Inhibitory effects of scorpion venom on the uptake of amino acids by synaptosomes and synaptosomal membrane vesicles

Scorpion (Tityus serrulatus) venom strongly inhibited the Na⁺-dependent uptake of [¹⁴C]proline by rat brain synaptosomal preparations. In addition, the efflux of proline was enhanced markedly by scorpion venom. The inhibitory effects of the venom were also demonstrated in synaptosomal vesicle preparations where proline uptake was energized by an artificially imposed Na⁺ gradient. In both preparations, the effect of scorpion venom was additive with the inhibitory effect of veratridine on Na⁺-dependent amino acid uptake. The inhibitory effects of both compounds were abolished by tetrodotoxin. The Na⁺-dependent uptakes of amino acids (e.g. proline, glutamic acid, and γ-aminobutyric acid) were much more sensitive to inhibition by the toxin than the Na⁺⁶-independent uptakes (e.g. leucine and phenylalanine). The results of the present study indicate that the scorpion venom mav exert its inhibitory effect on Na⁺-dependent transport by decreasing the transmembrane Na⁺ gradient. Efflux of accumulated proline, which is presumably controlled by maintenance of this Na⁺ gradient, was stimulated 3- to 4-fold by the scorpion venom.     

The Venom of the Spider Selenocosmia Jiafu Contains Various Neurotoxins Acting on Voltage-Gated Ion Channels in Rat Dorsal Root Ganglion Neurons

Selenocosmia jiafu is a medium-sized theraphosid spider and an attractive source of venom, because it can be bred in captivity and it produces large amounts of venom. We performed reversed-phase high-performance liquid chromatography (RP-HPLC) and matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analyses and showed that S. jiafu venom contains hundreds of peptides with a predominant mass of 3000–4500 Da. Patch clamp analyses indicated that the venom could inhibit voltage-gated Na⁺, K⁺ and Ca²⁺ channels in rat dorsal root ganglion (DRG) neurons. The venom exhibited inhibitory effects on tetrodotoxin-resistant (TTX-R) Na⁺ currents and T-type Ca²⁺ currents, suggesting the presence of antagonists to both channel types and providing a valuable tool for the investigation of these channels and for drug development. Intra-abdominal injection of the venom had severe toxic effects on cockroaches and caused death at higher concentrations. The LD₅₀ was 84.24 μg/g of body weight in the cockroach. However, no visible symptoms or behavioral changes were detected after intraperitoneal injection of the venom into mice even at doses up to 10 mg/kg body weight. Our results provide a basis for further case-by-case investigations of peptide toxins from this venom.     

Turkish scorpion Buthacus macrocentrus: General characterization of the venom and description of Bu1, a potent mammalian Na+-channel α-toxin

The venom of the scorpion Buthacus macrocentrus of Turkey was fractionated by high performance liquid chromatography (HPLC) and its mass finger print analysis was obtained by spectrometry. More than 70 different fractions were obtained, allowing the determination of the molecular masses of at least 60 peptides ranging between 648 and 44,336 Da. The venom is enriched with peptides containing molecular masses between 3200–4500 Da, and 6000–7500 Da. They very likely correspond to K⁺-channel and Na⁺-channel specific peptides, respectively, as expected from venoms of scorpions of the family Buthidae, already determined for other species. The major component obtained from HPLC was shown to be lethal to mice and was further purified and characterized. It contains 65 amino acid residues maintained closely packed by 4 disulfide bridges, and shows a molecular weight of 7263 Da. Additionally, a cDNA from the venomous glands of this scorpion was used in conjunction with sequence data from Edman degradation and mass spectrometry for cloning the gene that codes for Bu1 as we named this toxin. This gene codes for a 67 amino acid residues peptide, where the two last are eliminated post-translationally for production of an amidated C-terminal arginine. Its sequence is closely related to toxins from the species Leiurus quinquestriatus, as revealed by a phylogenetic tree analysis. Electrophysiological results conducted with Bu1 using patch-clamp techniques indicate that it modifies the Na⁺ currents, in a similar way as other well known α-scorpion toxins. These results support the conclusion that this species of scorpions is dangerous to humans, having an epidemiological interest for the country.     

Vejovine, a new antibiotic from the scorpion venom of Vaejovis mexicanus

Multidrug resistant bacterial infections are one of the most important health problems in recent years. Resistance to conventional antibiotics limits the therapeutic options causing increase rate in morbid-mortality in hospitals. Therefore, new antibacterial agents with new bacterial targets have been searched and found in many different sources, including scorpion venom and scorpion hemolymph. Here, we report a new anti-microbial peptide named Vejovine. This peptide was isolated from the venom of the Mexican scorpion Vaejovis mexicanus by two steps of reversed phase high performance liquid chromatography (RP-HPLC). It is composed of 47 amino acid residues with no cysteine residues in its sequence, with a molecular weight of 4873 Da. The chemical synthesis of Vejovine was performed by the solid phase method of Merrifield, using fluoren-9-ylmethoxycarbonyl (Fmoc)-amino acids. Both the native and synthetic peptides were shown to have essentially the same activity. Vejovine inhibits growth of clinical isolates of Gram-negative multidrug resistant (Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae and Acinetobacter baumanii) causing nosocomial infections with a minimum inhibitory concentration (MIC) of 4.4 μM up to 50 μM. This peptide has also hemolytic activity against human erythrocytes with a HC₅₀ value of 100 μM. A cDNA library of the venomous gland of this scorpion provided material for cloning the gene encoding Vejovine. This peptide is a new type of antibiotic, showing less than 50% similarity to other known scorpion peptides. Vejovine is a candidate to be used as a leading compound for future development of an effective peptide against multidrug resistant bacteria.     

Negative-shift activation, current reduction and resurgent currents induced by β-toxins from Centruroides scorpions in sodium channels

The β-toxins purified from the New World scorpion venoms of the Centruroides species affect several voltage-gated sodium channels (VGSCs) and thus are essential tools not only for the discrimination of different channel sub-types but also for studying the structure-function relationship between channels and toxins. This communication reports the results obtained with four different peptides purified from three species of Centruroides scorpions and assayed on seven distinct isoforms of VGSC (Na_v1.1-Na_v1.7) by specific functional analysis conducted through single cell electrophysiology. The toxins studied were CssII from Centruroides suffusus suffusus, Cll1 and Cll2 from Centruroides limpidus limpidus and a novel toxin from Centruroides noxius, which was characterized for the first time here. It has 67 amino acid residues and four disulfide bridges with a molecular mass of 7626 Da. Three different functional features were identified: current reduction of macroscopic conductance, left shift of the voltage-dependent activation and induction of resurgent currents at negative voltages following brief, strong depolarizations. The isoforms which revealed to be more affected resulted to be Na_v1.6 > 1.1 > 1.2 and, for the first time, a β-toxin is here shown to induce resurgent current also in isoforms different from Na_v1.6. Additionally, these results were analyzed with molecular modelling. In conclusion, although the four toxins have a high degree of identity, they display tri-modal function, each of which shows selectivity among the different sub-types of Na⁺-channels. Thus, they are invaluable as tools for structure-function studies of β-toxins and offer a basis for the design of novel ion channel-specific drugs.     

Purification and characterization of Ts15, the first member of a new α-KTX subfamily from the venom of the Brazilian scorpion Tityus serrulatus

Voltage-gated potassium channel toxins (KTxs) are basic short chain peptides comprising 23-43 amino acid residues that can be cross-linked by 3 or 4 disulfide bridges. KTxs are classified into four large families: α-, β-, γ- and κ-KTx. These peptides display varying selectivity and affinity for K_v channel subtypes. In this work, a novel toxin from the Tityus serrulatus venom was isolated, characterized and submitted to a wide electrophysiological screening on 5 different subtypes of Na_V channels (Na_V1.4; Na_V1.5; Na_V1.6; Na_V1.8 and DmNa_V1) and 12 different subtypes of K_V channels (K_V1.1 - K_V1.6; K_V2.1; K_V3.1; K_V4.2; K_V4.3; Shaker IR and ERG). This novel peptide, named Ts15, has 36 amino acids, is cross-linked by 3 disulfide bridges, has a molecular mass of 3956 Da and pI around 9. Electrophysiological experiments using patch clamp and the two-electrode voltage clamp techniques show that Ts15 preferentially blocks K_V1.2 and K_V1.3 channels with an IC₅₀ value of 196 ± 25 and 508 ± 67 nM, respectively. No effect on Na_V channels was observed, at all tested concentrations. Since Ts15 shows low amino acid identity with other known KTxs, it was considered a bona fide novel type of scorpion toxin. Ts15 is the unique member of the new α-Ktx21 subfamily and therefore was classified as α-Ktx21.1.     

General biochemical and immunological characteristics of the venom from Peruvian scorpion Hadruroides lunatus

This communication describes the general biochemical properties and some immunological characteristics of the venom from the Peruvian scorpion Hadruroides lunatus, which is the most medically relevant species in Peru. The soluble venom of this scorpion is toxic to mice, the LD₅₀ determined was 0.1 mg/kg and 21.55 mg/kg when the venom was injected intracranial or intraperitoneally, respectively. The soluble venom displayed proteolytic, hyaluronidasic, phospholipasic and cardiotoxic activities. High performance liquid chromatography of the soluble venom resulted in the separation of 20 fractions. Two peptides with phospholipasic activity were isolated to homogeneity and their molecular masses determined by mass spectrometry (MALDI TOF). Anti-H. lunatus venom sera were produced in rabbits. Western blotting analysis showed that most of the protein content of this venom is immunogenic. H. lunatus anti-venom displayed consistent cross-reactivity with venom antigens from the new World-scorpions Tityus serrulatus and Centruroides sculpturatus venoms; however, a weaker reactivity was observed against the venom antigens from the old World-scorpion Androctonus australis Hector.     

A covalent peptide inhibitor of RGS4 identified in a focused one-bead, one compound library screen

Background

K⁺ and Na⁺ channel toxins constitute a large set of polypeptides, which interact with their ion channel targets. These polypeptides are classified in two different structural groups. Recently a new structural group called birtoxin-like appeared to contain both types of toxins has been described. We hypothesized that peptides of this group may contain two conserved structural motifs in K⁺ and/or Na⁺ channels scorpion toxins, allowing these birtoxin-like peptides to be active on K⁺ and/or Na⁺ channels.

Results

Four multilevel motifs, overrepresented and specific to each group of K⁺ and/or Na⁺ ion channel toxins have been identified, using GIBBS and MEME and based on a training dataset of 79 sequences judged as representative of K⁺ and Na⁺ toxins. Unexpectedly birtoxin-like peptides appeared to present a new structural motif distinct from those present in K⁺ and Na⁺ channels Toxins. This result, supported by previous experimental data, suggests that birtoxin-like peptides may exert their activity on different sites than those targeted by classic K⁺ or Na⁺ toxins. Searching, the nr database with these newly identified motifs using MAST, retrieved several sequences (116 with e-value < 1) from various scorpion species (test dataset). The filtering process left 30 new and highly likely ion channel effectors. Phylogenetic analysis was used to classify the newly found sequences. Alternatively, classification tree analysis, using CART algorithm adjusted with the training dataset, using the motifs and their 2D structure as explanatory variables, provided a model for prediction of the activity of the new sequences.

Conclusion

The phylogenetic results were in perfect agreement with those obtained by the CART algorithm. Our results may be used as criteria for a new classification of scorpion toxins based on functional motifs.     

Overview of Scorpion Species from China and Their Toxins

Scorpions are one of the most ancient groups of terrestrial animals. They have maintained a steady morphology over more than 400 million years of evolution. Their venom arsenals for capturing prey and defending against predators may play a critical role in their ancient and conservative appearance. In the current review, we present the scorpion fauna of China: 53 species covering five families and 12 genera. We also systematically list toxins or genes from Chinese scorpion species, involving eight species covering four families. Furthermore, we review the diverse functions of typical toxins from Chinese scorpion species, involving Na⁺ channel modulators, K⁺ channel blockers, antimicrobial peptides and protease inhibitors. Using scorpion species and their toxins from China as an example, we build the bridge between scorpion species and their toxins, which helps us to understand the molecular and functional diversity of scorpion venom arsenal, the dynamic and functional evolution of scorpion toxins, and the potential relationships of scorpion species and their toxins.     

Tst26, a novel peptide blocker of Kv1.2 and Kv1.3 channels from the venom of Tityus stigmurus

Using high-performance liquid chromatography Tst26, a novel potassium channel blocker peptide, was purified from the venom of the Brazilian scorpion Tityus stigmurus. Its primary structure was determined by means of automatic Edman degradation and mass spectrometry analysis. The peptide is composed of 37 amino acid residues and tightly folded through three disulfide bridges, similar to other K⁺ channel blocking peptides purified from scorpion venoms. It contains the “essential dyad” for K⁺ channel recognition comprised of a lysine at position 27 and a tyrosine at position 36. Electrophysiological assays revealed that Tst26 blocked hKv1.2 and hKv1.3 channels with high affinity (K_d = 1.9 nM and 10.7 nM, respectively) while it did not affect several other ion channels (mKv1.1, hKv1.4, hKv1.5, hERG, hIKCa1, hBK, hNav1.5) tested at 10 nM concentration. The voltage-dependent steady-state parameters of K⁺ channel gating were unaffected by the toxin in both channels, but due to the fast association and dissociation kinetics Tst26 slowed the rate of inactivation of Kv1.3 channels. Based on the primary structure, the systematic nomenclature proposed for this peptide is α-KTx 4.6.     

Purification and pharmacological characterization of BmKK2 (α-KTx 14.2), a novel potassium channel-blocking peptide, from the venom of Asian scorpion Buthus martensi Karsch

BmKK2 (α-KTx 14.2) is one of the novel short-chain peptides found in molecular cloning of a venom gland cDNA library from Asian scorpion Buthus martensi Karsch. Based upon its amino acid sequence, the peptide was proposed to adopt a classical α/β-scaffold for α-KTxs. In the present study, we purified BmKK2 from the venom of B. martensi Karsch, and investigated its action on voltage-dependent K⁺ currents in dissociated hippocampal neurons from neonatal rats. BmKK2 (10–100 μM) selectively inhibited the delayed rectifier K⁺ current, but did not affect the fast transient K⁺ current. The inhibition of BmKK2 on the delayed rectifier K⁺ current was reversible and voltage-independent. The peptide did not affect the steady-state activation of the current, but caused a depolarizing shift (about 9 mV) of its steady-state inactivation curve. The results demonstrate that BmKK2 is a novel K⁺ channel-blocking scorpion peptide.     

Cloning and characterization of cDNA sequences encoding for new venom peptides of the Brazilian scorpion Opisthacanthus cayaporum

Scorpion venom glands produce a large variety of bioactive peptides. This communication reports the identification of venom components obtained by sequencing clones isolated from a cDNA library prepared with venomous glands of the Brazilian scorpion Opisthacanthus cayaporum (Ischnuridae). Two main types of components were identified: peptides with toxin-like sequences and proteins involved in cellular processes. Using the expressed sequence tag (EST) strategy 118 clones were identified, from which 61 code for unique sequences (17 contigs and 44 singlets) with an average length of 531 base-pairs (bp). These results were compared with those previously obtained by the proteomic analysis of the same venom, showing a considerable degree of similarity in terms of the molecular masses expected and DNA sequences found. About 36% of the ESTs correspond to toxin-like peptides and proteins with identifiable open reading frames (ORFs). The cDNA sequencing results also show the presence of sequences whose putative products correspond to a scorpine-like component; three short antimicrobial peptides; three K⁺-channel blockers; and an additional peptide containing 78 amino acid residues, whose sequence resembles peptide La1 from another Ischnuridae scorpion Liocheles australiasiae, thus far with unknown function.     

Underlying mechanism of actions of tefluthrin,a pyrethroid insecticide,on voltage-gated ion currents and on action currents in pituitary tumor (GH3) cells and GnRH-secreting (GT1-7) neurons

Tefluthrin is a synthetic pyrethroid and involved in acute neurotoxic effects. How this compound affects ion currents in endocrine or neuroendocrine cells remains unclear. Its effects on membrane ion currents in pituitary tumor (GH₃) cells and in hypothalamic (GT1-7) neurons were investigated. Application of Tef (10 μM) increased the amplitude of voltage-gated Na⁺ current (I_Na), along with a slowing in current inactivation and deactivation in GH₃ cells. The current–voltage relationship of I_Na was shifted to more negative potentials in the presence of this compound. Tef increased I_Na with an EC₅₀ value of 3.2 ± 0.8 μM. It also increased the amplitude of persistent I_Na. Tef reduced the amplitude of L-type Ca²⁺ current. This agent slightly inhibited K⁺ outward current; however, it had no effect on the activity of large-conductance Ca²⁺-activated K⁺ channels. Under cell-attached voltage-clamp recordings, Tef (10 μM) increased amplitude and frequency of spontaneous action currents, along with appearance of oscillatory inward currents. Tef-induced inward currents were suppressed after further application of tetrodotoxin, riluzole or ranolazine. In GT1-7 cells, Tef also increased the amplitude and frequency of action currents. Taken together, the effects of Tef and its structural related pyrethroids on ion currents can contribute to the underlying mechanisms through which they affect endocrine or neuroendocrine function in vivo.     

Actions of sea anemone type 1 neurotoxins on voltage-gated sodium channel isoforms

As voltage-gated Na⁺ channels are responsible for the conduction of electrical impulses in most excitable tissues in the majority of animals (except nematodes), they have become important targets for the toxins of venomous animals, from sea anemones to molluscs, scorpions, spiders and even fishes. During their evolution, different animals have developed a set of cysteine-rich peptides capable of binding different extracellular sites of this channel protein. A fundamental question concerning the mechanism of action of these toxins is whether they act at a common receptor site in Na⁺ channels when exerting their different pharmacological effects, or at distinct receptor sites in different Na_v channels subtypes whose particular properties lead to these pharmacological differences. The α-subunits of voltage-gated Na⁺ channels (Na_v1.x) have been divided into at least nine subtypes on the basis of amino acid sequences. Sea anemones have been extensively studied from the toxinological point of view for more than 40 years. There are about 40 sea anemone type 1 peptides known to be active on Na_v1.x channels and all are 46-49 amino acid residues long, with three disulfide bonds and their molecular weights range between 3000 and 5000 Da. About 12 years ago a general model of Na_v1.2-toxin interaction, developed for the α-scorpion toxins, was shown to fit also to action of sea anemone toxin such as ATX-II. According to this model these peptides are specifically acting on the type 3 site known to be between segments 3 and 4 in domain IV of the Na⁺ channel protein. This region is indeed responsible for the normal Na⁺ currents fast inactivation that is potently slowed by these toxins. This fundamental “gain-of-function” mechanism is responsible for the strong increase in the action potential duration. They constitute a class of tools by means of which physiologists and pharmacologists can study the structure/function relationships of channel proteins. As most of the structural and electrophysiological studies were performed on type 1 sea anemone sodium channel toxins, we will present a comprehensive and updated review on the current understanding of the physiological actions of these Na channel modifiers.     

Drosotoxin, a selective inhibitor of tetrodotoxin-resistant sodium channels

The design of animal toxins with high target selectivity has long been a goal in protein engineering. Based on evolutionary relationship between the Drosophila antifungal defensin (drosomycin) and scorpion depressant Na⁺ channel toxins, we exploited a strategy to create a novel chimeric molecule (named drosotoxin) with high selectivity for channel subtypes, which was achieved by using drosomycin to substitute the structural core of BmKITc, a depressant toxin acting on both insect and mammalian Na⁺ channels. Recombinant drosotoxin selectively inhibited tetrodotoxin-resistant (TTX-R) Na⁺ channels in rat dorsal root ganglion (DRG) neurons with a 50% inhibitory concentration (IC₅₀) of 2.6 ± 0.5 μM. This chimeric peptide showed no activity on K⁺, Ca²⁺ and TTX-sensitive (TTX-S) Na⁺ channels in rat DRG neurons and Drosophila para/tipE channels at micromolar concentrations. Drosotoxin represents the first chimeric toxin and example of a non-toxic core scaffold with high selectivity on mammalian TTX-R Na⁺ channels.     

Effect of maurotoxin,a four disulfide‐bridged toxin from the chactoid scorpion Scorpio maurus,on Shaker K+ channels

Abstract: Maurotoxin is a 34‐residue toxin isolated from the venom of the Tunisian chactoid scorpion Scorpio maurus palmatus and contains four disulfide bridges that are normally found in long‐chain toxins of 60–70 amino acid residues, which affect voltage‐gated sodium channels. However, despite the unconventional disulfide‐bridge pattern of maurotoxin, the conformation of this toxin remains similar to that of other toxins acting on potassium channels. Here, we analyzed the effects of synthetic maurotoxin on voltage‐gated Shaker potassium channels (ShB) expressed in Xenopus oocytes. Maurotoxin produces a strong, but reversible, inhibition of the ShB K⁺ current with an IC₅₀ of 2 nm . Increasing concentrations of the toxin induce a progressively higher block at saturating concentrations. At nonsaturating concentrations of the toxin (5–20 nm ), the channel block appears slightly more pronounced at threshold potentials suggesting that the toxin may have a higher affinity for the closed state of the channel. At the single channel level, the toxin does not modify the unitary current amplitude, but decreases ensemble currents by increasing the number of depolarizing epochs that failed to elicit any opening. A point mutation of Lys²³ to alanine in maurotoxin produces a 1000‐fold reduction in the IC₅₀ of block by the toxin suggesting the importance of this charged residue for the interaction with the channel. Maurotoxin does not affect K⁺ currents carried by Kir2.3 channels in oocytes or Na⁺ currents carried by the α_IIa channel expressed in CHO cells.     

ImKTx88, a novel selective Kv1.3 channel blocker derived from the scorpion Isometrus maculates

Scorpion toxins are useful in the structure-function research of ion channels and valuable resources for drug design. The Kv1.3 channel is an important pharmacological target for the therapy of T cell-mediated autoimmune diseases, and many toxin peptides targeting Kv1.3 have been identified as good drug candidates in recent years. In this study, a novel toxin gene ImKTx88 was isolated from the venom of the scorpion Isometrus maculates through the construction of the cDNA library method, and the recombinant toxin peptide was purified and characterized physiologically. The mature peptide of ImKTx88 contained 39 amino acid residues including six cysteines and was predicted to be a new member of α-KTx scorpion family by sequence analysis. The electrophysiological experiments further indicated that the rImKTx88 peptide had a novel pharmacological profile: it inhibited Kv1.3 channel current with an IC₅₀ of 91 ± 42 pM, and exhibited very good selectivity for Kv1.3 over Kv1.1 (4200-fold) and Kv1.2 (93000-fold) channels, respectively. All these results suggested that, as a new selective Kv1.3 channel blocker, the ImKTx88 peptide may serve as a potential drug candidate in the therapy of autoimmune diseases.     

Isolation and molecular cloning of beta-neurotoxins from the venom of the scorpion Centruroides suffusus suffusus

This communication reports the identification and characterization of two new toxins from the venom of the scorpion Centruroides suffusus suffusus, named: CssVIII and CssIX, according to the original nomenclature of toxins previously described for this scorpion. The isolation was obtained by means of two chromatographic steps, and a cDNA library was used to fully identify their precursors. CssVIII and CssIX contain signal peptides of 19 and 17 amino acid residues, and mature peptides of 66 and 65 residues, respectively. Intracranial injections into mice of both purified toxins showed toxicity results similar to those found for toxins CssII and CssIV. Additionally, they compete with the parent toxin CssIV, in binding and displacement experiments, conducted with brain synaptosomes showing nanomolar affinities. These results strongly support the conclusion that they are new β-neurotoxins and certainly would be of the interest of researchers in the field of venomics for studying sodium channels.