Peptide toxins that target vertebrate voltage-gated sodium channels underly the painful stings of harvester ants.

Harvester ants (genus Pogonomyrmex) are renowned for their stings which cause intense, long-lasting pain, and other neurotoxic symptoms in vertebrates. Here, we show that harvester ant venoms are relatively simple and composed largely of peptide toxins. One class of peptides is primarily responsible for the long-lasting local pain of envenomation via activation of peripheral sensory neurons. These hydrophobic, cysteine-free peptides potently modulate mammalian voltage-gated sodium (NaV) channels, reducing the voltage threshold for activation and inhibiting channel inactivation. These toxins appear to have evolved specifically to deter vertebrates.

Bioisosteric Modification of To042: Synthesis and Evaluation of Promising Use-Dependent Inhibitors of Voltage-Gated Sodium Channels.

Three analogues of To042, a tocainide-related lead compound recently reported for the treatment of myotonia, were synthesized and evaluated in vitro as skeletal muscle sodium channel blockers possibly endowed with enhanced use-dependent behavior. Patch-clamp experiments on hNav1.4 expressed in HEK293 cells showed that N-[(naphthalen-1-yl)methyl]-4-[(2,6-dimethyl)phenoxy]butan-2-amine, the aryloxyalkyl bioisostere of To042, exerted a higher use-dependent block than To042 thus being able to preferentially block the channels in over-excited membranes while preserving healthy tissue function. It also showed the lowest active transport across BBB according to the results of P-glycoprotein (P-gp) interacting activity evaluation and the highest cytoprotective effect on HeLa cells. Quantum mechanical calculations and dockings gave insights on the most probable conformation of the aryloxyalkyl bioisostere of To042 in solution and the target residues involved in the binding, respectively. Both approaches indicated the conformations that might be adopted in both the unbound and bound state of the ligand. Overall, N-[(naphthalen-1-yl)methyl]-4-[(2,6-dimethyl)phenoxy]butan-2-amine exhibits an interesting toxico-pharmacological profile and deserves further investigation.

Potency-Enhancing Mutations of Gating Modifier Toxins for the Voltage-Gated Sodium Channel NaV1.7 Can Be Predicted Using Accurate Free-Energy Calculations.

Gating modifier toxins (GMTs) isolated from venomous organisms such as Protoxin-II (ProTx-II) and Huwentoxin-IV (HwTx-IV) that inhibit the voltage-gated sodium channel NaV1.7 by binding to its voltage-sensing domain II (VSDII) have been extensively investigated as non-opioid analgesics. However, reliably predicting how a mutation to a GMT will affect its potency for NaV1.7 has been challenging. Here, we hypothesize that structure-based computational methods can be used to predict such changes. We employ free-energy perturbation (FEP), a physics-based simulation method for predicting the relative binding free energy (RBFE) between molecules, and the cryo electron microscopy (cryo-EM) structures of ProTx-II and HwTx-IV bound to VSDII of NaV1.7 to re-predict the relative potencies of forty-seven point mutants of these GMTs for NaV1.7. First, FEP predicted these relative potencies with an overall root mean square error (RMSE) of 1.0 ± 0.1 kcal/mol and an R2 value of 0.66, equivalent to experimental uncertainty and an improvement over the widely used molecular-mechanics/generalized born-surface area (MM-GB/SA) RBFE method that had an RMSE of 3.9 ± 0.8 kcal/mol. Second, inclusion of an explicit membrane model was needed for the GMTs to maintain stable binding poses during the FEP simulations. Third, MM-GB/SA and FEP were used to identify fifteen non-standard tryptophan mutants at ProTx-II[W24] predicted in silico to have a at least a 1 kcal/mol gain in potency. These predicted potency gains are likely due to the displacement of high-energy waters as identified by the WaterMap algorithm for calculating the positions and thermodynamic properties of water molecules in protein binding sites. Our results expand the domain of applicability of FEP and set the stage for its prospective use in biologics drug discovery programs involving GMTs and NaV1.7.

Flecainide-induced QRS complex widening correlates with negative inotropy.

BACKGROUND: The negative inotropic effect of Class IC antiarrhythmic drugs limits their use for acute cardioversion of atrial fibrillation (AF). OBJECTIVE: The purpose of this study was to examine, in an intact porcine model, the effects of pulmonary and intravenous (IV) administration of flecainide on left ventricular (LV) contractility and QRS complex width at doses that are effective in converting new-onset AF to sinus rhythm. METHODS: Flecainide (1.5 mg/kg bolus) was delivered by intratracheal administration and compared to 2.0 mg/kg 10-minute IV administration (European Society of Cardiology guideline) and to 0.5 and 1.0 mg/kg 2-minute IV doses in 40 closed-chest, anesthetized Yorkshire pigs. Catheters were fluoroscopically positioned in the LV to monitor QRS complex width and contractility and at the bifurcation of the main bronchi to deliver intratracheal flecainide. RESULTS: Peak flecainide plasma concentrations (Cmax) were similar, but the 30-minute area under the curve (AUC) of plasma levels was 1.4- to 2.8-fold greater for 2.0 mg/kg 10-minute IV infusion than for the lower, more rapidly delivered intratracheal and IV doses. AUC for LV contractility (ie, negative inotropic burden) was 2.2- to 3.6-fold greater for 2.0 mg/kg 10-minute IV dose than for the lower, more rapidly delivered doses. QRS complex widening by flecainide was highly correlated with the decrease in LV contractility (r2 = 0.890, P <.0001, for all IV doses; r2 = 0.812, P = .01, for intratracheal flecainide). CONCLUSION: QRS complex widening in response to flecainide is strongly correlated with decrease in LV contractility. Rapid pulmonary or IV flecainide delivery reduces the negative inotropic burden while quickly achieving Cmax levels associated with conversion of AF.

Fast and furious: flecainide toxicity presenting as monomorphic ventricular tachycardia.

A 63-year-old woman on flecainide, furosemide, and triamterene-hydrochlorothiazide presented with weakness and diarrhoea. She had profound hyponatraemia, hypokalaemia and a pre-renal acute kidney injury (AKI). Her ECG showed a regular wide complex tachycardia concerning for monomorphic ventricular tachycardia. She was haemodynamically stable and treated with aggressive electrolyte repletion and amiodarone. Flecainide toxicity can present as a variety of arrhythmias and early recognition is crucial. This case focuses on flecainide toxicity from multiple concomitant insults: diuretic use, diarrhoea, hypokalaemia, hyponatraemia and pre-renal AKI. We emphasise the importance of close outpatient monitoring of electrolytes in a patient on diuretics and flecainide to prevent life-threatening arrhythmias. We discourage use of multiple diuretics in patients taking flecainide.

Recombinant PaurTx-3, a spider toxin, inhibits sodium channels and decreases membrane excitability in DRG neurons.

Voltage-gated sodium channels are critical for the generation and propagation of action potentials. Gating modifier toxins from spider venom can modulate the gating mechanism of sodium channels and thus have potential as drug leads. Here, we established expression of the gating modifier toxin PaurTx-3, a sodium channel inhibitor found in the venom of the spider Phrixotrichus auratus. Whole-cell voltage-clamp recordings indicated that recombinant PaurTx-3 (rPaurTx-3) inhibited Nav1.4, Nav1.5, and Nav1.7 currents with IC50 values of 61 nM, 72 nM, and 25 nM, respectively. Furthermore, rPaurTx-3 irreversibly inhibited Nav1.7 currents, but had 60-70% recovery in Nav1.4 and Nav1.5 after washing with a bath solution. rPaurTx-3 also hyperpolarized the voltage-dependent steady-state inactivation curve and significantly slowed recovery from fast inactivation of Nav1.7. Current-clamp recordings showed that rPaurTx-3 suppressed small DRG neuron activity. The biological activity assay findings for rPaurTx-3 support its potent pharmacological effect in Nav1.7 and small DRG neurons.

GiTx1(ß/κ-theraphotoxin-Gi1a), a novel toxin from the venom of Brazilian tarantula Grammostola iheringi (Mygalomorphae, Theraphosidae): Isolation, structural assessments and activity on voltage-gated ion channels.

Spider venoms, despite their toxicity, represent rich sources of pharmacologically active compounds with biotechnological potential. However, in view of the large diversity of the spider species, the full potential of their venom molecules is still far from being known. In this work, we report the purification and structural and functional characterization of GiTx1 (ß/κ-TRTX-Gi1a), the first toxin purified from the venom of the Brazilian tarantula spider Grammostola iheringi. GiTx1 was purified by chromatography, completely sequenced through automated Edman degradation and tandem mass spectrometry and its structure was predicted by molecular modeling. GiTx1 has a MW of 3.585 Da, with the following amino acid sequence: SCQKWMWTCDQKRPCCEDMVCKLWCKIIK. Pharmacological activity of GiTx1 was characterized by electrophysiology using whole-cell patch clamp on dorsal root ganglia neurons (DRG) and two-electrode voltage-clamp on voltage-gated sodium and potassium channels subtypes expressed in Xenopus laevis oocytes. GiTx1, at 2 µM, caused a partial block of inward (∼40%) and outward (∼20%) currents in DRG cells, blocked rNav1.2, rNav1.4 and mNav1.6 and had a significant effect on VdNav, an arachnid sodium channel isoform. IC50 values of 156.39 ± 14.90 nM for Nav1.6 and 124.05 ± 12.99 nM for VdNav, were obtained. In addition, this toxin was active on rKv4.3 and hERG potassium channels, but not Shaker IR or rKv2.1 potassium channels. In summary, GiTx1 is a promiscuous toxin with multiple effects on different types of ion channels.

Pilsicainide Intoxication with Neuropsychiatric Symptoms Treated with Continuous Hemodiafiltration.

A 72-year-old lady with atrial fibrillation and chronic renal failure was hospitalized due to bradycardic shock with electrocardiographic QRS prolongation. She had experienced limb shaking two days before hospitalization, and additionally developed hallucinations one day before admission. Pilsicainide intoxication was diagnosed from a review of her medications and electrocardiographic findings. Consequently, continuous hemodiafiltration was performed resulting in a resolution of the hallucinations and the QRS prolongation. This is a rare case of psychiatric symptoms caused by pilsicainide intoxication. It is important to know the mode of excretion of a drug and to adjust its dose, so that such drug-related incidents can be avoided.

Discovery of DS-1971a, a Potent, Selective NaV1.7 Inhibitor.

A highly potent, selective NaV1.7 inhibitor, DS-1971a, has been discovered. Exploration of the left-hand phenyl ring of sulfonamide derivatives (I and II) led to the discovery of novel series of cycloalkane derivatives with high NaV1.7 inhibitory potency in vitro. As the right-hand heteroaromatic ring affected the mechanism-based inhibition liability of CYP3A4, replacement of this moiety resulted in the generation of 4-pyrimidyl derivatives. Additionally, GSH adducts formation, which can cause idiosyncratic drug toxicity, was successfully avoided by this modification. An additional optimization led to the discovery of DS-1971a. In preclinical studies, DS-1971a demonstrated highly potent selective in vitro profile with robust efficacy in vivo. DS-1971a exhibited a favorable toxicological profile, which enabled multiple-dose studies of up to 600 mg bid or 400 mg tid (1200 mg/day) administered for 14 days to healthy human males. DS-1971a is expected to exert potent efficacy in patients with peripheral neuropathic pain, with a favorable safety profile.

Digitalis Promotes Ventricular Arrhythmias in Flecainide- and Ranolazine-Pretreated Hearts.

A post hoc analysis of the PALLAS trial suggested life-threatening interactions of digitalis and dronedarone. Thus, there is concern about an interplay between digitalis and other drugs that influence cardiac electrophysiology. We therefore investigated the interaction between digitalis and flecainide or ranolazine. Twenty-five rabbit hearts were Langendorff-perfused and treated with flecainide (2 µM, 12 hearts) or ranolazine (10 µM, 13 hearts). Infusion of flecainide prolonged mean action potential duration [APD90, from 153 ms (interquartile range (IQR): 29.7 ms) to 159 ms (IQR: 24.9 ms, p = 0.04)] and effective refractory period [ERP, 170 ms (IQR: 40 ms) vs. 200 ms (IQR: 32.5 ms, p < 0.01)]. Administration of ranolazine prolonged APD90 [144 ms (IQR: 34.3 ms)) vs. 157 ms (IQR: 31.2 ms, p < 0.01)] and ERP [180 ms (IQR: 40 ms) vs. 200 ms (IQR: 30 ms, p < 0.01)]. Additional infusion of the digitalis glycoside ouabain (0.2 µM) abbreviated APD90 and ERP in both groups (flecainide: APD90: to 128 ms (IQR: 19 ms), ERP: to 170 ms (IQR: 20 ms), p < 0.01 each; ranolazine: APD90: to 141 ms (IQR: 40 ms), ERP: to 160 ms (IQR: 30 ms), p < 0.01 each). Ventricular vulnerability was assessed by a pacing protocol employing premature extra stimuli and burst stimulation. No proarrhythmic effect was observed with flecainide (1 vs. 3 episodes at baseline) or ranolazine (3 vs. 11 episodes at baseline). However, further infusion of ouabain had a proarrhythmic effect for both drugs (flecainide: 15 episodes, p = 0.04; ranolazine: 21 episodes, p = 0.09). Concomitant treatment of the sodium channel blockers flecainide or ranolazine with digitalis seems to be proarrhythmic. Abbreviation of repolarization and refractoriness that can facilitate re-entry was found as underlying mechanism.

Effects of Lidocaine and Articaine on Neuronal Survival and Recovery.

The local anesthetics lidocaine and articaine are among the most widely used drugs in the dentist's arsenal, relieving pain by blocking voltage-dependent Na+ channels and thus preventing transmission of the pain signal. Given reports of infrequent but prolonged paresthesias with 4% articaine, we compared its neurotoxicity and functional impairment by screening cultured neural SH-SY5Y cells with formulations used in patients (2% lidocaine + 1:100,000 epinephrine or 4% articaine + 1:100,000 epinephrine) and with pure formulations of the drugs. Voltage-dependent sodium channels Na(v)1.2 and Na(v)1.7 were expressed in SH-SY5Y cells. To test the effects on viability, cells were exposed to drugs for 5 minutes, and after washing, cells were treated with the ratiometric Live/Dead assay. Articaine had no effect on the survival of SH-SY5Y cells, while lidocaine produced a significant reduction only when used as pure powder. To determine reversibility of blockage, wells were exposed to drugs for 5 minutes and returned for medium for 30 minutes, and the calcium elevation induced by depolarizing cells with a high-potassium solution was measured using the calcium indicator Fura-2. High potassium raised calcium in control SH-SY5Y cells and those treated with articaine, but lidocaine treatment significantly reduced the response. In conclusion, articaine does not damage neural cells more than lidocaine in this in vitro model. While this does not question the safety of lidocaine used clinically, it does suggest that articaine is no more neurotoxic, at least in the in vitro setting.

Arizona bark scorpion venom resistance in the pallid bat, Antrozous pallidus.

The pallid bat (Antrozous pallidus), a gleaning bat found in the western United States and Mexico, hunts a wide variety of ground-dwelling prey, including scorpions. Anecdotal evidence suggests that the pallid bat is resistant to scorpion venom, but no systematic study has been performed. Here we show with behavioral measures and direct injection of venom that the pallid bat is resistant to venom of the Arizona bark scorpion, Centruroides sculpturatus. Our results show that the pallid bat is stung multiple times during a hunt without any noticeable effect on behavior. In addition, direct injection of venom at mouse LD50 concentrations (1.5 mg/kg) has no effect on bat behavior. At the highest concentration tested (10 mg/kg), three out of four bats showed no effects. One of the four bats showed a transient effect suggesting that additional studies are required to identify potential regional variation in venom tolerance. Scorpion venom is a cocktail of toxins, some of which activate voltage-gated sodium ion channels, causing intense pain. Dorsal root ganglia (DRG) contain nociceptive neurons and are principal targets of scorpion venom toxins. To understand if mutations in specific ion channels contribute to venom resistance, a pallid bat DRG transcriptome was generated. As sodium channels are a major target of scorpion venom, we identified amino acid substitutions present in the pallid bat that may lead to venom resistance. Some of these substitutions are similar to corresponding amino acids in sodium channel isoforms responsible for reduced venom binding activity. The substitution found previously in the grasshopper mouse providing venom resistance to the bark scorpion is not present in the pallid bat, indicating a potentially novel mechanism for venom resistance in the bat that remains to be identified. Taken together, these results indicate that the pallid bat is resistant to venom of the bark scorpion and altered sodium ion channel function may partly underlie such resistance.

Modulation of the heart's electrical properties by the anticonvulsant drug retigabine.

Retigabine, currently used as antiepileptic drug, has a wide range of potential medical uses. Administration of the drug in patients can lead to QT interval prolongation in the electrocardiogram and to cardiac arrhythmias in rare cases. This suggests that the drug may perturb the electrical properties of the heart, and the underlying mechanisms were investigated here. Effects of retigabine on currents through human cardiac ion channels, heterologously expressed in tsA-201 cells, were studied in whole-cell patch-clamp experiments. In addition, the drug's impact on the cardiac action potential was tested. This was done using ventricular cardiomyocytes isolated from Langendorff-perfused guinea pig hearts and cardiomyocytes derived from human induced pluripotent stem cells. Further, to unravel potential indirect effects of retigabine on the heart which might involve the autonomic nervous system, membrane potential and noradrenaline release from sympathetic ganglionic neurons were measured in the absence and presence of the drug. Retigabine significantly inhibited currents through hKv11.1 potassium, hNav1.5 sodium, as well as hCav1.2 calcium channels, but only in supra-therapeutic concentrations. In a similar concentration range, the drug shortened the action potential in both guinea pig and human cardiomyocytes. Therapeutic concentrations of retigabine, on the other hand, were sufficient to inhibit the activity of sympathetic ganglionic neurons. We conclude that retigabine- induced QT interval prolongation, and the reported cases of cardiac arrhythmias after application of the drug in a typical daily dose range, cannot be explained by a direct modulatory effect on cardiac ion channels. They are rather mediated by indirect actions at the level of the autonomic nervous system.

Voltage gated ion channels blockade is the underlying mechanism of BIMU8 induced cardiotoxicity.

BIMU8 is a 5-HT4a receptor agonist and used as an experimental drug to counteract opioid induced respiratory depression. In preliminary experiments serious disturbances in ECG were observed in anesthetized rabbits which prompted us to explore the underlying cause of BIMU8 induced abnormal changes in ECG recordings. Electrophysiological experiments were performed on HEK-293 cells expressing hERG, CaV1.2 and NaV1.5 ion channels. In whole-cell recordings BIMU8 effectively blocked these three channels, with IC50 values of 0.06±0.05, 1.46±0.26 and 4.66±0.58µM for hERG, NaV1.5 and CaV1.2, respectively. Additionally it also produced a hyperpolarizing shift of 3.27mV in half maximal activation and 12.87mV in fast inactivation of NaV1.5 channel. These experimental findings indicate that BIMU8 is a potent blocker of hERG, NaV1.5 and CaV1.2 cardiac ion channels thus revealing its proarrhythmic potential.

Dexmedetomidine Protects PC12 Cells from Lidocaine-Induced Cytotoxicity Through Downregulation of COL3A1 Mediated by miR-let-7b.

Safety concerns of some local anesthetics, such as lidocaine, have been raised in recent years due to potential neurological impairment. Dexmedetomidine may protect humans from neurotoxicity, and miR-let-7b is activated by nerve injury; however, the roles of miR-let-7b and its target gene in lidocaine-induced cytotoxicity are not well known. Through bioinformatics and a luciferase reporter assay, COL3A1 was suggested as a direct target gene of miR-let-7b. Here, we confirmed by measuring mRNA and protein levels that miR-let-7b was downregulated and COL3A1 was upregulated in lidocaine-treated cells, an observation that was reversed by dexmedetomidine. Similar to miR-let-7b mimics or knockdown of COL3A1, dexmedetomidine treatment reduced the expression of COL3A1, suppressed cell apoptosis and cell migration/invasion ability, and induced cell cycle progression and cell proliferation in PC12 cells, effects that were reversed by the miR-let-7b inhibitor. Meanwhile, proteins involved in cell apoptosis, such as Bcl2 and caspase 3, were impacted as well. Taken together, dexmedetomidine may protect PC12 cells from lidocaine-induced cytotoxicity through miR-let-7b and COL3A1, while also increasing Bcl2 and inhibiting caspase 3. Therefore, miR-let-7b and COL3A1 might play critical roles in neuronal injury, and they are potential therapeutic targets.

Cardiotoxic Antiemetics Metoclopramide and Domperidone Block Cardiac Voltage-Gated Na+ Channels.

BACKGROUND: Metoclopramide and domperidone are prokinetic and antiemetic substances often used in clinical practice. Although domperidone has a more favorable side effect profile and is considered the first-line agent, severe cardiac side effects were reported during the administration of both substances. Cardiac Na channels are common targets of therapeutics inducing cardiotoxicity. Therefore, the aim of this study was to investigate whether the differential cardiotoxicities of metoclopramide and domperidone correlate with the block of Na channels. METHODS: Effects of metoclopramide and domperidone on the human α-subunit Nav1.5 expressed in human embryonic kidney 293 cells and on Na currents in neonatal rat cardiomyocytes were investigated by means of whole-cell patch clamp recordings. RESULTS: Tonic block of resting Nav1.5 channels was more potent for domperidone (IC50 85 ± 25 µM; 95% confidence interval [CI], 36-134) compared with metoclopramide (IC50 458 ± 28 µM; 95% CI, 403-513). Both agents induced use-dependent block at 10 and 1 Hz, stabilized fast and slow inactivation, and delayed recovery from inactivation. However, metoclopramide induced considerably smaller effects compared with domperidone. Na currents in rat cardiomyocytes displayed tonic and use-dependent block by both substances, and in this system, domperidone (IC50 312 ± 15 µM; 95% CI, 22-602) and metoclopramide (IC50 250 ± 30 µM; 95% CI, 191-309) induced a similar degree of tonic block. CONCLUSIONS: Our data demonstrate that the clinically relevant cardiotoxicity of domperidone and metoclopramide corresponds to a rather potent and local anesthetic-like inhibition of cardiac Na channels including Nav1.5. These data suggest that Nav1.5 might be a hitherto unrecognized molecular mechanism of some cardiovascular side effects, for example, malignant arrhythmias of prokinetic and antiemetic agents.

Proarrhythmic mechanisms of the common anti-diarrheal medication loperamide: revelations from the opioid abuse epidemic.

Loperamide is a µ-opioid receptor agonist commonly used to treat diarrhea and often available as an over-the-counter medication. Recently, numerous reports of QRS widening accompanied by dramatic QT interval prolongation, torsades de pointe arrhythmia, and death have been reported in opioid abusers consuming large amounts of the drug to produce euphoria or prevent opiate withdrawal. The present study was undertaken to determine the mechanisms of this cardiotoxicity. Using whole-cell patch clamp electrophysiology, we tested loperamide on the cloned human cardiac sodium channel (Nav1.5) and the two main repolarizing cardiac K(+) channels cloned from the human heart: KvLQT1/minK and the human ether-a-go-go-related gene (hERG) channel. Loperamide inhibited Nav1.5 with IC50 values of 297 and 239 nM at holding potentials of -90 and -70 mV, respectively. Loperamide was weakly active on KvLQT1/minK producing 17 and 65 % inhibition at concentrations of 1 and 10 µM, respectively. Conversely, loperamide was found to be a very high affinity inhibitor of the hERG channel with an IC50 value of 89 nM at room temperature and 33 nM when measured at physiological temperature. The QRS and QT interval prolongation and the attending arrhythmias, produced by loperamide, derive from high affinity inhibition of Nav1.5 and especially hERG. Since the drug has been widely available and safely used as directed for many years, we believe that the potent inhibition loperamide possesses for cardiac ion channels has only been uncovered because of the excessive misuse of the drug as a consequence of the recent opioid abuse epidemic.

The tarantula toxin jingzhaotoxin-XI (κ-theraphotoxin-Cj1a) regulates the activation and inactivation of the voltage-gated sodium channel Nav1.5.

Specific peptide toxins interact with voltage-gated sodium channels by regulating the activation or inactivation of targeted channels. However, few toxins possessing dual effects have been identified. In the present study, we showed that jingzhaotoxin-XI/κ-theraphotoxin-Cj1a (JZTX-XI), a 34-residue peptide from the venom of the Chinese spider Chilobrachys jingzhao, inhibits the sodium conductance (IC50 = 124 ± 26 nM) and slows the fast inactivation (EC50 = 1.18 ± 0.2 µM) of Nav1.5 expressed in Chinese hamster ovary (CHO-K1) cells. JZTX-XI significantly shifted the activation to more depolarized voltages and decreased the deactivation of Nav1.5 currents upon extreme depolarization, but only slightly affected voltage-dependence of steady-state inactivation. In addition, JZTX-XI caused an approximately five-fold decrease in the rate of recovery from inactivation and an approximately 1.9-fold reduction in the closed-state inactivation rate. Our data suggest that JZTX-XI integrates the functions of site 3 toxins (α-scorpion toxins) with site 4 toxins (ß-scorpion and spider toxins) by targeting multiple sites on Nav1.5. The unique properties displayed by JZTX-XI in its inhibitory activity on Nav1.5 suggest that its mechanism of action is distinct from those of site 3 and site 4 toxins, making JZTX-XI a useful probe for investigating the gating mechanism of Nav1.5 and toxin-channel interactions.

Methemoglobinemia associated with metaflumizone poisoning.

CONTEXT: Metaflumizone is a voltage-dependent sodium channel blocker insecticide, which is chemically similar to indoxacarb. Although indoxacarb poisoning is known as a cause of methemoglobinemia, the effect of metaflumizone poisoning in humans is still unknown. CASE DETAILS: A 57-year-old man presented with a decreased mentality following ingestion of 100 ml of metaflumizone, 150 ml of glyphosate and alcohol. Although initial methemoglobin (MetHb) level was slightly higher than the normal limit, it gradually rose to reach a maximum level of 27.8%, on the 19 h after ingestion. After hemodialysis, MetHb level was reduced to 15.8%, which decreased further to the level of 6%, following methylene blue administration. DISCUSSION: Metaflumizone shares a similar chemical structure to indoxacarb, which is known to be a cause of methemoglobinemia. Physicians should be alert for the development of methemoglobinemia in symptomatic patients when facing potential pesticide poisoning such as metaflumizone poisoning.

A disulfide tether stabilizes the block of sodium channels by the conotoxin µO§-GVIIJ.

A cone snail venom peptide, µO§-conotoxin GVIIJ from Conus geographus, has a unique posttranslational modification, S-cysteinylated cysteine, which makes possible formation of a covalent tether of peptide to its target Na channels at a distinct ligand-binding site. µO§-conotoxin GVIIJ is a 35-aa peptide, with 7 cysteine residues; six of the cysteines form 3 disulfide cross-links, and one (Cys24) is S-cysteinylated. Due to limited availability of native GVIIJ, we primarily used a synthetic analog whose Cys24 was S-glutathionylated (abbreviated GVIIJSSG). The peptide-channel complex is stabilized by a disulfide tether between Cys24 of the peptide and Cys910 of rat (r) NaV1.2. A mutant channel of rNaV1.2 lacking a cysteine near the pore loop of domain II (C910L), was >10(3)-fold less sensitive to GVIIJSSG than was wild-type rNaV1.2. In contrast, although rNaV1.5 was >10(4)-fold less sensitive to GVIIJSSG than NaV1.2, an rNaV1.5 mutant with a cysteine in the homologous location, rNaV1.5[L869C], was >10(3)-fold more sensitive than wild-type rNaV1.5. The susceptibility of rNaV1.2 to GVIIJSSG was significantly altered by treating the channels with thiol-oxidizing or disulfide-reducing agents. Furthermore, coexpression of rNaVß2 or rNaVß4, but not that of rNaVß1 or rNaVß3, protected rNaV1.1 to -1.7 (excluding NaV1.5) against block by GVIIJSSG. Thus, GVIIJ-related peptides may serve as probes for both the redox state of extracellular cysteines and for assessing which NaVß- and NaVα-subunits are present in native neurons.