A tissue culture assay for tetrodotoxin, saxitoxin and related toxins

In the presence of ouabain, veratridine enhances sodium influx in the mouse neuroblastoma cell line Neuro-2A (ATCC, CCL131), causing cellular swelling and subsequent death. Tetrodotoxin (puffer fish toxin) or saxitoxin (paralytic shellfish poison), both of which block the sodium channel of excitable membranes, antagonize this effect, enabling cell growth to continue. This phenomenon was used as the basis of a new assay for these toxins. It is also possible to estimate the quantity of TTX from the relationship between TTX concentration and percentage of living cells. This new method is simple, inexpensive, and sensitive, and may replace the conventional mouse bioassay.     

Binding characteristics of BmK I,an α‐like scorpion neurotoxic polypeptide,on cockroach nerve cord synaptosomes

Abstract: In this study, the binding characteristics of BmK I, an α‐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 (K_d = 16.5 ± 4.4 nm ) and low binding capacity (B_max= 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 α‐like scorpion neurotoxic polypeptides bind to a distinct receptor site on sodium channels, which might be similar to the binding receptor site of α‐type insect toxins, and also related to those of BmK AS type and insect‐selective scorpion toxins on insect sodium channels.     

Interaction of brevetoxin A with a new receptor site on the sodium channel

Measurements of neurotoxin-activated 22Na influx in neuroblastoma cells and neurotoxin binding in synaptosomes were used to define the site and mechanism of action of brevetoxins on sodium channels. Brevetoxin A alone did not enhance the sodium permeability of neuroblastoma cells, but markedly enhanced persistent activation of sodium channels by veratridine, aconitine and batrachotoxin, which act at neurotoxin receptor site 2. Enhancement of batrachotoxin action was accompanied by a 4.3-fold increase in the binding of [3H]batrachotoxinin A 20-alpha-benzoate to receptor site 2 on sodium channels in synaptosomes. These results point to an allosteric mechanism of brevetoxin action involving preferential binding to active states of sodium channels which have high affinity for neurotoxins, causing persistent activation of sodium channels at receptor site 2. Brevetoxin A does not block [3H]saxitoxin binding at neurotoxin receptor site 1 or 125I-labeled scorpion toxin binding at neurotoxin receptor site 3. The brevetoxins appear to act at a new neurotoxin receptor site on the sodium channel.     

New insight on scorpion divergence inferred from comparative analysis of toxin structure, pharmacology and distribution.

The divergence of Buthidae, the most abundant family of scorpions, has relied thus far on anatomical and morphological features, but still remains controversial. However, much information has accumulated on Buthidae long-chain scorpion toxins affecting neuronal sodium channel conductance (alpha- and beta-toxins) and their pharmacology. Therefore, we constructed a toxin evolutionary tree, which together with recent data on toxin gene organization, toxin structures, and worldwide dispersion, sheds light on toxin and hence, scorpion divergence. Based on these data, we suggest that in the ancient world, the ancestral long-chain toxins affecting sodium channels developed into beta-like toxins, which most likely developed into alpha- and beta-toxins before the separation of South America from Africa. Subsequently, in the Old World, mostly excitatory and depressant toxins developed from the ancestral beta-like toxin and in the New World a new type of toxin group with beta-toxin structure but alpha-toxin activity developed from the beta-toxins. Assisted by the worldwide distribution of toxins and the zoogeographical dispersion of the studied genera in Asia and Africa (Old World) and in South and North America (New World), we suggest a route of divergence for some of the Buthidae scorpions, a task that has reached a standstill when morphological and anatomical features were used.     

Mammalian skeletal muscle voltage-gated sodium channels are affected by scorpion depressant "insect-selective" toxins when preconditioned

Among scorpion beta- and alpha-toxins that modify the activation and inactivation of voltage-gated sodium channels (Na(v)s), depressant beta-toxins have traditionally been classified as anti-insect selective on the basis of toxicity assays and lack of binding and effect on mammalian Na(v)s. Here we show that the depressant beta-toxins LqhIT2 and Lqh-dprIT3 from Leiurus quinquestriatus hebraeus (Lqh) bind with nanomolar affinity to receptor site 4 on rat skeletal muscle Na(v)s, but their effect on the gating properties can be viewed only after channel preconditioning, such as that rendered by a long depolarizing prepulse. This observation explains the lack of toxicity when depressant toxins are injected in mice. However, when the muscle channel rNa(v)1.4, expressed in Xenopus laevis oocytes, was modulated by the site 3 alpha-toxin LqhalphaIT, LqhIT2 was capable of inducing a negative shift in the voltage-dependence of activation after a short prepulse, as was shown for other beta-toxins. These unprecedented results suggest that depressant toxins may have a toxic impact on mammals in the context of the complete scorpion venom. To assess whether LqhIT2 and Lqh-dprIT3 interact with the insect and rat muscle channels in a similar manner, we examined the role of Glu24, a conserved "hot spot" at the bioactive surface of beta-toxins. Whereas substitutions E24A/N abolished the activity of both LqhIT2 and Lqh-dprIT3 at insect Na(v)s, they increased the affinity of the toxins for rat skeletal muscle channels. This result implies that depressant toxins interact differently with the two channel types and that substitution of Glu24 is essential for converting toxin selectivity.     

Two recombinant depressant scorpion neurotoxins differentially affecting mammalian sodium channels

The scorpion depressant toxins are a group of evolutionarily conserved polypeptides targeting sodium channels, which show preferential ability to induce flaccid paralysis in insects, making them attractive candidates for the construction of transgenic plants or viral vectors to control pests. In this study, two new depressant toxin-like peptides (BmKITc and BmKITc2) differing only at position 52 (Lys for Thr) were produced in Escherichia coli. Circular dichroism analysis indicated that these two recombinant peptides display a typical structural feature similar to native scorpion toxins. They both cause a maintained current component at the last phase of inactivation of the insect sodium channel DmNav1/tipE expressed in Xenopus oocytes and interestingly, they do not produce a beta effect despite of their primary structure as beta-toxins. Furthermore, an inhibitory effect with BmKITc but not with BmKITc2 was observed on TTX-R sodium currents in rat DRG neurons. We hypothesize that such differential potency highlights a crucial role of lysine 52 in channel selectivity. Our results therefore indicate that, in spite of the general idea, not all scorpion depressant toxins interact with mammalian and/or insect sodium channels in the same manner.     

Immunological relationships of phospholipase A2 neurotoxins from snake venoms

Polyclonal rabbit antisera were raised against ten snake phospholipase A2 neurotoxins and one snake phospholipase A2 cytotoxin. Immunological cross-reactivities between these toxins, two other snake phospholipase A2 enzymes and pancreatic phospholipase A2 were studied using ELISA technology. All snake phospholipase A2 neurotoxins fell into two main antigenic classes. One antigenic class was composed of all the elapid toxins tested (textilotoxin, taipoxin, notexin, pseudexin and beta-bungarotoxin), the cytotoxic phospholipase A2 from Naja naja atra and pancreatic phospholipase A2. beta-Bungarotoxin seemed to be in an immunological subclass of its own compared to the rest of the elapid toxins. The second antigenic class was comprised of crotalid and viperid phospholipase A2 neurotoxins (crotoxin, concolor toxin, Mojave toxin, vegrandis toxin, ammodytoxin and caudoxin). Our data indicated that the viperid toxins, caudoxin and ammodytoxin, were an immunological subclass apart from the crotalid toxins.     

SCORPION2: a database for structure-function analysis of scorpion toxins.

Scorpion toxins are important experimental tools for characterization of vast array of ion channels and serve as scaffolds for drug design. General public database entries contain limited annotation whereby rich structure-function information from mutation studies is typically not available. SCORPION2 contains more than 800 records of native and mutant toxin sequences enriched with binding affinity and toxicity information, 624 three-dimensional structures and some 500 references. SCORPION2 has a set of search and prediction tools that allow users to extract and perform specific queries: text searches of scorpion toxin records, sequence similarity search, extraction of sequences, visualization of scorpion toxin structures, analysis of toxic activity, and functional annotation of previously uncharacterized scorpion toxins. The SCORPION2 database is available at http://sdmc.i2r.a-star.edu.sg/scorpion/.     

Decrease in toxicity of microcystins LA and LR in drinking water by ozonation

Unchlorinated treated waters from two Australian reservoirs were spiked with microcystin-LA and -LR extracted from a toxic scum of Microcystis aeruginosa. The two waters had considerably different water quality and therefore ozone demands. The spiked sample waters were ozonated using the batch method of ozonation at a range of doses and the samples were analysed for toxins using high-performance liquid chromatography (HPLC). The toxin content of the samples was also determined using a protein phosphatase type 2A inhibition assay (PP2A) and toxicity via the standard mouse bioassay. The HPLC results correlated well with the PP2A results and toxicity tests for both waters. A loss of both toxins and toxicity was observed with increasing ozone dose, resulting in a complete loss of toxicity for both waters once an ozone residual had been achieved. At this ozone residual no toxin was detected using HPLC. The results indicate that microcystins are not transformed into toxic by-products.     

Positively selected sites of scorpion depressant toxins: possible roles in toxin functional divergence.

Scorpion depressant toxins represent a distinct pharmacological group of sodium channel neurotoxins, identified by their preferential ability in induction of depressant and flaccid paralysis of insects. However, recent observations that some members in this group exhibit anti-mammal activity raise an interesting evolutionary question of whether it is a consequence of adaptive evolution to the early radiation of mammals on earth. By employing the maximum likelihood method, we provided convincing statistical evidence in favor of positive selection driving the evolution of the depressant toxins, and found that two of three positively selected sites are located on the functional surface of the toxins. A complex model of the scorpion depressant toxin LqhIT2 binding to insect sodium channel alpha-subunit (DmNav1) was constructed by structural bioinformatics approaches which highlights a possible direct interaction between these two sites and insect sodium channels. Our work presented here thus suggests that accelerated substitutions in these site residues could offer an evolutionary advantage for these toxins to adapt different channels from diverse origins.     

A rapid hemolysis assay for the detection of sodium channel-specific marine toxins

Current methods of detection for fish and shellfish biotoxins in monitoring and research purposes are either labor intensive, expensive, require specialized techniques or all of the above. This paper reports on the development of a fairly sensitive, rapid, and inexpensive assay which detects the presence of compounds that affect the sodium channel. It is based on the principles of the mouse neuroblastoma tissue culture assay for sodium channel specific-biotoxins using red blood cells (RBCs) from the red tilapia (Sarotherodon mossambicus). This assay has the potential to complement the use of live animal bioassay testing for marine toxins. Veratridine, a sodium channel activator and ouabain, an inhibitor of Na(+)/K(+) ATPase, both react with the tilapia RBCs by affecting the permeability of the cell's membrane. Saxitoxin (STX), its analogs, and tetrodotoxin (TTX) can inhibit the action of veratridine and ouabain leaving the cell morphologically normal. By sequencing the addition of veratridine and ouabain, with either the extracted samples, saxitoxin, tetrodotoxin, or ciguatoxin (CTX-a sodium channel activator) to the RBCs a sodium channel antagonist or activator can be detected. Results using pure concentrations of a sodium channel-specific toxin could be detected to inhibit hemolysis at a concentration of 0.3 microg/ml STX, 3.5 microg/ml for neo-STX, 3.0 microg/ml for GTX, and 5.0 microgl for TTX in the presence of ouabain and veratridine. CTX was detected at a concentration of 50 microg/ml. The RBCs from the red tilapia was used due to the fish's ability to osmoregulate its internal environment to survive in both fresh and saltwater. In addition, with growing opposition to live animal testing, this assay has been designed as a non-lethal means of testing for sodium channel affecting marine toxins. No test animals are sacrificed and blood may be drawn from the same fish for continued sample testing.     

Purification, molecular cloning and functional characterization of HelaTx1 (Heterometrus laoticus): the first member of a new κ-KTX subfamily

Given their medical importance, most attention has been paid toward the venom composition of scorpions of the Buthidae family. Nevertheless, research has shown that the venom of scorpions of other families is also a remarkable source of unique peptidyl toxins. The κ-KTx family of voltage-gated potassium channel (VGPC) scorpion toxins is hereof an example. From the telson of the scorpion Heterometrus laoticus (Scorpionidae), a peptide, HelaTx1, with unique primary sequence was purified through HPLC and sequenced by Edman degradation. Based on the amino acid sequence, the peptide could be cloned and the cDNA sequence revealed. HelaTx1 was chemically synthesized and functionally characterized on VGPCs of the Shaker-related, Shab-related, Shaw-related and Shal-related subfamilies. Furthermore, the toxin was also tested on small- and intermediate conductance Ca(2+)-activated K(+) channels. From the channels studied, K(v)1.1 and K(v)1.6 were found to be the most sensitive (K(v)1.1 EC(50)=9.9±1.6 μM). The toxin did not alter the activation of the channels. Competition experiments with TEA showed that the toxin is a pore blocker. Mutational studies showed that the residues E353 and Y379 in the pore of K(v)1.1 act as major interaction points for binding of the toxin. Given the amino acid sequence, the predicted secondary structure and the biological activity on VGPCs, HelaTx1 should be included in the κ-KTX family. Based on a phylogenetic study, we rearranged this family of VGPC toxins into five subfamilies and suggest that HelaTx1 is the first member of the new κ-KTx5 subfamily.     

Biochemical and molecular characterization of the venom from the Cuban scorpion Rhopalurus junceus

This communication describes the first general biochemical, molecular and functional characterization of the venom from the Cuban blue scorpion Rhopalurus junceus, which is often used as a natural product for anti-cancer therapy in Cuba. The soluble venom of this arachnid is not toxic to mice, injected intraperitoneally at doses up to 200 μg/20 g body weight, but it is deadly to insects at doses of 10 μg per animal. The venom causes typical alpha and beta-effects on Na⁺ channels, when assayed using patch-clamp techniques in neuroblastoma cells in vitro. It also affects K⁺ currents conducted by ERG (ether-a-go-go related gene) channels. The soluble venom was shown to display phospholipase, hyaluronidase and anti-microbial activities. High performance liquid chromatography of the soluble venom can separate at least 50 components, among which are peptides lethal to crickets. Four such peptides were isolated to homogeneity and their molecular masses and N-terminal amino acid sequence were determined. The major component (RjAa12f) was fully sequenced by Edman degradation. It contains 64 amino acid residues and four disulfide bridges, similar to other known scorpion toxins. A cDNA library prepared from the venomous glands of one scorpion allowed cloning 18 genes that code for peptides of the venom, including RjA12f and eleven other closely related genes. Sequence analyses and phylogenetic reconstruction of the amino acid sequences deduced from the cloned genes showed that this scorpion contains sodium channel like toxin sequences clearly segregated into two monophyletic clusters. Considering the complex set of effects on Na⁺ currents verified here, this venom certainly warrant further investigation.     

The differential preference of scorpion alpha-toxins for insect or mammalian sodium channels: implications for improved insect control.

Receptor site-3 on voltage-gated sodium channels is targeted by a variety of structurally distinct toxins from scorpions, sea anemones, and spiders whose typical action is the inhibition of sodium current inactivation. This site interacts allosterically with other topologically distinct receptors that bind alkaloids, lipophilic polyether toxins, pyrethroids, and site-4 scorpion toxins. These features suggest that design of insecticides with specificity for site-3 might be rewarding due to the positive cooperativity with other toxins or insecticidal agents. Yet, despite the central role of scorpion alpha-toxins in envenomation and their vast use in the study of channel functions, molecular details on site-3 are scarce. Scorpion alpha-toxins vary greatly in preference for sodium channels of insects and mammals, and some of them are highly active on insects. This implies that despite its commonality, receptor site-3 varies on insect vs. mammalian channels, and that elucidation of these differences could potentially be exploited for manipulation of toxin preference. This review provides current perspectives on (i) the classification of scorpion alpha-toxins, (ii) their mode of interaction with sodium channels and pharmacological divergence, (iii) molecular details on their bioactive surfaces and differences associated with preference for channel subtypes, as well as (iv) a summary of the present knowledge about elements involved in constituting receptor site-3. These details, combined with the variations in allosteric interactions between site-3 and the other receptor sites on insect and mammalian sodium channels, may be useful in new strategies of insect control and future design of anti-insect selective ligands.     

Full characterization of three toxins from the Androctonus amoreuxi scorpion venom

In this study, we have characterized the immunological and pharmacological properties of the three major α-type toxins from the scorpion Androctonus amoreuxi, AamH1, AamH2 and AamH3, which were previously described as putative toxins from cDNAs [Chen, T. et al., 2003. Regul. Pept. 115, 115-121].The immunological tests (ELISA, RIA) have demonstrated that AamH1, AamH2 and AamH3 belong to the immunological groups 3 and 4 of α-type toxins. Analysis of the three toxin effects on currents through rat brain (rNav1.2), rat muscle (rNav1.4) and Drosophila (DmNav1) sodium channels expressed in Xenopus oocytes revealed that AamH1 and AamH2, but not AamH3, have anti-insect and anti-mammal activities and can be classified as α-like toxins. While AamH1 removes fast inactivation only in neuronal rNav1.2 channel and has no effect on muscular rNav1.4 channel, AamH2 affects both neuronal rNav1.2 and muscular rNav1.4 channels. AamH3 was lethal to mice by intracerebroventricular injection despite its lack of activity on the neuronal rNav1.2 channel. Finally, we have shown that the A. amoreuxi venom was better neutralized by the antiserum raised against the venom of Buthus occitanus tunetanus than by the antisera raised against scorpion venoms from the same genus Androctonus.     

Effects of beta-bungarotoxin and Naja naja atra snake venom phospholipase A2 on acetylcholine release and choline uptake in synaptosomes

Beta-bungarotoxin is a potent presynaptically acting snake venom toxin that exhibits phospholipase A2 activity. We compared the effects of beta-bungarotoxin and a less toxic snake venom phospholipase A2 on synaptosomal 3H-acetylcholine release and 3H-choline uptake. The purpose of these experiments was to study the mode by which beta-bungarotoxin inhibits 3H-acetylcholine release in this preparation. Under non-depolarizing conditions, both beta-bungarotoxin and Naja naja atra phospholipase A2 stimulated 3H-acetylcholine release from a synaptosomal fraction preloaded with 3H-choline. Beta-bungarotoxin was more potent, but less efficacious, than N. naja atra phospholipase A2. In contrast, both toxins inhibited 3H-acetylcholine release from the synaptosomal fraction incubated with 3H-choline after toxin exposure. In agreement with the results obtained by monitoring acetylcholine release, beta-bungarotoxin and N. naja atra phospholipase A2 appeared to block 3H-choline uptake into the synaptosomal fraction non-competitively. Although the toxins may cause the release of unlabeled choline from synaptosomes, the block of labeled choline uptake could not be explained by decreased specific activity of 3H-choline in the bathing medium. Therefore, beta-bungarotoxin and N. naja atra phospholipase A2 block 3H-acetylcholine release from synaptosomes indirectly by inhibiting the uptake of 3H-choline necessary for 3H-acetylcholine synthesis. In comparing these results using 3H-choline to those in the literature obtained with deuterated choline, there appears to be a difference in apparent toxin action that relates to the type of label (3H or 2H) attached to choline.     

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.