism of pyrethrum action on sodium channels and reveled differences in the modes of action of the six bioactive constitutes of pyrethrum.

Lacosamide protects striatal and hippocampal neurons from in vitro ischemia without altering physiological synaptic plasticity

Lacosamide ([(R)-2-acetamido-N-benzyl-3-methoxypropanamide], LCM), is an antiepileptic that exerts anticonvulsant activity by selectively enhancing slow sodium channel inactivation. By inhibiting seizures and neuronal excitability it might therefore be a good candidate to stabilize neurons and protect them from energetic insults. Using electrophysiological analyses, we have investigated in mice the possible neuroprotective effect of LCM against in vitro ischemia obtained by oxygen and glucose deprivation (ODG), in striatal and hippocampal tissues, two brain structures particularly susceptible to ischemic injury and of pivotal importance for different form of learning and memory. We also explored in these regions the influence of LCM on firing discharge and on long-term synaptic plasticity. We found that in both areas LCM reduced the neuronal firing activity in a use-dependent manner without influencing the physiological synaptic transmission, confirming its anticonvulsant effects. Moreover, we found that this AED is able to protect, in a dose dependent manner, striatal and hippocampal neurons from energy metabolism failure produced by OGD. This neuroprotective effect does not imply impairment of long-term potentiation of striatal and hippocampal synapses and suggests that LCM might exert additional beneficial therapeutic effects beyond its use as antiepileptic.

Lacosamida asociada a bloqueo de alto grado en un paciente con neuralgia del trigémino

Lacosamide is an antiepileptic drug whose exact mechanism of action remains unknown. It acts by increasing the slow inactivation of the voltage-dependent sodium channels of the cell membranes. It is indicated in the treatment of focal seizures with or without secondary generalisation and is occasionally used as adjunct treatment in neuropathic pain. Although the most frequent side effects are mild (dizziness, diplopia, blurred vision, headache, tremor, etc.), others such as supraventricular tachyarrhythmias, changes in repolarisation, atrioventricular blocks and even cardiac arrest or sudden death have been reported. A 74-year-old male, diagnosed with classic trigeminal neuralgia treated with 200 mg/12 h of carbamazepine, who visited due to a worsening of the pain in the trigeminal V1-V2 region. On the sixth day after admission, after adjusting the carbamazepine treatment to a descending regime, 400 mg/24 h of eslicarbazepine and 100 mg/12 h of intravenous lacosamide, he presented a complete atrioventricular block with extreme bradycardia that required the placement of a pacemaker. Voltage-dependent sodium channel blockade mainly affects non-sinusal cardiac tissue. An alteration in the atrioventricular or infrahisian node is more consistent with its mechanism of action. Other cases of atrioventricular block in this kind of polytherapy have been reported. Precaution is advised in the concomitant use of antiepileptic drugs, above all among those that prolong the PR interval, and they should be contraindicated in patients with a history of atrioventricular block, ischaemic heart disease or heart failure. Before starting, a baseline electrocardiogram and regular electrocardiographic monitoring are advised during the first few weeks.

Short-Term Depression of Axonal Spikes at the Mouse Hippocampal Mossy Fibers and Sodium Channel-Dependent Modulation.

Axonal spike is an important upstream process of transmitter release, which directly impacts on release probability from the presynaptic terminals. Despite the functional significance, possible activity-dependent modulation of axonal spikes has not been studied extensively, partly due to inaccessibility of the small structures of axons for electrophysiological recordings. In this study, we tested the possibility of use-dependent changes in axonal spikes at the hippocampal mossy fibers, where direct recordings from the axon terminals are readily feasible. Hippocampal slices were made from mice of either sex, and loose-patch clamp recordings were obtained from the visually identified giant mossy fiber boutons located in the stratum lucidum of the CA3 region. Stimulation of the granule cell layer of the dentate gyrus elicited axonal spikes at the single bouton which occurred in all or none fashion. Unexpected from the digital nature of spike signaling, the peak amplitude of the second spikes in response to paired stimuli at a 50-ms interval was slightly but reproducibly smaller than the first spikes. Repetitive stimuli at 20 or 100 Hz also caused progressive use-dependent depression during the train. Notably, veratridine, an inhibitor of inactivation of sodium channels, significantly accelerated the depression with minimal effect on the initial spikes. These results suggest that sodium channels contribute to use-dependent depression of axonal spikes at the hippocampal mossy fibers, possibly by shaping the afterdepolarization (ADP) following axonal spikes. Prolonged depolarization during ADP may inactivate a fraction of sodium channels and thereby suppresses the subsequent spikes at the hippocampal mossy fibers.

Natural meroterpenoids isolated from the plant pathogenic fungus Verticillium albo-atrum with noteworthy modification action against voltage-gated sodium channels of central neurons of Helicoverpa armigera

A new meroterpenoid, named acetoxydehydroaustin A (1) and the known meroterpenoid austin (2) were isolated from the plant pathogenic fungus Verticillium albo-atrum. Their structures were established based on general spectroscopic techniques and the relative configuration of compound 1 was determined by single-crystal X-ray diffraction analysis. We first investigated and identified their significant electrophysiological effects on the gating kinetics of voltage-gated sodium channels in central neurons acutely dissociated from Helicoverpa armigera using whole-cell patch clamp technique. Similar to the effects of pyrethroids on sodium late currents, both compounds produced concentration-dependent modification of sodium channels, prolonging the kinetics of channel inactivation to generate large persistent late currents during depolarization. However, different from the effects of tefluthrin and deltamethrin on sodium channels, two meroterpenoids did not induce tail currents during deactivation. Compounds 1 and 2 also caused depolarizing shifts in the voltage dependence of channel activation. The V0.5 shifted about 5.02mV and 6.32mV in the depolarizing direction by 50μM 1 and 50μM 2. The V0.5 of voltage-dependent inactivation shifted about 11.42mV and 11.62mV respectively in the hyperpolarizing direction by 50μM 1 and 100μM 2. In addition, they prolonged the time course of recovery from fast-inactivation for sodium channels. The effects of two compounds on the voltage-dependent gating substantially increased the size of sodium window currents. The overlapped area of window currents increased about 89.69% and 44.51% respectively by 10μM compound 1 and 10μM compound 2. These findings show that both compounds have effects on sodium channel activation, inactivation and window currents. The voltage-gated sodium channels in central neurons of H. armigera are the target sites of two meroterpenoid natural products.

Muscle membrane properties in A pig sepsis model: Effect of norepinephrine.

Sepsis-induced myopathy and critical illness myopathy are common causes of muscle weakness in intensive care patients. This study investigated the effect of different mean arterial blood pressure (MAP) levels on muscle membrane properties following experimental sepsis. Sepsis was induced with fecal peritonitis in 12 of 18 anesthetized and mechanically ventilated pigs. Seven were treated with a high (75-85 mmHg) and 5 were treated with a low (≥60 mmHg) MAP target for resuscitation. In septic animals, resuscitation was started 12 h after peritonitis induction, and muscle velocity recovery cycles were recorded 30 h later. Muscles in the sepsis/high MAP group showed an increased relative refractory period and reduced early supernormality compared with the remaining septic animals and the control group, indicating membrane depolarization and/or sodium channel inactivation. The membrane abnormalities correlated positively with norepinephrine dose. Norepinephrine may contribute to sepsis-induced abnormalities in muscle by impairing microcirculation. Muscle Nerve 57: 808-813, 2018.

Axonal excitability changes and acute symptoms of oxaliplatin treatment: In vivo evidence for slowed sodium channel inactivation

ObjectiveNeurotoxicity is the most frequent dose-limiting side effect of the anti-cancer agent oxaliplatin, but the mechanisms are not well understood. This study used nerve excitability testing to investigate the pathophysiology of the acute neurotoxicity. MethodsQuestionnaires, quantitative sensory tests, nerve conduction studies and nerve excitability testing were undertaken in 12 patients with high-risk colorectal cancer treated with adjuvant oxaliplatin and in 16 sex- and age-matched healthy controls. Examinations were performed twice for patients: once within 3 days after oxaliplatin treatment (post-infusion examination) and once shortly before the following treatment (recovery examination). ResultsThe most frequent post-infusion symptoms were tingling paresthesias and cold allodynia. The most prominent nerve excitability change was decreased superexcitability of motor axons which correlated with the average intensity of abnormal sensations (Spearman Rho = 0.80, p < .01). The motor nerve excitability changes were well modeled by a slowing of sodium channel inactivation, and were proportional to dose/m2 with a half-life of about 10d. ConclusionsOxaliplatin induces reversible slowing of sodium channel inactivation in motor axons, and these changes are closely related to the reversible cold allodynia. However, further studies are required due to small sample size in this study. SignificanceNerve excitability data provide an index of sodium channel dysfunction: an objective biomarker of acute oxaliplatin neurotoxicity.

In vivo assessment of muscle membrane properties in the sodium channel myotonias.

The gain-of-function mutations that underlie sodium channel myotonia (SCM) and paramyotonia congenital (PMC) produce differing clinical phenotypes. We used muscle velocity recovery cycles (MVRCs) to investigate membrane properties. MVRCs and responses to trains of stimuli were compared in patients with SCM (n = 9), PMC (n = 8), and normal controls (n = 26). The muscle relative refractory period was reduced in SCM, consistent with faster recovery of the mutant sodium channels from inactivation. Both SCM and PMC showed an increased early supernormality and increased mean supernormality following multiple conditioning stimuli, consistent with slowed sodium channel inactivation. Trains of fast impulses caused a loss of amplitude in PMC, after which only half of the muscle fibers recovered, suggesting that the remainder stayed depolarized by persistent sodium currents. The differing effects of mutations on sodium channel function can be demonstrated in human subjects in vivo using this technique. Muscle Nerve 57: 586-594, 2018.

Pro-excitatory alterations in sodium channel activity facilitate subiculum neuron hyperexcitability in temporal lobe epilepsy

Temporal lobe epilepsy (TLE) is a common form of adult epilepsy involving the limbic structures of the temporal lobe. Subiculum neurons act to provide a major output from the hippocampus and consist of a large population of endogenously bursting excitatory neurons. In TLE, subiculum neurons are largely spared, become hyperexcitable and show spontaneous epileptiform activity. The basis for this hyperexcitability is unclear, but is likely to involve alterations in the expression levels and function of various ion channels. In this study, we sought to determine the importance of sodium channel currents in facilitating neuronal hyperexcitability of subiculum neurons in the continuous hippocampal stimulation (CHS) rat model of TLE. Subiculum neurons from TLE rats were hyperexcitable, firing a higher frequency of action potentials after somatic current injection and action potential (AP) bursts after synaptic stimulation. Voltage clamp recordings revealed increases in resurgent (INaR) and persistent (INaP) sodium channel currents and pro-excitatory shifts in sodium channel activation and inactivation parameters that would facilitate increases in AP generation. Attenuation of INaR and INaP currents with 4,9-anhydro-tetrodotoxin (4,9-ah TTX; 100nM), a toxin with increased potency against Nav1.6 channels, suppressed neuronal firing frequency and inhibited AP bursting induced by synaptic stimulation in TLE neurons. These findings support an important role of sodium channels, particularly Nav1.6, in facilitating subiculum neuron hyperexcitability in TLE and provide further support for the importance of INaR and INaP currents in establishing epileptiform activity of subiculum neurons.

Eslicarbazepine Acetate: A New Improvement on a Classic Drug Family for the Treatment of Partial-Onset Seizures.

Eslicarbazepine acetate is a new anti-epileptic drug belonging to the dibenzazepine carboxamide family that is currently approved as adjunctive therapy and monotherapy for partial-onset (focal) seizures. The drug enhances slow inactivation of voltage-gated sodium channels and subsequently reduces the activity of rapidly firing neurons. Eslicarbazepine acetate has few, but some, drug–drug interactions. It is a weak enzyme inducer and it inhibits cytochrome P450 2C19, but it affects a smaller assortment of enzymes than carbamazepine. Clinical studies using eslicarbazepine acetate as adjunctive treatment or monotherapy have demonstrated its efficacy in patients with refractory or newly diagnosed focal seizures. The drug is generally well tolerated, and the most common side effects include dizziness, headache, and diplopia. One of the greatest strengths of eslicarbazepine acetate is its ability to be administered only once per day. Eslicarbazepine acetate has many advantages over older anti-epileptic drugs, and it should be strongly considered when treating patients with partial-onset epilepsy.

Valproic acid inhibits TTX-resistant sodium currents in prefrontal cortex pyramidal neurons

Valproic acid is frequently prescribed and used to treat epilepsy, bipolar disorder and other conditions. However, the mechanism of action of valproic acid has not been fully elucidated. The aim of this study was to assess the influence of valproic acid (200 μM) on TTX-resistant sodium currents in mPFC pyramidal neurons. Valproic acid inhibited the maximal amplitude and did not change the activation parameters of TTX-resistant sodium currents. Moreover, valproic acid (2 μM and 200 μM) shifted the TTX-resistant sodium channel inactivation curve towards hyperpolarisation. In the presence of valproic acid, TTX-resistant sodium currents recovered from inactivation more slowly. Valproic acid did not influence the use-dependent blockade of TTX-resistant sodium currents.This study suggests that a potential new mechanism of the antiepileptic action of valproic acid is, among others, inhibition of TTX-resistant sodium currents.

Readthrough of SCN5A Nonsense Mutations p.R1623X and p.S1812X Questions Gene-therapy in Brugada Syndrome.

Nonsense mutation readthrough is used as a gene-specific treatment in some genetic diseases. The response to readthrough treatment is determined by the readthrough efficiency of various nonsense mutations. In this manuscript, we aimed to explore the harmful effects of nonsense mutation suppression. HEK293 cells were transfected with two SCN5A (encode cardiac Na+ channel) nonsense mutations, p.R1623X and p.S1812X. We applied two readthrough-enhancing methods (either aminoglycosides or a siRNA-targeting eukaryotic release factor eRF3a (a GTPase that binds eRF1)) to suppress these SCN5A nonsense mutations. When either of readthrough methods was used, the sodium channel proteins were examined by western blot and immunoblotting and recorded by whole cell patch-clamp to observe the functional characterization of the restored channels. Upon readthrough treatment, the sodium currents were restored to the mutant cDNAs. These mutations reduced full-length sodium channel protein levels, and the sodium currents were reduced to 3% of wild-type. The mutant cDNA sodium currents were increased to 30% of wild-type, and the fulllength proteins also increased. However, the functional characterization of these channels from cDNAs carrying p.R1623X and p.S1812X exhibited abnormal biophysical properties, including a negative shift in steady-state sodium channel inactivation, a positive shift in sodium channel activation and robust late sodium currents. The ramp test showed prolonged QT intervals. These results demonstrated that readthrough-enhancing methods effectively suppressed nonsense mutations in SCN5A and restored the expression of full-length channels. However, the restored channels may increase the risk of arrhythmia.

Isopimaric acid - a multi-targeting ion channel modulator reducing excitability and arrhythmicity in a spontaneously beating mouse atrial cell line.

Atrial fibrillation is the most common persistent cardiac arrhythmia, and it is not well controlled by present drugs. Because some resin acids open voltage-gated potassium channels and reduce neuronal excitability, we explored the effects of the resin acid isopimaric acid (IPA) on action potentials and ion currents in cardiomyocytes. Spontaneously beating mouse atrial HL-1 cells were investigated with the whole-cell patch-clamp technique. 1-25 μmol L-1 IPA reduced the action potential frequency by up to 50%. The effect of IPA on six different voltage-gated ion channels was investigated; most voltage-dependent parameters of ion channel gating were shifted in the negative direction along the voltage axis, consistent with a hypothesis that a lipophilic and negatively charged compound binds to the lipid membrane close to the positively charged voltage sensor of the ion channels. The major finding was that IPA inactivated sodium channels and L- and T-type calcium channels and activated the rapidly activating potassium channel and the transient outward potassium channel. Computer simulations of IPA effects on all of the ion currents were consistent with a reduced excitability, and they also showed that effects on the Na channel played the largest role to reduce the action potential frequency. Finally, induced arrhythmia in the HL-1 cells was reversed by IPA. Low concentrations of IPA reduced the action potential frequency and restored regular firing by altering the voltage dependencies of several voltage-gated ion channels. These findings can form the basis for a new pharmacological strategy to treat atrial fibrillation.

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.

Heterogeneous firing responses predict diverse couplings to presynaptic activity in mice layer V pyramidal neurons.

In this study, we present a theoretical framework combining experimental characterizations and analytical calculus to capture the firing rate input-output properties of single neurons in the fluctuation-driven regime. Our framework consists of a two-step procedure to treat independently how the dendritic input translates into somatic fluctuation variables, and how the latter determine action potential firing. We use this framework to investigate the functional impact of the heterogeneity in firing responses found experimentally in young mice layer V pyramidal cells. We first design and calibrate in vitro a simplified morphological model of layer V pyramidal neurons with a dendritic tree following Rall's branching rule. Then, we propose an analytical derivation for the membrane potential fluctuations at the soma as a function of the properties of the synaptic input in dendrites. This mathematical description allows us to easily emulate various forms of synaptic input: either balanced, unbalanced, synchronized, purely proximal or purely distal synaptic activity. We find that those different forms of dendritic input activity lead to various impact on the somatic membrane potential fluctuations properties, thus raising the possibility that individual neurons will differentially couple to specific forms of activity as a result of their different firing response. We indeed found such a heterogeneous coupling between synaptic input and firing response for all types of presynaptic activity. This heterogeneity can be explained by different levels of cellular excitability in the case of the balanced, unbalanced, synchronized and purely distal activity. A notable exception appears for proximal dendritic inputs: increasing the input level can either promote firing response in some cells, or suppress it in some other cells whatever their individual excitability. This behavior can be explained by different sensitivities to the speed of the fluctuations, which was previously associated to different levels of sodium channel inactivation and density. Because local network connectivity rather targets proximal dendrites, our results suggest that this aspect of biophysical heterogeneity might be relevant to neocortical processing by controlling how individual neurons couple to local network activity.

Phoneutria nigriventer spider toxin Tx2-6 induces priapism in mice even after cavernosal denervation

The Phoneutria nigriventer spider toxin Tx2-6 causes priapism in humans and mice. This toxin produces a delay in Sodium channel inactivation, generalized vascular congestion and death by respiratory failure. NO-Synthase inhibitors seem to abolish toxin-induced priapism. The understanding of the ultimate molecular mechanism involved in toxin-induced priapism may shed light on aspects of erectile function/dysfunction. This study investigates if cavernosal denervation can abolish the toxin-induced priapism. Surgical cavernosal nerve excision/denervation was performed in mice and confirmed by infertility, histological assessment of fibrosis and immunohistochemical staining for synaptophysin. Denervated mice showed intense fibrosis of the cavernosal tissue as well as absence of synaptophysin IHC staining; surprisingly mice showed toxin-induced priapism when tested 15, 30 or 60 days after denervation. While sham-operated mice presented full priapism, denervated animals showed only partial priapism possibly due to the fibrosis. These results reveal that erection caused by Tx2-6 toxin may not depend on cavernosal nerves integrity. The effect of this toxin on sodium channels seem not directly involved in priapism as many toxins have identical effects but do not induce priapism. Discussion approaches the many different potential sites of intervention listed in the signaling cascades of NO/cGMP, RhoA/Rho-Kinase, as well as the emerging new gasotransmitter H2S. The pharmacological inhibition of Rho-kinase and toxin Tx2-6 have similar effects in vivo.

Zn2+ reduction induces neuronal death with changes in voltage-gated potassium and sodium channel currents

In the present study, cultured rat primary neurons were exposed to a medium containing N,N,N’,N’-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), a specific cell membrane-permeant Zn2+ chelator, to establish a model of free Zn2+ deficiency in neurons. The effects of TPEN-mediated free Zn2+ ion reduction on neuronal viability and on the performance of voltage-gated sodium channels (VGSCs) and potassium channels (Kvs) were assessed. Free Zn2+ deficiency 1) markedly reduced the neuronal survival rate, 2) reduced the peak amplitude of INa, 3) shifted the INa activation curve towards depolarization, 4) modulated the sensitivity of sodium channel voltage-dependent inactivation to a depolarization voltage, and 5) increased the time course of recovery from sodium channel inactivation. In addition, free Zn2+ deficiency by TPEN notably enhanced the peak amplitude of transient outward K+ currents (IA) and delayed rectifier K+ currents (IK), as well as caused hyperpolarization and depolarization directional shifts in their steady-state activation curves, respectively. Zn2+ supplementation reversed the effects induced by TPEN. Our results indicate that free Zn2+ deficiency causes neuronal damage and alters the dynamic characteristics of VGSC and Kv currents. Thus, neuronal injury caused by free Zn2+ deficiency may correlate with its modulation of the electrophysiological properties of VGSCs and Kvs.

A novel μ-conotoxin from worm-hunting Conus tessulatus that selectively inhibit rat TTX-resistant sodium currents

μ-conotoxins are a group of marine Conus peptides that inhibit sodium currents, so μ-conotoxins are valuable in sodium channel research and new analgesic drug discovery. Here, a novel μ-conotoxin TsIIIA was identified from a worm-hunting Conus tessulatus. TsIIIA was chemical synthesized according to its amino acid sequence GCCRWPCPSRCGMARCCSS and identified by mass spectrum. Patch clamp on rat dorsal root ganglion cells showed that 10 μM TsIIIA specifically inhibit TTX-resistant sodium currents but has no effect on TTX-sensitive sodium currents. TsIIIA inhibits TTX-resistant sodium currents by a dose-dependent mode with an IC50 of 2.61 μM. Further study showed 10 μM TsIIIA has no obvious effect on the current-voltage relationships, conductance-voltage relationships and voltage-dependence of steady-state inactivation of TTX-resistant sodium channels. Mice hotplate analgesic assay indicated that TsIIIA obviously increase the pain threshold at 0.5–4 h. In addition, TsIIIA has better analgesic effects than Ziconotide, indicating that TsIIIA was a valuable lead compound for development of new analgesic drug.