A scorpion venom neurotoxin paralytic to insects that affects sodium current inactivation: purification, primary structure, and mode of action

A new toxin, Lqh alpha IT, which caused a unique mode of paralysis of blowfly larvae, was purified from the venom of the scorpion Leiurus quinquestriatus hebraeus, and its structural and pharmacological properties were compared to those of three other groups of neurotoxins found in Buthinae scorpion venoms. Like the excitatory and depressant insect-selective neurotoxins, Lqh alpha IT was highly toxic to insects, but it differed from these toxins in two important characteristics: (a) Lqh alpha IT lacked strict selectivity for insects; it was highly toxic to crustaceans and had a measurable but low toxicity to mice. (b) It did not displace an excitatory insect toxin, 125I-AaIT, from its binding sites in the insect neuronal membrane; this indicates that the binding sites for Lqh alpha IT are different from those shared by the excitatory and depressant toxins. However, in its primary structure and its effect on excitable tissues, Lqh alpha IT strongly resembled the well-characterized alpha scorpion toxins, which affect mammals. The amino acid sequence was identical with alpha toxin sequences in 55%-75% of positions. This degree of similarity is comparable to that seen among the alpha toxins themselves. Voltage- and current-clamp studies showed that Lqh alpha IT caused an extreme prolongation of the action potential in both cockroach giant axon and rat skeletal muscle preparations as a result of the slowing and incomplete inactivation of the sodium currents. These observations indicate that Lqh alpha IT is an alpha toxin which acts on insect sodium channels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Localization of receptor sites for insect-selective toxins on sodium channels by site-directed antibodies.

Site-directed antibodies corresponding to conserved putative extracellular segments of sodium channels, coupled with binding studies of radiolabeled insect-selective scorpion neurotoxins, were employed to clarify the relationship between the toxins' receptor sites and the insect sodium channel. (1) The depressant insect toxin LqhIT2 was shown to possess two noninteracting binding sites in locust neuronal membranes: a high-affinity (KD1 = 0.9 +/- 0.6 nM) and low-capacity (Bmax1 = 0.1 +/- 0.07 pmol/mg) binding site as well as a low-affinity (KD2 = 185 +/- 13 nM) and high-capacity (Bmax2 = 10.0 +/- 0.6 pmol/mg) binding site. (2) The high-affinity site serves as a target for binding competition by the excitatory insect toxin AaIT. (3) The binding of LqhIT2 was significantly inhibited in a dose-dependent manner by each of four site-directed antibodies. The binding inhibition resulted from reduction in the number of binding sites. (4) The antibody-mediated inhibition of [125I]AaIT binding differs from that of LqhIT2: three out of the four antibodies which inhibited LqhIT2 binding only partially affected AaIT binding. Two antibodies, one corresponding to extracellular and one to intracellular segments of the channel, did not affect the binding of either toxin. These data suggest that the receptors to the depressant and excitatory insect toxins (a) comprise an integral part of the insect sodium channel, (b) are formed by segments of external loops in domains I, III, and IV of the sodium channel, and (c) are localized in close proximity but are not identical in spite of the competitive interaction between these toxins.     

An excitatory and a depressant insect toxin from scorpion venom both affect sodium conductance and possess a common binding site

Two insect selective toxins were purified by gel-permeation and ion-exchange chromatographies from the venom of the scorpion, Leiurus quinquestriatus quinquestriatus, and their chemical and pharmacological properties were studied. The first toxin (LqqIT1) induces a fast excitatory contraction paralysis of fly larvae and is about 40 times more toxic than the crude venom. It is a polypeptide composed of 71 amino acids, including 8 half-cystines and devoid of methionine and tryptophan, with an estimated molecular weight of 8189 and a pI value of 8.5. The second toxin (LqqIT2) induces a slow depressant, flaccid paralysis of fly larvae. It is composed of 72 amino acids, including 8 half-cystines, is devoid of proline methionine and histidine, and has an estimated molecular weight of 7990 and a pI value of 8.3. The contrasting symptomatology of these toxins is interpreted in terms of their effects on an isolated axonal preparation of the cockroach in current and voltage clamp conditions. LqqIT1 (0.5-4 microM) induced repetitive firing of the axon which was attributable to two changes in the sodium conductance, a small increase in the peak conductance and a slowing of its turning off. LqqIT2 (1-8 microM) caused a blockage of the evoked action potentials, attributable to both a strong depolarization of the axonal membrane and a progressive suppression of the sodium current. Neither toxin affected potassium conductance. The two toxins differ mainly in their opposite effects on the activatable sodium permeability. In binding assays to a preparation of insect synaptosomal membrane vesicles, the two toxins were shown to competitively displace the radioiodinated excitatory insect toxin derived from the venom of the scorpion, Androctonus australis [( 125I]AaIT), which strongly resembles, in its chemistry and action, the LqqIT1 toxin. The present two toxins have demonstrated a strong affinity closely resembling the AaIT, with KD values of 0.4, 1.9, and 1.0 nM for LqqIT1, LqqIT2, and AaIT, respectively. These data suggest the possibility that the excitatory and depressant insect toxins share a common binding site associated with sodium channels in insect neuronal membranes.     

A new approach to insect-pest control—combination of neurotoxins interacting with voltage sensitive sodium channels to increase selectivity and specificity

Voltage-sensitive sodium channels are responsible for the generation of electrical signals in most excitable tissues and serve as specific targets for many neurotoxins. At least seven distinct classes of neurotoxins have been designated on the basis of physiological activity and competitive binding studies. Although the characterization of the neurotoxin receptor sites was predominantly performed using vertebrate excitable preparations, insect neuronal membranes were shown to possess similar receptor sites. We have demonstrated that the two mutually competing antiinsect excitatory and depressant scorpion toxins, previously suggested to occupy the same receptor site, bind to two distinct receptors on insect sodium channels. The latter provides a new approach to their combined use in insect control strategy. Although the sodium channel receptor sites are topologically separated, there are strong allosteric interactions among them. We have shown that the lipid-soluble sodium channel activators, veratridine and brevetoxin, reveal divergent allosteric modulation on scorpion α-toxins binding at homologous receptor sites on mammalian and insect sodium channels. The differences suggest a functionally important structural distinction between these channel subtypes. The differential allosteric modulation may provide a new approach to increase selective activity of pesticides on target organisms by simultaneous application of allosterically interacting drugs, designed on the basis of the selective toxins. Thus, a comparative study of neurotoxin receptor sites on mammalian and invertebrate sodium channels may elucidate the structural features involved in the binding and activity of the various neurotoxins, and may offer new targets and approaches to the development of highly selective pesticides.     

cDNA cloning, sequence analysis and molecular modeling of a new peptide from the scorpion Buthotus saulcyi venom

In this study, the cDNA of a new peptide from the venom of the scorpion, Buthotus saulcyi, was cloned and sequenced. It codes for a 64 residues peptide (Bsaul1) which shares high sequence similarity with depressant insect toxins of scorpions. The differences between them mainly appear in the loop1 which connects the beta-strand1 to the alpha-helix and seems to be functionally important in long chain scorpion neurotoxins. This loop is three amino acids longer in Bsaul1 compared to other depressant toxins. A comparative amino acid sequence analysis done on Bsaul1 and some of alpha-, beta-, excitatory and depressant toxins of scorpions showed that Bsaul1 contains all the residues which are highly conserved among long chain scorpion neurotoxins. Structural model of Bsaul1 was generated using Ts1 (a beta-toxin that competes with the depressant insect toxins for binding to Na(+) channels) as template. According to the molecular model of Bsaul1, the folding of the polypeptide chain is being composed of an anti-parallel three-stranded beta-sheet and a stretch of alpha- helix, tightly bound by a set of four disulfide bridges. A striking similarity in the spatial arrangement of some critical residues was shown by superposition of the backbone conformation of Bsaul1 and Ts1.     

Binding characteristics of BmK I, an alpha-like scorpion neurotoxic polypeptide, on cockroach nerve cord synaptosomes.

In this study, the binding characteristics of BmK I, an alpha-like neurotoxic polypeptide purified from the venom of the Chinese scorpion Buthus martensi Karsch, were investigated on rat brain and cockroach nerve cord synaptosomes. The results showed that BmK I can bind to a single class of noninteracting binding sites on cockroach nerve cord synaptosomes with medium affinity (Kd = 16.5 +/ - 4.4 nM) and low binding capacity (Bmax = 1.05 +/- 0.23 pmol/mg protein), but lacks specific binding on rat brain synaptosomes. BmK AS, BmK AS-1 (two novel sodium channel-blocking ligands), BmK IT (an excitatory insect-selective toxin) and BmK IT2 (a depressant insect-selective toxin) from the same venom were found to be capable of depressing BmK I binding in cockroach nerve cord synaptosomes, which might be attributed to either allosteric modulation of voltage-gated Na+ channels by these toxic polypeptides or partial overlapping between the receptor binding sites of BmK I and these toxins. This thus supported the notion that alpha-like scorpion neurotoxic polypeptides bind to a distinct receptor site on sodium channels, which might be similar to the binding receptor site of alpha-type insect toxins, and also related to those of BmK AS type and insect-selective scorpion toxins on insect sodium channels.     

BjalphaIT: a novel scorpion alpha-toxin selective for insects--unique pharmacological tool

Long-chain neurotoxins derived from the venom of the Buthidae scorpions, which affect voltage-gated sodium channels (VGSCs) can be subdivided according to their toxicity to insects into insect-selective excitatory and depressant toxins (beta-toxins) and the alpha-like toxins which affect both mammals and insects. In the present study by the aid of reverse-phase HPLC column chromatography, RT-PCR, cloning and various toxicity assays, a new insect selective toxin designated as BjalphaIT was isolated from the venom of the Judean Black Scorpion (Buthotus judaicus), and its full primary sequence was determined: MNYLVVICFALLLMTVVESGRDAYIADNLNCAYTCGSNSYCNTECTKNGAVSGYCQWLGKYGNACWCINLPDKVPIRIPGACR (leader sequence is underlined). Despite its lack of toxicity to mammals and potent toxicity to insects, BjalphaIT reveals an amino acid sequence and an inferred spatial arrangement that is characteristic of the well-known scorpion alpha-toxins highly toxic to mammals. BjalphaITs sharp distinction between insects and mammals was also revealed by its effect on sodium conductance of two cloned neuronal VGSCs heterloguously expressed in Xenopus laevis oocytes and assayed with the two-electrode voltage-clamp technique. BjalphaIT completely inhibits the inactivation process of the insect para/tipE VGSC at a concentration of 100 nM, in contrast to the rat brain Na(v)1.2/beta1 which is resistant to the toxin. The above categorical distinction between mammal and insect VGSCs exhibited by BjalphaIT enables its employment in the clarification of the molecular basis of the animal group specificity of scorpion venom derived neurotoxic polypeptides and voltage-gated sodium channels.     

Purification of two depressant insect neurotoxins and their gene cloning from the scorpion Buthus martensi Karsch.

Insect-specific neurotoxins are important components of scorpion venoms. In this study, two toxins from the scorpion Buthus martensi Karsch (BmK) were purified. They shared high sequence homology with other depressant insect toxins and were designated BmK ITa and BmK ITb, respectively. They were able to suppress the action potential of cockroach isolated axon, which is due to a decrease in the peak sodium current. Furthermore, the effect of BmK ITb was lower than that of BmK ITa, and some of the electrophysiological characteristics of BmK ITb even resemble that of excitatory insect toxins. Their primary structures were determined by N-terminal partial sequence determination and cDNA cloning. The differences in their structures, especially the 31st residues, may result in the unique activity of BmK ITb.     

Action of different toxins from the scorpion Androctonus australis on a locust nerve-muscle preparation

The neuromuscular effects of four purified toxins and crude venom from the scorpion Androctonus australis were investigated in the extensor tibiae nerve-muscle preparation of the locust Locusta migratoria. Insect and crustacean toxin and the mammal toxins I and II which have previously been shown to act on fly larvae, isopods, and mice all paralyse locust larvae. The paralytic potencies decrease in the following order: insect toxin → mammal toxin I → crustacean toxin → mammal toxin II.The toxins and crude venom cause repetitive activity of the motor axons. This leads to long spontaneous trains of junction potentials in the case of crude venom and insect toxin. The other toxins chiefly cause short bursts of action and junction potentials following single stimuli.The ‘slow’ excitatory motor axon invariably is affected sooner than the inhibitory or the ‘fast’ excitatory one. The minimal doses of toxins required to affect the ‘slow’ motor axon decrease in an order somewhat different from that established for their paralytic potencies: insect toxin → crustacean toxin → mammal toxin I → mammal toxin II.Crude venom depolarises and destabilises the muscle membrane potential at low doses. At high doses it decreases the membrane resistance, whereas insect toxin leads to an increase.Crude venom and insect toxin enhance the frequency of mejps, whereas mammal toxin I leads to the occurrence of ‘giant’ mejps.The pattern of axonal activities indicates that the various peripheral branches of the motor nerve are the primary target of the toxins.The time course of nerve action potentials is affected by mammal toxin I and crustacean toxin which cause anomalous shapes and prolongations not caused by insect toxin.The results with other animals suggest that only the insect toxin is selective in its activity. The way it affects the axon might be quite different from that previously reported for scorpion venoms or toxins.     

Molecular cloning and nucleotide sequence analysis of encoded anti-insect toxin BotIT2 from the scorpion Buthus occitanus tunetanus venom

Numerous toxins from scorpion venoms are much more toxic to insects than to other animal classes, and possess high affinity to Na+ channels. Many of them active on insects were purified from the venom of Buthus occitanus tunetanus. Using amino acid sequences of BotIT2 and RACE-PCR amplification (Rapid amplification of cDNA ends) technique, we isolated, identified and sequenced the nucleotide sequence from the venom glands of the scorpion Buthus occitanus tunetanus. The cDNA encodes a precursor of an insect toxin of 60 amino acid residues. The deduced nucleotide sequence toxin was identical to the determined amino acid sequence of BotIT2. BotIT2 is more similar to the excitatory toxins in its mode of action and to the depressant toxins in its primary structure.     

Interactions of neurotoxins with the action potential Na+ ionophore

Four neurotoxins that activate the action potential Na⁺ ionophore of electrically excitable neuroblastoma cells interact with two distinct classes of sites, one specific for the alkaloids veratridine, batrachotoxin, and aconitine, and the second specific for scorpion toxin. Positive heterotropic cooperativity is observed between toxins bound at these two classes of sites. Tetrodotoxin, a specific inhibitor of the action potential Na⁺ current, inhibits activation by each of these toxins in a noncompetitive manner (K_I = 4–8 nM). These results suggest the existence of three functionally separable components of the action potential Na⁺ ionophore: two regulatory components, which bind activating neurotoxins and interact allosterically in controlling the activity of a third ion-transport component, which binds tetrodotoxin. The dissociation constant for scorpion toxin binding is increased 10-fold by depolarization of the cells with K⁺, suggesting that the scorpion toxin binding site is located on a voltage-sensitive regulatory component of the ionophore.     

Simultaneous modifications of sodium channel gating by two scorpion toxins.

The effects of purified scorpion toxins from two different species on the kinetics of sodium currents were evaluated in amphibian myelinated nerves under voltage clamp. A toxin from Leiurus quinquestriatus slowed and prevented sodium channel inactivation, exclusively, and a toxin from Centruroides sculpturatus Ewing reduced transient sodium currents during a maintained depolarization, and induced a novel inward current that appeared following repolarization, as previously reported by Cahalan (1975, J. Physiol. [Lond.]. 244:511-534) for the crude scorpion venom. Both of these effects were observed in fibers treated with both of these toxins, and the kinetics of the induced current were modified in a way that showed that the same sodium channels were modified simultaneously by both toxins. Although the toxins can act on different sites, the time course of the action of C. sculpturatus toxin was accelerated in the presence of the L. quinquestriatus toxin, indicating some form of interaction between the two toxin binding sites.     

Scorpion neurotoxin: Mode of action on neuromuscular junctions and synaptosomes

Electrophysiological analysis of the effects of scorpion toxin I, one of the neurotoxins from the venom of the scorpion Androctonus australis Hector, upon crayfish neuromuscular junctions has shown that the toxin strongly associates with the nerve terminal to stimulate release of neurotransmitters.The biochemical approach has shown that the binding of scorpion toxin I to rat brain synaptosomes is accompanied by a decrease in their capacity to accumulate γ-aminobutyric acid. The main effect of the toxin is to stimulate neurotransmitter release. The apparent dissociation constant of the toxin-receptor complex is 0.1–0.2 μM at 22 °C. The rate of dissociation is so slow that complex formation seems to be quasi-irreversible. The “quasi-irreversibility” has also been observed in electrophysiological experiments with the crayfish neuromuscular junction. Tetrodotoxin prevents scorpion toxin I action if it is incubated with synaptosomes or with crayfish neuromuscular junctions before scorpion toxin I application. Tetrodotoxin does not reverse scorpion toxin action if it is added to the preparation after scorpion toxin I. Prevention of scorpion toxin action by tetrodotoxin permits measurements of binding characteristics of this toxin to synaptosomes. The dissociation constant of the tetrodotoxin-receptor complex is 2.2 nM at 22 °C. No cooperativity is observed in the binding. Because of its high affinity for synaptosomes (and the “quasi-irreversibility” of the binding), scorpion toxin I appears to be a potentially excellent tool for further studies of the molecular mechanism of neurotransmitter secretion.     

Purification, characterization, and primary structure of four depressant insect-selective neurotoxin analogs from scorpion (Buthus sindicus) venom.

Four depressant insect-selective neurotoxin analogs (termed Bs-dprIT1 to 4) from the venom of the scorpion Buthus sindicus were purified to homogeneity in a single step using reverse-phase HPLC. The molecular masses of the purified toxins were 6820.9, 6892.4, 6714.7, and 6657.1 Da, respectively, as determined by mass spectrometry. These long-chain neurotoxins were potent against insects with half lethal dose values of 67, 81, 103, and 78 ng/100 mg larva and 138, 160, 163, and 142 ng/100 mg cockroach, respectively, but were not lethal to mice even at the highest applied dose of 10 microg/20 g mouse. When injected into blowfly larvae (Sarcophaga falculata), Bs-dprIT1 to 4 induced classical manifestations of depressant toxins, i.e., a slow depressant flaccid paralysis. The primary structures of Bs-dprIT 1 to 4 revealed high sequence homology (60-75%) with other depressant insect toxins isolated from scorpion venoms. Despite the high sequence conservation, Bs-dprIT1 to 4 showed some remarkable features such as (i) the presence of methionine (Met(6) in Bs-dprIT1 and Met(24) in Bs-dprIT2 to 4) and histidine (His(53) and His(57) in Bs-dprIT1) residues, i.e., amino acid residues that are uncommon to this type of toxin; (ii) the substitution of two highly conserved tryptophan residues (Trp43 --> Ala and Trp53 --> His) in the sequence of Bs-dprIT1; and (iii) the occurrence of more positively charged amino acid residues at the C-terminal end than in other depressant insect toxins. Multiple sequence alignment, sequence analysis, sequence-based structure prediction, and 3D homology modeling studies revealed a protein fold and secondary structural elements similar to those of other scorpion toxins affecting sodium channel activation. The electrostatic potential calculated on the surface of the predicted 3D model of Bs-dprIT1 revealed a significant positive patch in the region of the toxin that is supposed to bind to the sodium channel.     

An anti-insect toxin purified from the scorpion Androctonus australis Hector also acts on the alpha- and beta-sites of the mammalian sodium channel: sequence and circular dichroism study

A new anti-insect neurotoxin, AaH IT4, has been isolated from the venom of the North African scorpion Androctonus australis Hector. This polypeptide has a toxic effect on insects and mammals and is capable of competing with anti-insect scorpion toxins for binding to the sodium channel of insects; it also modulates the binding of alpha-type and beta-type anti-mammal scorpion toxins to the mammal sodium channel. This is the first report of a scorpion toxin able to exhibit these three kinds of activity. The molecule is composed of 65 amino acid residues and lacks methionine and, more unexpectedly, proline, which until now has been considered to play a role in the folded structure of all scorpion neurotoxins. The primary structure showed a poor homology with the sequences of other scorpion toxins; however, it had features in common with beta-type toxins. In fact, radioimmunoassays using antibodies directed to scorpion toxins representative of the main structural groups showed that there is a recognition of AaH IT4 via anti-beta-type toxin antibodies only. A circular dichroism study revealed a low content of regular secondary structures, particularly in beta-sheet structures, when compared to other scorpion toxins. This protein might be the first member of a new class of toxins to have ancestral structural features and a wide toxic range.     

Tetrodotoxin-insensitive sodium channels. Binding of polypeptide neurotoxins in primary cultures of rat muscle cells

The binding of 125I-labeled derivatives of scorpion toxin and sea anemone toxin to tetrodotoxin-insensitive sodium channels in cultured rat muscle cells has been studied. Specific binding of 125I-labeled scorpion toxin and 125I-labeled sea anemone toxin was each blocked by either native scorpion toxin or native sea anemone toxin. K0.5 for block of binding by several polypeptide toxins was closely correlated with K0.5 for enhancement of sodium channel activation in rat muscle cells. These results directly demonstrate binding of sea anemone toxin and scorpion toxin to a common receptor site on the sodium channel. Binding of both 125I-labeled toxin derivatives is enhanced by the alkaloids aconitine and batrachotoxin due to a decrease in KD for polypeptide toxin. Enhancement of polypeptide toxin binding by aconitine and batrachotoxin is precisely correlated with persistent activation of sodium channels by the alkaloid toxins consistent with the conclusion that there is allosteric coupling between receptor sites for alkaloid and polypeptide toxins on the sodium channel. The binding of both 125I-labeled scorpion toxin and 125I-labeled sea anemone toxin is reduced by depolarization due to a voltage-dependent increase in KD. Scorpion toxin binding is more voltage-sensitive than sea anemone toxin binding. Our results directly demonstrate voltage-dependent binding of both scorpion toxin and sea anemone toxin to a common receptor site on the sodium channel and introduce the 125I-labeled polypeptide toxin derivatives as specific binding probes of tetrodotoxin-insensitive sodium channels in cultured muscle cells.     

Genetic polymorphism and expression of a highly potent scorpion depressant toxin enable refinement of the effects on insect Na channels and illuminate the key role of Asn-58

We isolated from the venom of the scorpion Leiurus quinquestriatus hebraeus an extremely active anti-insect selective depressant toxin, Lqh-dprIT(3). Cloning of Lqh-dprIT(3) revealed a gene family encoding eight putative polypeptide variants (a-h) differing at three positions (37A/G, 50D/E, and 58N/D). All eight toxin variants were expressed in a functional form, and their toxicity to blowfly larvae, binding affinity for cockroach neuronal membranes, and CD spectra were compared. This analysis links Asn-58, which appears in variants a-d, to a toxin conformation associated with high binding affinity for insect sodium channels. Variants e-h, bearing Asp-58, exhibit a different conformation and are less potent. The importance of Asn-58, which is conserved in other depressant toxins, was further validated by construction and analysis of an N58D mutant of the well-characterized depressant toxin, LqhIT(2). Current and voltage clamp assays using the cockroach giant axon have shown that despite the vast difference in potency, the two types of Lqh-dprIT(3) variants (represented by Lqh-dprIT(3)-a and Lqh-dprIT(3)-e) are capable of blocking the action potentials (manifested as flaccid paralysis in blowfly larvae) and shift the voltage dependence of activation to more negative values, which typify the action of beta-toxins. Moreover, the stronger and faster shift in voltage dependence of activation and lack of tail currents observed in the presence of Lqh-dprIT(3)-a suggest an extremely efficient trapping of the voltage sensor compared to that of Lqh-dprIT(3)-e. The current clamp assays revealed that repetitive firing of the axon, which is reflected in contraction paralysis of blowfly larvae, can be obtained with either the less potent Lqh-dprIT(3)-e or the highly potent Lqh-dprIT(3)-a at more negative membrane potentials. Thus, the contraction symptoms in flies are likely to be dominated by the resting potential of neuronal membranes. This study clarifies the electrophysiological basis of the complex symptoms induced by scorpion depressant toxins in insects, and highlights for the first time molecular features involved in their activity.     

蝎β型昆虫毒素的结构及其与钠通道作用机制

蝎毒素是蝎为防卫的需要而产生的一系列活性短肽.其中蝎昆虫特异性毒素可特异性结合并调控昆虫可兴奋细胞膜上的钠离子通道,是研究离子通道结构与功能的首选探针,并在转基因抗虫植物及生物杀虫剂研究方面具有潜在的应用价值.本文对蝎β型昆虫毒素的结构与功能及其对钠离子通道的作用方式和β毒素的电压传感器捕获(voltage sensor-trapping)模型做一综述,为进一步揭示蝎β毒素的结构与功能的关系和在农作物抗虫领域的应用提供依据.     

Activation of the Voltage-Sensitive Sodium Channel by a β-Scorpion Toxin in Rat Brain Nerve-Ending Particles

Neurotoxins purified from scorpion venoms previously had been divided into two classes according to their binding properties in rat brain synaptosomes. However, the pharmacological action of beta-scorpion toxin (beta-ScTx) on this preparation has not yet been described. In this report we show that a beta-ScTx induced an increase in 22Na+ uptake through synaptosomal voltage-sensitive sodium channels since this stimulation was abolished by tetrodotoxin (TTX). The increase was smaller than with veratridine and no synergy was observed between beta-ScTx and veratridine, as is the case for alpha-scorpion toxin (alpha-ScTx) and veratridine. The effects of alpha- and beta-ScTx were additive and the concentration-effect curves for each type of toxin were not modified by the other, suggesting that these two types of toxins act through distinct and noninteracting receptor sites. This was confirmed by the absence of mutual modification of the equilibrium and kinetic binding properties. beta-ScTx was shown to inhibit the uptake and to stimulate the release of [3H]gamma-aminobutyric acid. These effects were blocked by TTX, and no synergy was observed with veratridine. It was concluded that all these effects are mediated by the activation of voltage-sensitive sodium channels induced by the binding of beta-ScTx to a receptor site (site 4) distinct from those for other neurotoxins acting on sodium channels.     

The complete amino acid sequence of toxin TsTX-VI isolated from the venom of the scorpionTityus serrulatus

The complete sequence of the toxin TsTX-VI from the venom of the scorpionTityus serrulatus Lutz and Mello is presented. The sequence has been determined by automated Edman analysis of the reduced and carboxymethylated protein as well as of the resulting peptides, obtained fromS. aureus protease and tryptic digestions. TsTX-VI is composed of 62 residues and has a calculated molecular weight of 6717. Homology studies with other scorpion toxins show that TsTX-VI is more similar to the Old World than to the North American scorpion toxins. The hydropathic index indicates that TsTX-VI is more hydrophobic than Ts-γ. Toxicity studies carried out in mice demonstrate that i.v. injection of TsTX-VI is unable to evoke the usual symptoms induced by the typical neurotoxins of this venom, but only a generalized allergic reaction. These properties are important in clarifying the relationship between primary structure and biological function of scorpion toxins.