Sea Anemone Toxin ATX-II Research Articles

Dataset of electrophysiological patch-clamp recordings of the effect of the compounds deltamethrin, ATx-II and β4-peptide on human cardiac Nav1.5 sodium channel gating properties.

This article describes the effect of the pyrethroid insecticide deltamethrin on the cardiac voltage-gated sodium channel Nav1.5. Two concentrations of deltamethrin were used and the effects were compared with those of the sea anemone toxin ATx-II and β4-peptide, which is the C-terminus of the Nav channel β-subunit. Activation, fast inactivation, deactivation, persistent currents and resurgent currents of Nav1.5 channels were assessed in the presence of these compounds. The data display not only the effect of separately applied compounds on Nav1.5 channels but also investigates how combinations of these substances affect Nav1.5 channel gating properties.The dataset presented in this article is related to the research article “Mechanism underlying hooked resurgent-like tail currents induced by an insecticide in human cardiac Nav1.5″ (Sarah Thull, Cristian Neacsu, Andrias O. O'Reilly, Stefanie Bothe, Ralf Hausmann, Tobias Huth, Jannis Meents, Angelika Lampert, doi: 10.1016/j.taap.2020.11501), that investigates the effect of the pyrethroid insecticide deltamethrin on Nav channel gating properties and explains the mechanism underlying hooked, resurgent-like tail currents induced by deltamethrin in Nav1.5 channels.

Mechanism underlying hooked resurgent-like tail currents induced by an insecticide in human cardiac Nav1.5

Voltage-gated sodium channels are responsible not only for the fast upstroke of the action potential, but they also modify cellular excitability via persistent and resurgent currents. Insecticides act via permanently opening sodium channels to immobilize the animals. Cellular recordings performed decades ago revealed distinctly hooked tail currents induced by these compounds. Here, we applied the classical type-II pyrethroid deltamethrin on human cardiac Nav1.5 and observed resurgent-like currents at very negative potentials in the absence of any pore-blocker, which resemble those hooked tail currents. We show that deltamethrin dramatically slows both fast inactivation and deactivation of Nav1.5 and thereby induces large persistent currents. Using the sea anemone toxin ATx-II as a tool to prevent all inactivation-related processes, resurgent-like currents were eliminated while persistent currents were preserved. Our experiments suggest that, in deltamethrin-modified channels, recovery from inactivation occurs faster than delayed deactivation, opening a brief window for sodium influx and leading to hooked, resurgent-like currents, in the absence of an open channel blocker. Thus, we now explain with pharmacological methods the biophysical gating changes underlying the deltamethrin induced hooked tail currents. SummaryThe pyrethroid deltamethrin induces hooked resurgent-like tail currents in human cardiac voltage-gated Nav1.5 channels. Using deltamethrin and ATx-II, we identify additional conducting channel states caused by a faster recovery from inactivation compared to the deltamethrin-induced delayed deactivation.

Inhibitory Profile of Clozapine AT CiPA Cardiac Ion Channels

The atypical antipsychotic clozapine is a prominent treatment for refractory schizophrenia. This drug inhibits the human ether-à-go-go related gene (hERG) and prolongs the QT-interval on the electrocardiogram. Although these biomarkers are sensitive predictors of the risk of Torsade de Pointe (TdP) arrhythmia, the long standing clinical use of clozapine did not show consistent association with increased incidence of TdP. It has recently been suggested that inhibition of other ion channels influencing the ventricular action potential can contribute refine true torsadogenic risk assessment. Here we have profiled clozapine on seven such currents using an automated electrophysiology platform. CHO or HEK cells expressing the human ion channels retained by the Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative were recorded in whole-cell mode at room temperature on a Qpatch 48X planar patch-clamp workstation (Sophion, Denmark). Cells were exposed to cumulative concentrations of clozapine in saline buffer containing 0.3% DMSO supplemented with 0.06% Pluronic F-68. Clozapine concentration-dependently inhibited currents through Kv11.1 (hERG), Cav1.2, Nav1.5 (peak), Kv7.1 and Kv4.3 channels with IC50‘s of 4.2 ± 0.6 µM, 11 ± 2.4, µM, 10 ± 1.8 µM, 12 ± 1.2 µM and 54 ± 5.5 µM, respectively. The late NaV1.5 current through persistently opened channels by the sea anemone toxin ATX-II was inhibited as strongly as hERG with an IC50 of 5.5 ± 0.3 µM. In contrast, outward currents through Kir2.1 channels were reduced by only 28 ± 2.5 % at 100 µM clozapine. These data suggest that simultaneous inhibition of multiple cardiac ion channels underlies the low pro-arrhythmia risk profile of clozapine, although the submicromolar therapeutic free plasma concentrations in patients should be taken into account. How this balanced ion-channel profile translates into QT prolongation deserves further studies in integrated systems.

GBR-12909: Potent Blocker of Peak and Late Nav1.5 Currents

GBR-12909 (vanoxerine) is a potent highly selective dopamine transporter antagonist. It was initially developed for treatment of Parkinson's disease and while safe in man, lacked efficacy. Although a potent hERG blocker, the drug exhibits multiple ion channel effects including frequency-dependence. The drug effectively terminated induced, sustained atrial fibrillation (AF) and atrial flutter in a canine model of sterile pericarditis. Subsequently the drug terminated AF in a Phase 2B ascending dose trial. Previous in vitro measurements used protocols that did not address the issue of the late Nav1.5 current which is important in understanding safety of a potent hERG blocker. Peak and late sodium currents were measured in HEK-293 cells stably expressing the human Nav1.5 channel. Peak currents were measured using a step-ramp protocol consisting of a 40 ms step to −15 mV (where the peak current was measured), a 180 ms step to +30 mV and a 200 ms ramp to the holding potential of −80 mV (1 Hz stimulating frequency). The effect of the drug was parameterized with a single binding isotherm having an IC50 of 29 ± 3 nM (95% CI: 22-37) and a Hill coefficient of 0.91 ± 0.10 (95% CI: 0.68-1.18) (seven cells, three concentrations). We also evaluated the effect of GBR-19209 on currents increased using the sea-anemone toxin ATX-II as a surrogate of the late sodium current. The calculated IC50 and Hill coefficients from five cells and five concentrations were 93 ± 9 nM (95% CI: 7-117) and 1.57 ± 0.24 (95% CI: 1.05-2.67), respectively. Thus, GBR-12909 block is at least sixty times more potent than ranolazine's (IC50 = 6 µM).The combination of potent peak and late sodium block may explain the antiarrhythmic and safe profile of the drug.

CrossTalk proposal: The late sodium current is an important player in the development of diastolic heart failure (heart failure with a preserved ejection fraction)

Approximately half of all patients with heart failure have an ejection fraction greater than 40–50% and may be diagnosed as having Heart Failure with preserved Ejection Fraction (HFpEF). Diastolic dysfunction is central to the pathophysiology of HFpEF (Borlaug & Paulus, 2011), and describes the slowing of ventricular relaxation and increased diastolic stiffness which ultimately impairs ventricular filling. The mechanistic basis of this impairment is complex and not yet well understood. Structural remodelling undoubtedly plays an important role in increasing left ventricular stiffness. However, the acute worsening of diastolic dysfunction during stress or exercise characteristic of HFpEF suggests an important contribution from dynamic changes in left ventricular (LV) functional properties. Frequency-dependent elevation of diastolic tension and intracellular Ca2+ ([Ca2+]i) has been observed in cardiac muscle strips from patients with left ventricular hypertrophy and diastolic dysfunction, or heart failure (Sossalla et al. 2008; Selby et al. 2011), implying that dysregulation of [Ca2+]i homeostasis of the cardiomyocyte contributes to diastolic dysfunction. Intracellular Ca2+ regulation is closely linked to intracellular Na+ homeostasis, through the Na+–Ca2+ exchanger (NCX). Intracellular Na+ of cardiomyocytes from failing hearts is increased and associated with elevated diastolic tension (Pieske et al. 2002). An important mechanism underlying this observation may be an increase in the late sodium current (INa,L). The Na+ conductance responsible for rapid depolarization of cardiomyocytes does not completely inactivate during the action potential. (Noble & Noble, 2006; Maier, 2012) Some channels continue to conduct, or even reactivate at relatively positive membrane potentials during the plateau and repolarization phases. This is INa,L (Zaza et al. 2008). Consequently, about half of the myocyte Na+ entry occurs during the initial 2–3 ms, and about half during the remainder of the action potential (Makielski & Farley, 2006). At the molecular level, INa,L results from channel reopening during sustained depolarization by two different modes of gating: burst openings and late scattered openings (Maltsev & Undrovinas, 2008). As outlined in Fig. 1, increased Na+ entry through INa,L increases intracellular Na+ ([Na+]i), which reduces the driving force for extrusion of Ca2+ and favours Ca2+ influx via the Na+–Ca2+ exchanger (NCX). This leads to increased [Ca2+]i. Elevated [Ca2+]i eventually increases actin–myosin filament interaction during diastole and thus increases diastolic tension. This mechanism of Ca2+ overload has been demonstrated in numerous animal studies, and in strips of ventricular muscle or myocytes isolated from patients with failing hearts (Valdivia et al. 2005; Makielski & Farley, 2006; Maltsev & Undrovinas, 2008; Sossalla et al. 2008; Selby et al. 2011; Coppini et al. 2013). Further, specific augmentation of INa,L with the sea anemone toxin ATXII in isolated myocytes and perfused hearts results in Na+ and Ca2+ overload (Fraser et al. 2006; Sossalla et al. 2008) and impaired diastolic function. Diastolic dysfunction with preserved systolic function has also been described in LQT syndrome type 3 patients, where INa,L is enhanced due to a Na+ channel mutation (Moss et al. 2008; Hummel et al. 2013). Figure 1 A pathological enhanced INa,L contributes to Na+-dependent Ca2+ overload, diastolic dysfunction We propose that a pathological increase in Na+ influx through cardiac Na+ channels, specifically due to enhanced INa,L is a major contributor to Ca2+ overload and diastolic dysfunction in HFpEF. Key evidence to support this hypothesis is outlined below.

Rebuttal from Marc Pourrier, Sarah Williams, Donald McAfee, Luiz Belardinelli and David Fedida

Papp et al. (2013) raise a number of salient points in disputing the importance of INa,L in diastolic dysfunction in HFpEF. First, they question the selective effect of enhanced INa,L on passive relaxation when one would expect increased INa,L to affect early active relaxation kinetics as well. While the passive phase of relaxation is dependent on diastolic Ca2+ accumulation and Ca2+ extrusion primarily via the Na+–Ca2+ exchanger, the active phase of relaxation, on the other hand is more related to the rate of Ca2+ reuptake into the sarcoplasmic reticulum (SR). Although specific enhancement of INa,L in cardiac myocytes using the sea anemone toxin ATXII slows Ca2+ reuptake into the SR (Sossalla et al. 2008), experimental and clinical data obtained with ranolazine indicate it has mixed effects on active relaxation (Moss et al. 2008; Sossalla et al. 2008; Undrovinas et al. 2010; Coppini et al. 2013; Maier et al. 2013). These discrepancies suggest that the contribution of INa,L to active relaxation may differ in various diseases. Papp et al. also argue that the role of CaMKII in increased INa,L-induced Na+-dependent Ca2+ accumulation in human cardiac myocytes has yet to be demonstrated to be directly associated with diastolic dysfunction and HFpEF. While experiments in human failing myocytes are lacking, there is experimental evidence that associates CaMKII with INa,L, Na+-dependent Ca2+ accumulation and diastolic dysfunction. Specifically, (1) activity of CaMKII is increased in human heart failure (HF) and diastolic dysfunction (Sossalla et al. 2010; Coppini et al. 2013) and (2) CaMKII phosphorylates Na+ channels, resulting in enhanced INa,L, and elevation of intracellular Na+ (Wagner et al. 2006). It is therefore reasonable to consider that CaMKII, as well as other modulators play an important role in INa,L-induced Na+-dependent Ca2+ accumulation and diastolic dysfunction in HFpEF (Zaza et al. 2008). Ranolazine is considered the prototypical INa,L inhibitor, and we agree with Papp et al. that it has other actions able to account for some of the effects observed on diastolic function. However, it is unlikely that the effects of ranolazine on myocardial relaxation are related to pFOX inhibition, as high concentrations (12% inhibition at 100 μm) are required, whereas cardiac function is improved in the presence of ∼10 μm ranolazine. More compelling is the fact that the specific Na+ channel inhibitor tetrodotoxin attenuates INa,L-induced Na+-dependent Ca2+ accumulation in failing ventricular myocytes (Undrovinas et al. 2010). Similarly, the novel highly specific and selective INa,L inhibitor GS967 decreases ATXII-induced increase in intracellular Na+ and diastolic Ca2+ in isolated rabbit ventricular myocytes (Belardinelli et al. 2013).

A use-dependent sodium current modification induced by type I pyrethroid insecticides in honeybee antennal olfactory receptor neurons

We studied the mode of action of type I pyrethroids on the voltage-dependent sodium current from honeybee olfactory receptor neurons (ORNs), whose proper function in antenna is crucial for interindividual communication in this species. Under voltage-clamp, tetramethrin and permethrin induce a long lasting TTX-sensitive tail current upon repolarization, which is the hallmark of an abnormal prolongation of the open channel configuration. Permethrin and tetramethrin also slow down the sodium current fast inactivation. Tetramethrin and permethrin both bind to the closed state of the channel as suggested by the presence of an obvious tail current after the first single depolarization applied in the presence of either compounds. Moreover, at first sight, channel opening seems to promote tetramethrin and permethrin binding as evidenced by the progressive tail current summation along with trains of stimulations, tetramethrin being more potent at modifying channels than permethrin. However, a use-dependent increase in the sodium peak current along with stimulations suggests that the tail current accumulation could also be a consequence of progressively unmasked silent channels. Experiments with the sea anemone toxin ATX-II that suppresses sodium channels fast inactivation are consistent with the hypothesis that these silent channels are either in an inactivated state at rest, or that they normally inactivate before they open so that they do not participate to the control sodium current. In honeybee ORNs, three processes lead to a use-dependent pyrethroid-induced tail current accumulation: (i) a recruitment of silent channels that produces an increase in the peak sodium current, (ii) a slowing down of the sodium current inactivation produced by prolongation of channels opening and (iii) a typical deceleration in current deactivation. The use-dependent recruitment of silent sodium channels in honeybee ORNs makes pyrethroids more potent at modifying neuronal excitability.

Using Patchxpress to Screen Blockers of the Cardiac Sodium Channel (Nav1.5) for Effects on Late INA, Peak INA, and Channel Kinetics

The Nav1.5 channel is responsible for the upstroke of the action potential in cardiac myocytes. Congenital mutations in hNav1.5 have been shown to impair fast inactivation, leading to an increase in persistent sodium current (late INa) and LQT3. Growing evidence suggests that an increase in late INa can contribute to the pathogenesis of several cardiac diseases. With the goal of finding small molecules that selectively target cardiac late INa over peak INa and have rapid unbinding kinetics, we developed three novel assays on PatchXpress using HEK-293 cells stably expressing hNav1.5. The first assay measures block of late INa generated by tefluthrin, a pyrethroid that impairs fast inactivation. We chose tefluthrin over another late INa activator, the sea anemone toxin ATX-II, because with tefluthrin late INa reaches steady state faster and more reliably. Importantly, block obtained with tefluthrin and ATX-II is usually similar. The second assay measures tonic and use-dependent block of peak INa stimulated at 0.1 Hz and 3 Hz, respectively. To reduce voltage clamp errors on PatchXpress due to large inward peak currents and access resistance, we used 50% series resistance compensation and reduced Na+ in the extracellular solution to 20 mM. The third assay is designed to determine the rate at which compounds dissociate from the channel and is calculated from the time-course of the decrease in peak INa when the stimulation frequency is reduced from 10 to 1 Hz. All three assays on PatchXpress were validated by comparing IC50s or off-rates for several standard Nav1.5 blockers (eg. flecainide, mexiletine, quinidine and lidocaine) to those obtained from manual patch clamp. IC50s agree within two-fold and off-rate rankings are identical.

Sea anemone ‘sting’ isolates IB4‐negative sensory neurones

Small diameter dorsal root ganglion cells can be broadly classified into those that bind Griffonia simplicifolia isolectin IB4 (IB4+) and those that do not bind this lectin (IB4−). These two populations can also be discriminated by the presence of pro-inflammatory peptides and by the expression of neurotrophin receptors; IB4− neurones have high levels of neuropeptides such as calcitonin gene-related peptide and substance P, and express receptors for nerve growth factor, whereas IB4+ neurones are neuropeptide poor and express receptors for glial cell line-derived neurotrophic factor. While both populations are composed of neurones which function as nociceptors, the functional significance of this sub-division remains largely unanswered. However, there is growing evidence that the sensory and biophysical properties IB4– and IB4+ sensory neurones differ between. For example, IB4+ neurones have longer-duration somatic action potentials and larger tetrodotoxin (TTX)-resistant Na+ currents attributable to the α-subunit NaV1.8 (Stucky & Lewin, 1999). It would be predicted that the differential expression of Na+ channel α-subunits between IB4− and IB4+ neurones would have important consequences for their excitability but this has not been extensively investigated. In this issue of The Journal of Physiology, Snape et al. (2010) provide further evidence for the differential expression of Na+ channel α-subunits between IB4− and IB4+ neurons. Firstly they demonstrate that the IB4− neurons are selectively sensitive to the sea anemone toxin ATX-II. As this toxin is relatively selective for the channels NaV1.1 and NaV1.2, it is suggested that one or both may be selectively expressed in IB4− neurons, but this remains to be directly demonstrated. Irrespective of the explanation for the sensitivity to ATX-II, this feature provides strong evidence that the expression of TTX-sensitive Na+ channel α-subunits differs between IB4− and IB4+ neurons and that this has consequences for their excitability (at least in response to this toxin). Secondly the authors demonstrate in IB4+ neurons that excitability increases as the membrane potential is shifted from the normal resting value to ∼−20 mV and attribute this to the high expression of NaV1.8 in these neurons. This conclusion fits well with evidence that sensory nerve endings with these channels are able to sustain the initiation of action potentials when substantially depolarized. In IB4− neurones, excitability peaked ∼10 mV positive of the resting membrane potential and then declined as the membrane was further depolarized, reflecting inactivation of the Na+ channels contributing to the Na+ current. Presumably this behaviour indicates that these neurones will not sustain action potentials during prolonged periods of depolarization (or maintained sensory stimulation). Finally they investigated the mechanisms that contribute to the slowing in conduction velocity that is observed during repetitive activation of C-fibre axons. This feature has been used in microneurography experiments to identify single sensory C-fibres projecting to skin. Importantly, the magnitude of conduction velocity slowing has been shown to differ between sensory neurons with differing modalities (indicating differences in the mechanisms that control excitability; Serra et al. 1999). Recently it has been debated whether the change in conduction velocity results from an increase in Na+/K+ ATPase activity that hyperpolarizes the membrane potential during trains of action potentials (decreasing excitability; Rang & Ritchie, 1968) or the transition of Na+ channels (in particular NaV1.8) into a slow inactivated state (De Col et al. 2008). As Na+ channels recover from slow inactivation slowly, the latter change produces a prolonged decrease in the numbers of Na+ channels available for activation. In IB4+ neurones (but not IB4− neurones), they demonstrated in current clamp that repetitive activation with depolarizing steps produces a pronounced reduction in the derived action current and a delay in action potential initiation. As this change occurred when the ‘resting’ membrane potential was held constant, it cannot be attributed to membrane hyperpolarization. Instead, the change is explained by slow inactivation of NaV1.8 in the IB4+ neurones, supporting the hypothesis that slow inactivation of Na+ channels explains the decrease in conduction velocity (as suggested by De Col et al. 2008). Whether this is the only mechanism remains to be resolved. Together the data presented in this paper provide further functional evidence that the mechanisms regulating excitability differ between IB4− and IB4+ neurones. This new knowledge will prove useful both in investigating and in explaining the differences in sensory phenotypes between these two populations of neurones. The findings also highlight the need to consider the consequences of differential expression of TTX-sensitive Na+ channel α-subunits between IB4− and IB4+ neurones. Recently NaV1.7 (which is relatively insensitive to ATX-II) has been shown to play a key role in nociceptor activation but other TTX (and ATX-II)-sensitive Na+ channel α-subunits must also play an important role in regulating the excitability of IB4− neurones.

Antiarrhythmic effect of I Kr activation in a cellular model of LQT3

Long QT syndrome type 3 (LQT3) is an inherited cardiac disorder caused by gain-of-function mutations in the cardiac voltage-gated sodium channel, Na(v)1.5. LQT3 is associated with the polymorphic ventricular tachycardia torsades de pointes (TdP), which can lead to syncope and sudden cardiac death. The sea anemone toxin ATX-II has been shown to inhibit the inactivation of Na(v)1.5, thereby closely mimicking the underlying cause of LQT3 in patients. The hypothesis for this study was that activation of the I(Kr) current could counteract the proarrhythmic effects of ATX-II. Two different activators of I(Kr), NS3623 and mallotoxin (MTX), were used in patch clamp studies of ventricular cardiac myocytes acutely isolated from guinea pig to test the effects of selective I(Kr) activation alone and in the presence of ATX-II. Action potentials were elicited at 1 Hz by current injection and the cells were kept at 32 degrees C to 35 degrees C. NS3623 significantly shortened action potential duration at 90% repolarization (APD(90)) compared with controls in a dose-dependent manner. Furthermore, it reduced triangulation, which is potentially antiarrhythmic. Application of ATX-II (10 nM) was proarrhythmic, causing a profound increase of APD(90) as well as early afterdepolarizations and increased beat-to-beat variability. Two independent I(Kr) activators attenuated the proarrhythmic effects of ATX-II. NS3623 did not affect the late sodium current (I(NaL)) in the presence of ATX-II. Thus, the antiarrhythmic effect of NS3623 is likely to be caused by selective I(Kr) activation. The present data show the antiarrhythmic potential of selective I(Kr) activation in a cellular model of the LQT3 syndrome.

Sinus node dysfunction in ATX-II-induced in-vitro murine model of long QT3 syndrome and rescue effect of ranolazine

The aim of this study was to characterize the role of the late Na+ current (INa,L) as a mechanism for induction of both tachy and bradyarrhythmias in murine heart and sino-atrial node tissue. The sea anemone toxin ATX-II and ranolazine were used to increase and inhibit, respectively, INa,L. In sixteen hearts studied, exposure to 1–10nM ATX-II caused a slowing of intrinsic heart rate and prolongations of the P–R and QT intervals, the duration of the monophasic action potential, and the sinus node recovery time, accompanied by frequent occurrences of early afterdepolarisations, delayed afterdepolarisations and rapid, repetitive ventricular tachy and sino-atrial bradyarrhythmias. ATX-II also slowed sinus node pacemaking, and induced bradycardic arrhythmias in isolated sino-atrial preparations (n=5). The ATX-II-induced alteration of electrophysiological properties and occurrence of arrhythmic events were significantly attenuated by 10μM ranolazine in intact hearts (n=11) and isolated sino-atrial preparations (n=5). In conclusion, the INa,L enhancer ATX-II causes both tachy and bradyarrhythmias in the murine heart, and these arrhythmias are markedly attenuated by the INa,L blocker, ranolazine (10μM). The results suggest that INa,L blockade may be the mechanism underlying the reductions of both brady and tachyarrhythmias by ranolazine that were observed during the MERLIN-TIMI clinical outcomes trial.

Role of late sodium current in modulating the proarrhythmic and antiarrhythmic effects of quinidine

Quinidine is used to treat atrial fibrillation and ventricular arrhythmias. However, at low concentrations, it can induce torsade de pointes (TdP). The purpose of this study was to examine the role of late sodium current (I(Na)) as a modulator of the arrhythmogenicity of quinidine in female rabbit isolated hearts and cardiomyocytes. Epicardial and endocardial monophasic action potentials (MAPs), ECG signals, and ion channel currents were measured. The sea anemone toxin ATX-II was used to increase late I(Na). Quinidine had concentration-dependent and often biphasic effects on measures of arrhythmogenicity. Quinidine increased the duration of epicardial MAP (MAPD(90)), QT interval, transmural dispersion of repolarization (TDR), and ventricular effective refractory period. Beat-to-beat variability of MAPD(90) (BVR), the interval from peak to end of the T wave (Tpeak-Tend) and index of Tpeak-Tend/QT interval were greater at 0.1 to 3 micromol/L than at 10-30 micromol/L quinidine. In the presence of 1 nmol/L ATX-II, quinidine caused significantly greater concentration-dependent and biphasic changes of Tpeak-Tend, TDR, BVR, and index of Tpeak-Tend/QT interval. Quinidine (1 micromol/L) induced TdP in 2 and 13 of 14 hearts in the absence and presence of ATX-II, respectively. Increases of BVR, index of Tpeak-Tend/QT interval, and Tpeak-Tend were associated with quinidine-induced TdP. Quinidine inhibited I(Kr), peak I(Na), and late I(Na) with IC(50)s of 4.5 +/- 0.3 micromol/L, 11.0 +/- 0.7 micromol/L, and 12.0 +/- 0.7 micromol/L. Quinidine had biphasic proarrhythmic effects in the presence of ATX-II, suggesting that late I(Na) is a modulator of the arrhythmogenicity of quinidine. Enhancement of late I(Na) increased proarrhythmia caused by low but not high concentrations of quinidine.

A Scorpion α-Like Toxin That Is Active on Insects and Mammals Reveals an Unexpected Specificity and Distribution of Sodium Channel Subtypes in Rat Brain Neurons

Several scorpion toxins have been shown to exert their neurotoxic effects by a direct interaction with voltage-dependent sodium channels. Both classical scorpion alpha-toxins such as Lqh II from Leiurus quiquestratus hebraeus and alpha-like toxins as toxin III from the same scorpion (Lqh III) competitively interact for binding on receptor site 3 of insect sodium channels. Conversely, Lqh III, which is highly toxic in mammalian brain, reveals no specific binding to sodium channels of rat brain synaptosomes and displaces the binding of Lqh II only at high concentration. The contrast between the low-affinity interaction and the high toxicity of Lqh III indicates that Lqh III binding sites distinct from those present in synaptosomes must exist in the brain. In agreement, electrophysiological experiments performed on acute rat hippocampal slices revealed that Lqh III strongly affects the inactivation of voltage-gated sodium channels recorded either in current or voltage clamp, whereas Lqh II had weak, or no, effects. In contrast, Lqh III had no effect on cultured embryonic chick central neurons and on sodium channels from rat brain IIA and beta1 subunits reconstituted in Xenopus oocytes, whereas sea anemone toxin ATXII and Lqh II were very active. These data indicate that the alpha-like toxin Lqh III displays a surprising subtype specificity, reveals the presence of a new, distinct sodium channel insensitive to Lqh II, and highlights the differences in distribution of channel expression in the CNS. This toxin may constitute a valuable tool for the investigation of mammalian brain function.

Dose-dependent modulation of the cardiac sodium channel by sea anemone toxin ATXII.

The effects of sea anemone toxin ATXII on single sodium channels were studied in cell-attached patches on rabbit ventricular myocytes at 20-22 degrees C. Exposure of patches to 1,000 nM ATXII induced long-lasting bursts of openings, which were more dramatically different from control at -20 mV than at -50 mV. Mean open duration, which had a biphasic dependence on voltage in control patches, was monotonically dependent on voltage in toxin-exposed patches, being 3.5 times longer than control at -20 mV and 4.5 times longer at -10 mV. Multiple mean open durations were detected at depolarized potentials. To test whether the multiple mean open durations resulted from a mixture of modified and unmodified openings, histograms of late openings (when unmodified channels would be inactivated) were constructed. Because in most cases these fit a single exponential with a mean open duration like that of modified channels, we conclude that voltage-dependent toxin unbinding produced a mixed population of unmodified and modified openings. Consistent with this hypothesis, lower concentrations of toxin most often produced open-duration histograms best fit with two exponentials. Ensembles revealed complex decay kinetics, which could be interpreted within the context of the toxin-induced increase in mean open duration and burst duration and the summation of modified and unmodified events. Analysis of the numbers of early versus late events at -20 mV for patches exposed to 20 nM, 100 nM, and 1,000 nM ATXII predicted the ED50 for ATXII block to be 285 nM at this potential. Using a five-state Markovian model, the action of ATXII could be explained as a reduction of the open-to-inactivated rate constant without effect on inactivation from closed states or other rate transitions.

Mechanical and electrophysiological effects of sea anemone (Anemonia sulcata) toxins on rat innervated and denervated skeletal muscle.

1 Some effects of the sea-anemone toxin ATX-II on mammalian nerve-muscle preparations have been described. 2 When ATX-II (10(-8)-10(-6) M) was applied to rat hemidiaphragm preparations, both directly and indirectly generated twitch responses were potentiated and prolonged. At the same time the resting tension of the preparations increased. 3 The increase in resting tension caused by ATX-II in innervated muscles was not prevented by curarization, but was reversed by exposure to tetrodotoxin. The increase in denervated muscles was not completely reversed by tetrodotoxin. 4 At concentrations exceeding 1 x 10(-7) M, ATX-II caused a sodium-dependent depolarization of both normal and denervated muscles. The depolarization of the denervated muscles was only partially reversed by tetrodotoxin. 5 In the presence of ATX-II repetitive endplate potentials (e.p.ps) were evoked by single shocks to the motor nerves in many fibres, and in those in which a single e.p.p. was still observed, the quantum content (m) was increased. Miniature e.p.p. frequency was not increased by ATX-II, even when muscle fibres were depolarized by 30 mV. 6 The indirectly and directly elicited action potentials of normal and denervated muscle fibres were much prolonged by ATX-II. The action potentials remained sodium-dependent. The sodium-dependent tetrodotoxin-resistant action potential of the denervated muscle fibre was also prolonged by ATX-II. 7 It is concluded that ATX-II both activates, and delays inactivation of, sodium channels in mammalian skeletal muscle fibres, probably in interacting with the channel "gate'.