Ion channel dysfunction is considered to be an important mechanism involved in various neurological diseases such as epilepsy. To better understand the mechanism by which these disorders occurs, a blocker of K+ channels, the Kaliotoxin (KTx), a neurotoxin isolated from scorpion venom is used to develop an experimental model of seizures. The effect of the cytokine interleukin-4 (IL-4), was used to modulate microglia homeostasis on the neurotoxicity induced by KTx. Results showed that injected KTx (25 ng/25g, i.c.v) is able to induce myoclonic seizure with neuronal loss and neuro-inflammatory process. The administration of IL-4 (100 ng/25 g, i.p.) enhanced the GFAP expression. IL-4 also promoted an increase of the anti-inflammatory cytokine IL-10 in the cerebral cortex. This treatment using IL-4 seems to reverse the observed oxidative damage induced in KTx model characterized by a decrease of NO, H2O2 levels and lipid peroxidation (MDA) accompanied by an increase of anti-oxidant markers (catalase and GSH) in brain and an improvement of mitochondria swelling. IL-4 seems to enhance the expression of anti-apoptotic protein Bcl-2 which would be responsible for the decrease in the number of neuronal cells death induced by KTx. The developed model using KTx could help the understanding of the cellular and molecular pathway involved in the neuropathological processes of epilepsy related to K+ channel dysfunctions. Interleukin-4 seems to blunt the toxic effects of the KTx. Exogenous interleukin-4 can be neuroprotective, it could represent one of key aspects for developing novel therapeutic strategy.

The availability of a large variety of specific blockers, which inhibit different K-1- currents, would help to elucidate their differences in physiological function. Short peptide toxins isolated from scorpion venoms are able to block voltage-dependent or Ca2+-activated K+ channels. Here, we have studied the venom of the Moroccan scorpion Buthus occitanus Paris (BoP) in order to find new peptides, which could enlarge our structure-function relationship knowledge on the Kv1.3 blocker Kaliotoxin (KTX) that belongs to the alpha-KTx3.1 family. Indeed and since more a decade, KTX is widely used by international investigators because it exhibits a quite sharp specificity and a high-affinity for the Kv1.3 channel, which is not only a neuronal channel but also a therapeutic target for diverse autoimmune diseases such as multiple sclerosis, type 1 diabetes, and rheumatoid arthritis.The BoP venom was first investigated using HPLC and MALDI-TOF/MS. Further, the HPLC fractions were screened by ELISA with antibodies raised against KI'X. These antibodies recognized at least three components toxic in mice by intracerebroventricular injection. They were further pharmacologically characterized by competition using I-125-KIX bound to its specific binding sites on rat brain synaptosomes. A single component (4161 Da) inhibited totally the I-125-KTX binding and with high-affinity (IC50 = 0.1 nM), while the two other components poorly competed with (IC50 > 100 nM). These toxins were sequenced in full by Edman's degradation. The high-affinity ligand (BoPKTX) shares 86% sequence identity with KTX and was classified as toxin alpha-KTx3.17. The two others peptides (O0P1 and B0P2, 4093 Da and 4121 Da, respectively) only differ by a Lys/Arg mutation. Their amino acid sequences were related to Martentoxin, which has been characterized from the Chinese scorpion Buthus martenzi Karch and described as both a BKCa and Kv1.3 blocker. Accordingly, they belong to the alpha-KTx16 family. (C) 2013 Elsevier Ltd. All rights reserved.

Using a proteomic approach, a new structural family of peptides was put in evidence in the venom of the yellow scorpion Tityus serrulatus. Tityus serrulatus Hypotensins (TsHpt) are random-coiled linear peptides and have a similar bradykinin-potentiating peptide (BPP) amino acid signature. TsHpt-I (2.7kDa), the first member of this family, was able to potentiate the hypotensive effects of bradykinin (BK) in normotensive rats. Using the C-terminal of this peptide as a template, a synthetic analog peptide (TsHpt-I[17–25]) was designed to held the BK-potentiating effect. A relevant hypotensive effect, independent on BK, was also observed on both TsHpt (native and synthetic). To better evaluate this hypotensive effect, we examined the vasorelaxation of aortic rings from male Wistar rats and the peptides were able to induce endothelium-dependent vasorelaxation dependent on NO release. Both TsHpt could not inhibit ACE activity. These peptides appear to exert their anti-hypertensive effect through NO-dependent and ACE-independent mechanisms.

. In recent years, several molecular engineering methods of designing bispecific antibodies in various formats have been developed. Tandem-scFvs comprising two scFvs fused together via a peptide are 55-kDa molecules, and are one of the most promising and most straightforward approaches to bispecific antibody production. We report an attempt to design more effective antivenoms to the Androctonus australis scorpion using murine scFvs as building blocks to create a unique bispecific molecule that neutralizes the potent neurotoxins Aah I and Aah II. The tandem-scFv was produced in recombinant bacteria, purified by immobilized metal ion affinity chromatography, and analyzed by polyacrylamide gel electrophoresis, Western blot, gel filtration, mass spectrometry, and direct and competitive radioimmunoassay. In vivo , it neutralized the binding of the Aah I and Aah II toxins to their receptor, and protected mice against experimental envenomation. The findings reported here highlight the potential of recombinant antibody fragments for protecting against scorpion venom toxicity.

The genomic DNA sequence encoding the scorpion toxin Amm VIII was amplified from genomic DNA of the scorpion Androctonus mauretanicus mauretanicus from Morocco, subcloned and sequenced. An intron, with a high A+T content (73.5%), split a Gly codon at the end of the precursor signal peptide and the consensus GT/AG splice junction was identified in the Amm VIII gene. This intron of only 166bp is the smallest intron described so far for a long-chain scorpion toxin gene. In addition, this study led to the identification of three new toxin-related genes. From the deduced amino acid sequences of the encoded precursor proteins, we found that the mature putative toxins were highly similar to the scorpion toxins Leiurus quinquestriatus quinquestriatus IV and Odonthobuthus doriae 1.

Interweaving strategies of electrophysiology, calcium imaging and immunocytochemistry bring new insights into the mode of action of the Brazilian scorpion Tityus serrulatus beta-toxin VII. Pacemaker dorsal unpaired median neurons isolated from the cockroach central nervous system were used to study the effects of toxin VII. In current-clamp, 50 nm toxin VII produced a membrane depolarization and reduced spiking. At 200 nm, depolarization associated with multiphasic effects was seen. After artificial hyperpolarization, plateau potentials on which spontaneous electrical activity appeared were observed. In voltage clamp, toxin VII induced a negative shift of the voltage dependence of sodium current activation without significant effect on steady-state inactivation. In addition, toxin VII produced a permanent TTX-sensitive holding inward current, indicating that background sodium channels were targeted by beta-toxin. Cell-attached patch recordings indicated that these channels were switched from unclustered single openings to current fluctuating between distinct subconductance levels exhibiting increased open probability and open-time distribution. Toxin VII also produced a TTX-sensitive [Ca2+](i) rise. Immunostaining with Cav2.2(alpha 1b) antibodies and calcium imaging data obtained with omega-CgTx GVIA indicated that N-type high-voltage-activated calcium channels initiated calcium influx and were an essential intermediate in the pathway linking toxin VII-modified sodium channels to the activation of an additional route for calcium entry. By using inhibitors of (i) noncapacitative calcium entry (inhibitor LOE-908), (ii) NO-sensitive guanylyl cyclase (ODQ) and (iii) phosphodiesterase 2 (EHNA), together with cGMP antibodies, we demonstrated that noncapacitative calcium entry was the final step in a complex combination of events that was initiated by toxin VII-alteration of sodium channels and then involved successive activation of other membrane ion channels.

In this study, we have used Amm VIII, a natural anatoxin from the scorpion Androctonus mauretanicus mauretanicus, to elicit specific polyclonal antibodies in rabbit. Using liquid-phase radioimmunoassay, we have studied its selectivity and its neutralizing activity both in vitro and in vivo for the most lethal scorpion alpha-toxins described, in particular the alpha-toxin of reference AaH II. We have shown that the anti-Amm VIII serum prevents the association of 125I-AaH II with its receptor and is able to remove 125I-AaH II already bound to its site (the half-life of the complex 125I-AaH II-receptor site was 12 min in the absence of anti-Amm VIII serum but decreased to only 2 min in the presence of anti-Amm VIII serum). In vivo, the serum also has a protective effect in mice: 42 LD50 of AaH II by millilitre are neutralized, measured by subcutaneous injection.

The AaH II toxin from the scorpion Androctonus australis Hector is considered to be the standard α-toxin because it selectively binds with the highest known affinity to site 3 of mammalian voltage-activated Na+ channels (Nav) on rat brain synaptosomes but does not bind to insect synaptosomes. We generated two different constructs in pMALp allowing us to produce AaH II fused with the maltose-binding protein (MBP) in E. coli. We obtained reasonable amounts of recombinant AaH II after cleavage by enterokinase at the site DDDDK. We show that the introduction of a net negative charge at the C-terminus by the suppression of H64 amidation and the addition of an extra residue to the C-terminus (G65) led to fully active AaH II mutants, exhibiting exactly the same affinity as the native toxin for its target on rat brain synaptosomes. In contrast, the mutation of residue K58 into V, I or E residues drastically reduced toxin activity.

LqqIT2 is a depressant neurotoxin present in the venom of the Leiurus quinquestriatus quinquestriatus scorpion, one of the world's most dangerous scorpions endemic to dry habitats in Africa and Asia. In order to determine its efficacy, potency and selectivity, LqqIT2 was subjected for the first time to an electrophysiological and pharmacological comparison between two different cloned sodium channels expressed in Xenopus laevis oocytes. Aside from typical beta-toxin effects, LqqIT2 also affected the inactivation process and ion selectivity of the insect voltage-gated sodium channel. The most interesting feature of LqqIT2 is its total insect-selectivity. At a concentration of 1 microM, the insect-voltage-gated sodium channel, para, was profoundly modulated while its mammalian counterpart, the rat brain Na(v)1.2 channel, was not affected. This trait offers excellent prospects for the development of novel insecticides.

Although it is well established that symptomatology, morbidity and death following scorpion envenomation are due to increases in neurotransmitter release secondary to toxins binding to voltage-sensitive sodium channels, the mechanism by which venom action is involved in damaging heart, liver, lungs and kidneys remains unclear. We hypothesized that scorpion toxins could induce the generation of high levels of free radicals responsible for membrane damage in organs targeted by venom action. We have investigated lipid peroxidation in different organs, through the evaluation of thiobarbituric acid reactive substances (TBARS), after experimental envenomation of rats by toxic fractions of Androctonus australis Hector venom. We have shown that scorpion toxins cause considerable lipid peroxidation in most vital organs. We also evaluated the protective effects of antioxidants in mice injected with lethal doses of toxins. Among the drugs tested, N-acetylcysteine (NAC) was effective in protecting the mice when injected prior to toxin application. However, the free radical scavenging properties of NAC seem less implicated in these protective effects than its ability to increase the fluidity of bronchial secretions. We therefore conclude that free radical generation only plays a minor role in the toxicity of scorpion venom.

BmTX3 is a toxin recently characterised from the venom of the Chinese scorpion Buthus martensi Karch, which specifically blocks a transient A-type K+ current in striatum neurons in culture and binds to rat brain synaptosomes with high affinity. With Aa1 and AmmTX3, it belongs to the new α-KTx15 subfamily from ‘short-chain’ scorpion toxins, which specifically block different types of K+ channels. Here, a highly specific polyclonal antiserum was raised in rabbit against a C-terminal deleted BmTX3 analogue (BmTX-del YP). Using liquid-phase radioimmunoassay, we have studied its selectivity for the toxins from the α-KTx15 subfamily. We have also demonstrated that no/or poor cross-reactivity was observed with a panel of ‘short-chain’ scorpion toxins representative of other structurally different subfamilies. These results suggest that a wide antigenic polymorphism, similar to that previously observed for ‘long-chain’ scorpion toxins acting as modulators of voltage-activated Na+ channels, is also the rule for the ‘short-chain’ scorpion toxins able to block K+ channels.

Small conductance calcium activated potassium channels (SK) are crucial in the regulation of cell firing frequency in the nervous system and other tissues. In the present work, a novel SK channel blocker, designated BmSKTx1, was purified from the scorpion Buthus martensi Karsh venom. The sequence of the N-terminal 22 amino acid residues was determined by Edman degradation. Using this sequence information, the full-length cDNA and genomic gene of BmSKTx1 were cloned and sequenced. By these analyses, BmSKTx1 was found to be a peptide composed of 31 amino acid residues with three disulfide bonds. It shared little sequence homology with other known scorpion alpha-KTxs but showed close relationship with SK channel blockers in the phylogenetic tree. According to the previous nomenclature, BmSKTx1 was classified as alpha-KTx14.1. We examined the effects of BmSKTx1 on different ion channels of rat adrenal chromaffin cells (RACC) and locust dorsal unpaired median (DUM) neurons. BmSKTx1 selectively inhibited apamin-sensitive SK currents in RACC with Kd of 0.72 microM and Hill coefficient of 2.2. And it had no effect on Na+, Ca2+, Kv, and BK currents in DUM neuron, indicating that BmSKTx1 was a selective SK toxin.

Venom variability in specimens of Tityus serrulatus scorpion was assessed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) analyses. An expanded time lag venom extraction protocol was carried out using ten scorpions to study individual variations that might occur due to different rates in protein expression and/or processing. The first extraction of venom was made from the animals after 20 days of starvation, which allowed the venom gland to be filled up. The second extraction event was carried out 24 hours after the first one. The third was 8 days after the first extraction. By means of MALDI-TOF analyses, important variations were observed in venoms of a single specimen extracted at different times, especially in latter extraction events. These variations are most probably related to dynamics in cell gland production. Since T. serrulatus is a parthenogenetic species, sexual variations are naturally excluded and we did not expect intra-specific variations, which was confirmed. Knowledge of individual venom variability is extremely important to avoid misunderstandings in the use of venom proteomic analysis as a taxonomic tool.

Enzymes with gelatinolytic activity were detected in Tityus bahiensis and Tityus serrulatus venom. Their activity was optimal at pH 8.0 in SDS–PAGE–gelatin. They were inhibited by PMSF but not by iodoacetamide, pepstatin or phenantrolin in the assay conditions used. This suggests that these enzymes are serine proteases. The presence of metal ions did not affect the proteolytic activity of these enzymes. Several possible functions may be envisaged for these enzymes: in tissue permeabilization, pancreatitis and toxin processing.

The bacterial potassium channel, KcsA, can be modified to express a high-affinity receptor site for the scorpion toxin kaliotoxin (KTX) by substituting subregion I in the P region of KcsA with the one present in the human voltage-gated potassium channel Kv1.3 [Legros, C., Pollmann, V., Knaus, H. G., Farrell, A. M., Darbon, H., Bougis, P. E., Martin-Eauclaire, M. F., and Pongs, O. (2000) J. Biol. Chem. 275, 16918-16924]. This approach opened the way to investigate whether sequence differences in subregion I of Kv1 channels correlate with the distinct pharmacological profiles of peptide inhibitors. A panel of six chimeras between KcsA and human Kv1.1-6 were constructed, expressed in Escherichia coli, purified to homogeneity, and assessed in filter binding assays using either monoiodo-tyrosine-KTX ([(125)I]KTX) or monoiodo-tyrosine-hongotoxin(1)(A19Y/Y37F) ([(125)I]HgTX(1)(A19Y/Y37F)). The KcsA-Kv1.X chimeras were found to have lower affinities for these ligands than the corresponding mammalian Kv1.X channels, indicating that other parts of the channels may contribute to binding or that subtle structural differences exist between these channels. The properties of the KcsA-Kv1.X chimeras were also characterized in surface plasmon resonance experiments. KcsA-Kv1.3 chimeras were immobilized on the surface of a sensor chip for determining, in real time, binding of the peptides. KTX binding properties to immobilized KcsA-Kv1.3 chimera were similar to those determined by filtration techniques. Taken together, our results demonstrate that the pharmacological profile of peptide toxins can be incorporated into KcsA-Kv1.X chimeras containing the subregion I of the corresponding mammalian Kv1.X channels. This innovative approach may facilitate the high-throughput screening of ligand libraries aimed at the discovery of novel potassium channel modulators.

Tx4(6-1) a neurotoxic peptide from the venom of the aggressive South American 'armed' spider Phoneutria nigriventer, has been previously isolated and sequenced. It shows no detectable activity in mice but affects the peripheral nervous system of insects by stimulating glutamate release at the neuromuscular junction. Here we investigate possible interactions of the toxin with voltage-activated sodium channels (Na(v)). We confirm that it is ineffective on mammalian Na(v) channels, and establish that it competes with the alpha-like toxin 125I-Bom IV, for binding on the site 3 of insect Na(v) channel (IC(50) value around 25nM). The physiological consequences of this binding to the insect Na(v) channel are shown by electrophysiology: Tx4(6-1) prolongs evoked axonal action potentials (APs) (<500&mgr;s duration in control). Prolonged 8-10ms or 'plateau' 500-800ms APs accompanied by repetitive firing at 80-150Hz are recorded after 4-8min of toxin action. This modification of evoked activity is due to a slowing down of sodium current inactivation. Effects of Tx4(6-1) on sodium current are compared with those of a typical scorpion alpha-toxin and of some other spider toxins active on insect Na(v) channels. At the end of long voltage pulses, the maintained inward sodium current may represent 50% of the peak current after scorpion alpha-toxin but only about 8-10% after spider toxins. To understand the slight differences in the effects of alpha-scorpion and spider toxins on the insect Na(v) channel, structural studies of toxin-channels interactions would be necessary.