Evaluation of neuroprotective effects of insulin on immuno-inflammatory and systemic disorders induced by kaliotoxin, a Kv1.3 channel blocker.

OBJECTIVE: Kaliotoxin2 (KTX2) is a highly selective blocker of voltage-dependent potassium channels Kv1.3 containing 37 amino acid residues. It is purified from Androctonus australis scorpion venom. The binding of KTX2 to its targets is able to alter the neuronal excitability leading to neurological disorders, accompanied by an inflammatory response. In brain, activation of insulin receptor signaling pathway by insulin induces current suppression and concomitant tyrosine phosphorylation of Kv1.3 channel. The aim of this study is to evaluate the effect of insulin injected by i.c.v. route on the neuro-pathophysiological and systemic disorders induced by KTX2. MATERIALS AND METHODS: Tissue damage, inflammatory response and oxidative stress biomarkers were assessed in NMRI mice at 24 h after co-injection of KTX2 and insulin by intracerebroventricular route. RESULTS: Obtained results revealed that the central administration of insulin prevents cerebral cortex injury, brain edema and blood-brain barrier alteration induced by KTX2, these are accompanied by significant decrease of systemic disorders including serum cytokines, inflammatory and oxidative stress markers and tissue damage. CONCLUSION: These results indicate that insulin is able to reduce neuro-immunological effects and systemic disorders induced by KTX2. The neuroprotective effect of insulin may be due to its crucial role in the regulation of inflammation response and its properties to modulate the activity of Kv1.3 channels in brain.

K(+) channel blocker-induced neuroinflammatory response and neurological disorders: immunomodulatory effects of astaxanthin.

OBJECTIVE: Channelopathies due to the brain ion channel dysfunction is considered to be an important mechanism involved in various neurodegenerative diseases. In this study, we evaluated the ability of kaliotoxin (KTX) as K(+) channel blocker to induce neuro-inflammatory response and neurodegenerative alteration. We also investigate the effects of astaxanthin (ATX) against KTX disorders. MATERIAL AND TREATMENT: NMRI mice were injected with KTX (1 pg/kg, by i.c.v route) with or without pretreatment using ATX (80 mg/kg, o.p route). RESULTS: Results showed that KTX was detected in cerebral cortex area due to its binding to the specific receptors (immunofluorescence analysis). It induced an activation of inflammatory cascade characterized by an increase of IL-6, TNFα, NO, MDA levels and NF-κB expression associated to a decrease of GSH level. The neuroinflammatory response is accompanied with cerebral alterations and blood-brain barrier (BBB) disruption. The use of ATX prior to the KTX exerts a preventive effect not only on the neuroinflammation but also on altered tissues and the BBB disruption. CONCLUSIONS: Kaliotoxin is able to induce neurological disorders by blocking the K(+) ion channel, and ATX suppresses this alterations with down regulation of IL-6, TNF-α and NF-κB expression in the brain.

Neuro-Modulation of Immuno-Endocrine Response Induced by Kaliotoxin of Androctonus Scorpion Venom.

Kaliotoxin (KTX), a specific blocker of potassium channels, exerts various toxic effects due to its action on the central nervous system. Its use in experimental model could help the understanding of the cellular and molecular mechanisms involved in the neuropathological processes related to potassium channel dysfunctions. In this study, the ability of KTX to stimulate neuro-immuno-endocrine axis was investigated. As results, the intracerebroventricular injection of KTX leads to severe structural-functional alterations of both hypothalamus and thyroid. These alterations were characterized by a massive release of hormones' markers of thyroid function associated with damaged tissue which was infiltrated by inflammatory cell and an imbalanced redox status. Taken together, these data highlight that KTX is able to modulate the neuro-endocrine response after binding to its targets leading to the hypothalamus and the thyroid stimulation, probably by inflammatory response activation and the installation of oxidative stress in these organs.

Kv4 channels underlie A-currents with highly variable inactivation time courses but homogeneous other gating properties in the nucleus tractus solitarii.

In the nucleus of the tractus solitarii (NTS), a large proportion of neurones express transient A-type potassium currents (I KA) having deep influence on the fidelity of the synaptic transmission of the visceral primary afferent inputs to second-order neurones. Up to now, the strong impact of I KA within the NTS was considered to result exclusively from its variation in amplitude, and its molecular correlate(s) remained unknown. In order to identify which Kv channels underlie I KA in NTS neurones, the gating properties and the pharmacology of this current were determined using whole cell patch clamp recordings in slices. Complementary information was brought by immunohistochemistry. Strikingly, two neurone subpopulations characterized by fast or slow inactivation time courses (respectively about 50 and 200 ms) were discriminated. Both characteristics matched those of the Kv4 channel subfamily. The other gating properties, also matching the Kv4 channel ones, were homogeneous through the NTS. The activation and inactivation occurred at membrane potentials around the threshold for generating action potentials, and the time course of recovery from inactivation was rapid. Pharmacologically, I KA in NTS neurones was found to be resistant to tetraethylammonium (TEA), sea anemone toxin blood-depressing substance (BDS) and dendrotoxin (DTX), whereas Androctonus mauretanicus mauretanicus toxin 3 (AmmTX3), a scorpion toxin of the α-KTX 15 family that has been shown to block all the members of the Kv4 family, inhibited 80 % of I KA irrespectively of its inactivation time course. Finally, immunohistochemistry data suggested that, among the Kv4 channel subfamily, Kv4.3 is the prevalent subunit expressed in the NTS.

Shal-type (Kv4.x) potassium channel pore blockers from scorpion venoms.

Voltage-gated potassium channels (Kv4.1, Kv4.2 and Kv4.3) encoded by the members of the KCND/Kv4 (Shal) channel family mediate the native, fast inactivating (A-type) K(+) current (IA) described both in heart and neurons. This IA current is specifically blocked by short scorpion toxins that belong to the α-KTx15 subfamily and which act as pore blockers, a different mode of action by comparison to spider toxins known as gating modifiers. This review summarizes our present chemical and pharmacological knowledge on the α-KTx15 toxins.

Deconstructing voltage sensor function and pharmacology in sodium channels.

Voltage-activated sodium (Na(v)) channels are crucial for the generation and propagation of nerve impulses, and as such are widely targeted by toxins and drugs. The four voltage sensors in Na(v) channels have distinct amino acid sequences, raising fundamental questions about their relative contributions to the function and pharmacology of the channel. Here we use four-fold symmetric voltage-activated potassium (K(v)) channels as reporters to examine the contributions of individual S3b-S4 paddle motifs within Na(v) channel voltage sensors to the kinetics of voltage sensor activation and to forming toxin receptors. Our results uncover binding sites for toxins from tarantula and scorpion venom on each of the four paddle motifs in Na(v) channels, and reveal how paddle-specific interactions can be used to reshape Na(v) channel activity. One paddle motif is unique in that it slows voltage sensor activation, and toxins selectively targeting this motif impede Na(v) channel inactivation. This reporter approach and the principles that emerge will be useful in developing new drugs for treating pain and Na(v) channelopathies.

Ca²âº/cAMP-sensitive covariation of I(A) and I(H) voltage dependences tunes rebound firing in dopaminergic neurons.

The level of expression of ion channels has been demonstrated to vary over a threefold to fourfold range from neuron to neuron, although the expression of distinct channels may be strongly correlated in the same neurons. We demonstrate that variability and covariation also apply to the biophysical properties of ion channels. We show that, in rat substantia nigra pars compacta dopaminergic neurons, the voltage dependences of the A-type (I(A)) and H-type (I(H)) currents exhibit a high degree of cell-to-cell variability, although they are strongly correlated in these cells. Our data also demonstrate that this cell-to-cell covariability of voltage dependences is sensitive to cytosolic cAMP and calcium levels. Finally, using dynamic clamp, we demonstrate that covarying I(A) and I(H) voltage dependences increases the dynamic range of rebound firing while covarying their amplitudes has a homeostatic effect on rebound firing. We propose that the covariation of voltage dependences of ion channels represents a flexible and energy-efficient way of tuning firing in neurons.

Dipeptidyl-peptidase-like-proteins confer high sensitivity to the scorpion toxin AmmTX3 to Kv4-mediated A-type K+ channels.

K+ channels containing Kv4.2 and Kv4.3 pore-forming subunits mediate most of the subthreshold-operating somatodendritic A-type K+ current in CNS neurons. These channels are believed to be important in regulating the frequency of repetitive firing, the backpropagation of action potential into dendrites, and dendritic integration and plasticity. Moreover, they have been implicated in several diseases from pain to epilepsy and autism spectrum disorders. The lack of toxins that specifically and efficiently block these channels has hampered studies aimed at confirming their functional role and their involvement in disease. AmmTX3 and other related members of the α-KTX15 family of scorpion toxins have been shown to block the A-type K+ current in cultured neurons, but their specificity has been questioned because the toxins do not efficiently block the currents mediated by Kv4.2 or Kv4.3 subunits expressed in heterologous cells. Here we show that the high-affinity blockade of Kv4.2 and Kv4.3 channels by AmmTX3 depends on the presence of the auxiliary subunits DPP6 and DPP10. These proteins are thought to be components of the Kv4 channel complex in neurons and to be important for channel expression in dendrites. These studies validate the use of AmmTX3 as a blocker of the Kv4-mediated A-type K+ current in neurons.

Structural insights into antibody sequestering and neutralizing of Na+ channel α-type modulator from old world scorpion venom.

The Old World scorpion Androctonus australis hector (Aah) produces one of the most lethal venoms for humans. Peptidic α-toxins AahI to AahIV are responsible for its potency, with AahII accounting for half of it. All four toxins are high affinity blockers of the fast inactivation phase of mammalian voltage-activated Na(+) channels. However, the high antigenic polymorphism of α-toxins prevents production of a polyvalent neutralizing antiserum, whereas the determinants dictating their trapping by neutralizing antibodies remain elusive. From an anti-AahII mAb, we generated an antigen binding fragment (Fab) with high affinity and selectivity for AahII and solved a 2.3 Å-resolution crystal structure of the complex. Sequestering of the C-terminal region of the bound toxin within a groove formed by the Fab combining loops is associated with a toxin orientation and main and side chain conformations that dictate the AahII antigenic specificity and efficient neutralization. From an anti-AahI mAb, we also preformed and crystallized a high affinity AahI-Fab complex. The 1.6 Å-resolution structure solved revealed a Fab molecule devoid of a bound AahI and with combining loops involved in packing interactions, denoting expulsion of the bound antigen upon crystal formation. Comparative analysis of the groove-like combining site of the toxin-bound anti-AahII Fab and planar combining surface of the unbound anti-AahI Fab along with complementary data from a flexible docking approach suggests occurrence of distinctive trapping orientations for the two toxins relative to their respective Fab. This study provides complementary templates for designing new molecules aimed at capturing Aah α-toxins and suitable for immunotherapy.

Diabody mixture providing full protection against experimental scorpion envenoming with crude Androctonus australis venom.

Androctonus australis is primarily involved in envenomations in North Africa, notably in Tunisia and Algeria, and constitutes a significant public health problem in this region. The toxicity of the venom is mainly due to various neurotoxins that belong to two distinct structural and immunological groups, group I (the AahI and AahIII toxins) and group II (AahII). Here, we report the use of a diabody mixture in which the molar ratio matches the characteristics of toxins and polymorphism of the venom. The mixture consists of the Db9C2 diabody (anti-group I) and the Db4C1op diabody (anti-AahII), the latter being modified to facilitate in vitro production and purification. The effectiveness of the antivenom was tested in vivo under conditions simulating scorpion envenomation. The intraperitoneal injection of 30 µg of the diabody mixture protected almost all the mice exposed to 3 LD(50) s.c. of venom. We also show that the presence of both diabodies is necessary for the animals to survive. Our results are the first demonstration of the strong protective power of small quantities of antivenom used in the context of severe envenomation with crude venom.

Neuropathophysiological effect and immuno-inflammatory response induced by kaliotoxin of androctonus scorpion venom.

OBJECTIVE: Kaliotoxin (KTX) is a neurotoxin purified from Androctonus scorpion venom. Purification and pharmacological and immunological characterization of this neurotoxin has been extensively studied, but its biological effects have not. The ability of KTX to induce neuropathophysiological and immuno-inflammatory effects was investigated. METHODS: NMRI mice were injected with a sublethal dose of KTX (20 ng/20 g of body weight) or saline solution via the intra-cerebro-ventricular route. Tissue damage and immunological biomarkers such as eosinophil peroxidase (EPO), myeloperoxidase (MPO), and nitric oxide (NO) were analyzed in serum, brain, lung, and heart tissue. Protein levels, LDH, and CPK activities were also determined in serum 24 h after injection. RESULTS: In this study, KTX injection induced severe alterations in the cerebral cortex, myocardium, and pulmonary parenchyma. Tissue damage was correlated with seric increase in creatine kinase and lactate dehydrogenase activities. KTX also induced an immuno-inflammatory response distinguished by cell infiltration characterized by a significant increase in EPO and MPO activities in the brain, heart, and lungs. This infiltration was also associated with an increase in albumin, α-, ß-, and γ-globulin fractions, and NO release. CONCLUSION: KTX binding to its targets in CNS (Kv1.1 and Kv1.3 channels) may induce severe modifications in the structure and function of various organs associated with the activation of immuno-inflammatory reactions.

Immunomodulation of the inflammatory response induced by Androctonus australis hector neurotoxins: biomarker interactions.

OBJECTIVE: Androctonus australis hector (Aah) is the most dangerous scorpion in the Maghreb countries. Its venom contains three major neurotoxins (Aah I, Aah II and Aah III), which are responsible for almost all the lethal effects caused in mammals. These toxins act on the voltage-gated sodium channels of excitable cells. The targets and the lethal effects of these toxins have been extensively studied. However, their effects on the induced immune response after envenoming have not deeply elicited. We therefore investigated the effects induced by Aah venom and its toxic components, mainly its main toxin Aah II, on the activation of the inflammatory process. METHODS: Wistar rats were injected by intraperitoneal route with a sublethal dose of Aah venom, FTox-G50, the purified Aah II toxin or with 400 µl of sterile physiological saline solution. Immunological biomarkers such as MPO, NO and ICAM-1 were analyzed in serum in lung tissue. Cytokine levels were also determined in serum at 3, 6 and 24 h after envenoming. RESULTS: We report in this study that intraperitoneal injection of the venom or its toxins (the whole toxic fraction or Aah II toxin) caused an inflammatory reaction involving increased neutrophil release into blood and neutrophil accumulation in lung tissue. This cell infiltration was associated with the release of NO, histamine, cytokines (IL-1, IL-6, IL-12, IL-4 and IL-5) and ICAM. CONCLUSION: Aah II binding to its targets, in this case Na⁺ channels, may induce a cascade of events such as inflammatory mediator release and neutrophil migration that could contribute to the exacerbation of the systemic inflammatory response and the development of lung injury following scorpion envenoming.

Toxin-induced conformational changes in a potassium channel revealed by solid-state NMR.

The active site of potassium (K+) channels catalyses the transport of K+ ions across the plasma membrane--similar to the catalytic function of the active site of an enzyme--and is inhibited by toxins from scorpion venom. On the basis of the conserved structures of K+ pore regions and scorpion toxins, detailed structures for the K+ channel-scorpion toxin binding interface have been proposed. In these models and in previous solution-state nuclear magnetic resonance (NMR) studies using detergent-solubilized membrane proteins, scorpion toxins were docked to the extracellular entrance of the K+ channel pore assuming rigid, preformed binding sites. Using high-resolution solid-state NMR spectroscopy, here we show that high-affinity binding of the scorpion toxin kaliotoxin to a chimaeric K+ channel (KcsA-Kv1.3) is associated with significant structural rearrangements in both molecules. Our approach involves a combined analysis of chemical shifts and proton-proton distances and demonstrates that solid-state NMR is a sensitive method for analysing the structure of a membrane protein-inhibitor complex. We propose that structural flexibility of the K+ channel and the toxin represents an important determinant for the high specificity of toxin-K+ channel interactions.

MeuTXKbeta1, a scorpion venom-derived two-domain potassium channel toxin-like peptide with cytolytic activity.

Recent studies have demonstrated that scorpion venom contains unique two-domain peptides with the peculiarity of possessing different functions, i.e. neurotoxic and cytolytic activities. Here we report systematic characterization of a new two-domain peptide (named MeuTXKbeta1) belonging to the TsTXKbeta molecular subfamily from the scorpion Mesobuthus eupeus by molecular cloning, biochemical purification, recombinant expression, functional assays, CD and NMR studies. Its full-length bioactive form as well as 1-21 and 22-72 fragments (named N(1-21) and C(22-72), respectively) was produced in Escherichia coli by an on-column refolding approach. Recombinant peptide (rMeuTXKbeta1) exhibited a low affinity for K(+) channels and cytolytic effects against bacteria and several eukaryotic cells. N(1-21) was found to preserve anti-Plasmodium activity in contrast to haemolytic activity, whereas C(22-72) retains these two activities. Circular dichroism analysis demonstrates that rMeuTXKbeta1 presents a typical scorpion toxin scaffold in water and its alpha-helical content largely increases in a membrane-mimicking environment, consistent with the NMR structure of N(1-21) and an ab initio structure model of MeuTXKbeta1 predicted using I-TASSER algorithm. Our structural and functional data clearly indicate an evolutionary link between TsTXKbeta-related peptides and antiparasitic scorpines which both comprise the betaSPN (beta-KTxs and scorpines) family.

Chitosan nanoparticles as a delivery platform for neurotoxin II from Androctonus australis hector scorpion venom: Assessment of toxicity and immunogenicity.

In recent years, biodegradable polymers based nanoparticles received high interest for the development of vaccine delivery vehicles. In this study, chitosan nanoparticles encapsulating Aah II toxin (AahII-CNPs) isolated from Androctonus australis hector venom, were investigated as vaccine delivery system. Particles obtained by ionotropic gelation were characterized for their size, surface charge, morphology and toxin release profile from Aah II-CNPs. Toxin-nanoparticles interactions were assessed by Fourier Transform Infrared Spectrometry and X-Ray Diffraction. An immunization protocol was designed in mice to investigate anti-toxin immunity and the protective status induced by different Aah II immune formulations. Unloaded chitosan nanoparticles presenting a spherical shape and smooth surface, were characterized by a size of 185 nm, a dispersion index (PDI) of 0.257 and a zeta potential of +34.6 mV. Aah II toxin was successfully entrapped into chitosan nanoparticles as revealed by FTIR and XRD data. Entrapment efficiency (EE) and Loading capacity (LC) were respectively of 96.66 and 33.5%. Aah II-CNPs had a diameter of 208 nm, a PDI of 0.23 and a zeta potential of +30 mV. Encapsulation of Aah II reduced its toxicity and protected mice until 10 LD50. Mice were immunized via a dual prime-boost scheme. Nanoentrapped Aah II immunogen elicited systemic innate and humoral immune responses as well as local spleen parenchyma hyperplasic alterations. Aah II-CNPs immunized mice withstood high lethal doses of native Aah II, one-month post-boost inoculation. This study provided encouraging and promising results for the development of preventive therapies against scorpion envenoming mainly for the populations at-risk.

Serotherapy against Voltage-Gated Sodium Channel-Targeting αToxins from Androctonus Scorpion Venom.

Because of their venom lethality towards mammals, scorpions of the Androctonus genus are considered a critical threat to human health in North Africa. Several decades of exploration have led to a comprehensive inventory of their venom components at chemical, pharmacological, and immunological levels. Typically, these venoms contain selective and high affinity ligands for the voltage-gated sodium (Nav) and potassium (Kv) channels that dictate cellular excitability. In the well-studied Androctonusaustralis and Androctonusmauretanicus venoms, almost all the lethality in mammals is due to the so-called α-toxins. These peptides commonly delay the fast inactivation process of Nav channels, which leads to increased sodium entry and a subsequent cell membrane depolarization. Markedly, their neutralization by specific antisera has been shown to completely inhibit the venom's lethal activity, because they are not only the most abundant venom peptide but also the most fatal. However, the structural and antigenic polymorphisms in the α-toxin family pose challenges to the design of efficient serotherapies. In this review, we discuss past and present accomplishments to improve serotherapy against Androctonus scorpion stings.

Structural basis of α-scorpion toxin action on Nav channels.

Fast inactivation of voltage-gated sodium (Nav) channels is essential for electrical signaling, but its mechanism remains poorly understood. Here we determined the structures of a eukaryotic Nav channel alone and in complex with a lethal α-scorpion toxin, AaH2, by electron microscopy, both at 3.5-angstrom resolution. AaH2 wedges into voltage-sensing domain IV (VSD4) to impede fast activation by trapping a deactivated state in which gating charge interactions bridge to the acidic intracellular carboxyl-terminal domain. In the absence of AaH2, the S4 helix of VSD4 undergoes a ~13-angstrom translation to unlatch the intracellular fast-inactivation gating machinery. Highlighting the polypharmacology of α-scorpion toxins, AaH2 also targets an unanticipated receptor site on VSD1 and a pore glycan adjacent to VSD4. Overall, this work provides key insights into fast inactivation, electromechanical coupling, and pathogenic mutations in Nav channels.

A new Kaliotoxin selective towards Kv1.3 and Kv1.2 but not Kv1.1 channels expressed in oocytes.

In this paper were described the purification, the sequencing, and the immunological and biological characterization of a new Kaliotoxin analog, Aam-KTX, from the venom of the scorpion Androctonus amoreuxi. The toxin effects on three cloned Kv channels (Kv1.1, Kv1.2, and Kv1.3) were investigated in Xenopus oocytes using electrophysiology experiments. The Aam-KTX preference for Kv1.3 channel versus Kv1.2 was expected (EC(50) values, 1.1+/-0.02 and 10.4+/-1.5 nM, respectively) but its total inefficacy on Kv1.1 was very surprising. 3D molecular modeling of Aam-KTX brought putative answers to this difference in selectivity.

Combination of two antibody fragments F(ab')(2)/Fab: an alternative for scorpion envenoming treatment.

Immunotherapy is the only effective treatment for scorpion stings. However the efficiency of this treatment varies depending on the forms of the antibodies and route of administration used. The antibodies are mostly injected as F(ab )(2) fragments. In this study, we investigated damage to the heart and lung tissue and the inflammatory response caused by Androctonus australis hector venom, its toxic fraction after molecular filtration or the isolated main alpha toxin (Aah II) in the presence or absence of different antibody molecules. A mixture of antibody fragments, F(ab )(2) and Fab, significantly reduced local leukocytosis, hemorrhage and inflammatory oedema induced by the A. australis hector venom and its toxins.

New analysis of the toxic compounds from the Androctonus mauretanicus mauretanicus scorpion venom.

Scorpion venoms are very complex mixtures of molecules, most of which are peptides displaying different kinds of biological activity. Indeed, these peptides specifically bind to a variety of pharmacological targets, in particular ionic channels located in prey tissues, resulting in neurotoxic effects. Toxins modulating Na+, K+, Ca2+ and Cl(-) currents have been described in scorpion venoms. In this work, we have used several specific antibodies raised against the most lethal scorpion toxins already described to screen the Moroccan scorpion Androctonus mauretanicus mauretanicus venom in order to characterize new compounds. This immunological screening was also implemented by toxicity tests in mice and with mass spectrometry study, providing new informations on the molecular composition of this venom. In fine, we were able to determine the molecular masses of 70-80 different compounds. According to the immunological data obtained, many toxins cross-react with three sera raised against the most lethal alpha-toxins found in North African scorpion venoms, but not at all with those raised against the main beta-toxins from South and North American venoms. Some of the previously described toxins from Androctonus mauretanicus mauretanicus venom could thus be detected by combining immunological tests, toxicity in mice and molecular masses. Among these toxins, one of them, which showed a mild cross-reaction with the serum raised against AaH I (a highly potent toxin from the venom of Androctonus australis), was identified as Amm III and fully sequenced.