HgeTx1, the first K-channel specific toxin characterized from the venom of the scorpion Hadrurus gertschi Soleglad

A novel toxin was identified, purified and characterized from the venom of the Mexican scorpion Hadrurus gertschi (abbreviated HgeTx1). It has a molecular mass of 3950 atomic mass units (a.m.u.) and contains 36 amino acids with four disulfide bridges established between Cys1–Cys5, Cys2–Cys6, Cys3–Cys7 and Cys4–Cys8. It blocks reversibly the Shaker B K⁺-channels with a Kd of 52 nM. HgeTx1 shares 60%, 45% and 40% sequence identity, respectively, with Heterometrus spinnifer toxin1 (HsTX1), Scorpio maurus K⁺-toxin (maurotoxin) and Pandinus imperator toxin1 (Pi1), all four-disulfide bridged toxins. It is 57–58% identical with the other scorpion K⁺-channel toxins that contain only three disulfide bridges. Sequence comparison, chain length and number of disulfide bridges analysis classify HgeTx1 into subfamily 6 of the -KTx scorpion toxins (systematic name: -KTx 6.14).     

Identification of a mammalian target of κM-conotoxin RIIIK

Despite the great variability of the conus peptides characterized until now only relatively few have been identified that interact with K⁺ channels. κM-conotoxin RIIIK (κM-RIIIK) is a 24 amino acid peptide from Conus radiatus, which is structurally similar to μ-conotoxin GIIIA, a peptide known to block specifically skeletal muscle Na⁺ channels. Recently, it has been shown that κM-RIIIK does not interact with Na⁺ channels, but inhibits Shaker potassium channels expressed in Xenopus oocytes. It was demonstrated that κM-RIIIK binds to the pore region of Shaker channels and a teleost homologue of the Shaker channel TSha1 was identified as a high affinity target of the toxin. In contrast the mammalian Shaker-homologues Kv1.1, Kv1.3, Kv1.4 are not affected by the toxin. In this study the activity of κM-RIIIK on other mammalian Kv1 K⁺ channels expressed in Xenopus oocytes was investigated. We demonstrate that κM-conotoxin RIIIK up to 5 μM exhibits no significant effect on Kv1.5 and Kv1.6 mediated currents, but the human Kv1.2 K⁺ channel is blocked by this peptide. The binding of κM-RIIIK to Kv1.2 channels is state dependent with an IC₅₀ for the closed state of about 200 nM and for the open state of about 400 nM at a test potential of 0 mV. κM-conotoxin RIIIK is the first conotoxin described to block human Kv1.2 potassium channels.     

The effect of the venom of the yellow Iranian scorpion Odontobuthus doriae on skeletal muscle preparations in vitro

The yellow Iranian scorpion Odontobuthus doriae can cause fatal envenoming, but its mechanism of action is unclear. One of the reported manifestations of envenoming is moderate to severe involuntary tremor of skeletal muscle. In order to understand better the mechanism of action of this venom on skeletal muscle function, we examined the effects of the venom in vitro on chick biventer cervicis (CBC) and mouse hemidiaphragm (MHD) nerve muscle preparations. O. doriae venom (0.3–10 μg/ml) initially increased and then decreased twitch height. The venom also caused contracture in both preparations. In mouse triangularis sterni preparations, used for all intracellular recording techniques, the venom enhanced the release of acetylcholine and induced repetitive firing of nerve action potentials and endplate potentials in response to single-shock stimulation. With extracellular recording techniques, scorpion venom (1 μg/ml) was found to cause changes to the perineural waveform associated with nerve terminal action potentials consistent with effects on Na⁺ and K⁺ currents. The main facilitatory effects of O. doriae venom are likely to be due to toxins that affect Na⁺ channels in nerve–muscle preparations similar to most Old World scorpion venoms, but blocking effects on K⁺ channels are also possible. Such effects could lead to initial enhancement of transmitter release that could underlie the muscle tremors seen in victims. Toxins acting on Na⁺ and K+ currents have been isolated from the venom [Jalali, A., Bosmans, F., Amininasab, M., Clynen, E., Cuypers, E., Zaremirakabadi, A., Sarbolouki, M.N., Schoofs, L., Vatanpour, H., Tytgat, J., 2005. OD1, the first toxin isolated from the venom of the scorpion Odontobuthus doriae active on voltage-gated Na⁺ channels. FEBS Lett. 579, 4181–4186; Abdel-Mottaleb, Y., Clynen, E., Jalali, A., Bosmans, F., Vatanpour, H., Schoofs, L., Tytgat, J., 2006. The first potassium channel toxin from the venom of the Iranian scorpion Odontobuthus doriae. FEBS Lett. 580, 6254–6258]; however, the muscle paralysis seen at higher concentrations of venom may be due to additional, as yet uncharacterised, components of the venom.     

Definition of the alpha-KTx15 subfamily

Three novel scorpion toxins, Aa1 from Androctonus australis, BmTX3 from Buthus martensi and AmmTX3 from Androctonus mauretanicus were shown able to selectively block A-type K⁺ currents in cerebellum granular cells or cultured striatum neurons from rat brain. In electrophysiology experiments, the transient A-current completely disappeared when 1 μM of the toxins was applied to the external solution whereas the sustained K⁺ current was unaffected.

The three toxins shared high sequence homologies (more than 94%) and constituted a new ‘short-chain’ scorpion toxin subfamily: -KTx₁₅. Monoiododerivative of ¹²⁵I-sBmTX3 specifically bound to rat brain synaptosomes. Under equilibrium binding conditions, maximum binding was 14 fmol/mg of protein and the dissociation constant (K_d) was 0.21 nM. This K_d value was confirmed by kinetic experiments (k_on=6.0×10⁶ M⁻¹ s⁻¹ and k_off=6.0×10⁻⁴ s⁻¹). Competitions with AmmTX3 and Aa1 with ¹²⁵I-sBmTX3 bound to its receptor on rat brain synaptosomes showed that they fully inhibited the ¹²⁵I-sBmTX3 binding (K_i values of 20 and 44 pM, respectively), demonstrating unambiguously that the three molecules shared the same target in rat brain. A panel of toxins described as specific ligands for different K⁺, Na⁺ and Ca²⁺ channels were not able to displace ¹²⁵I-sBmTX3 from its binding site. Thus, ¹²⁵I-sBmTX3 is a new ligand for a still unidentified target in rat brain. In autoradiography, the distribution of ¹²⁵I-sBmTX3 binding sites in the adult rat brain indicated a high density of ¹²⁵I-sBmTX3 receptors in the striatum, hippocampus, superior colliculus, and cerebellum.     

Partial structural determination of hepatotoxic peptides from Microcystis aeruginosa (cyanobacterium) collected in ponds of central China

, , , and . Partial structural determination of hepatotoxic peptides from Microcystis aeruginosa (cyanobacterium) collected in ponds of central China. Toxicon 26, 1213–1217, 1988.—Waterbloom samples of the colonial cyanobacterium Microcystis aeruginosa, collected in fish ponds at the Hydrobiological Institute, Wuhan, People's Republic of China, were hepatotoxic to mice. Lyophilized cells had an ₅₀ (i.p. mouse; 40 mg/kg) and signs of poisoning similar to that reported for other cyanobacterial hepatotoxic peptides. Two toxins, with an ₅₀ (i.p. mouse) of 40 and 150 μg/kg, were isolated using gel filtration and high performance liquid chromatography. The amino acid composition and mol.wt (994) of the 40 μg/kg toxin was the same as that for microcystin-LR, while the 150 μg/kg toxin had an amino acid composition and mol.wt (1048) different from any of the reported cyanobacteria heptapeptide toxins reported to date.     

A novel scorpion toxin blocking small conductance Ca activated K channel

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 -KTxs but showed close relationship with SK channel blockers in the phylogenetic tree. According to the previous nomenclature, BmSKTx1 was classified as -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 K_d of 0.72 μM 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.     

Transfer and metabolism of paralytic shellfish poisoning from scallop (Chlamys nobilis) to spiny lobster (Panulirus stimpsoni)

The transfer and transformation of paralytic shellfish poisoning (PSP) from scallop Chlamys nobilis to spiny lobster Panulirus stimpsoni were investigated in the present study. The results demonstrate that transfer and transformation of PSP toxins occurred when Panulirus stimpsoni were fed with toxic viscera of Chlamys nobilis, but depurated with non-toxic squids. Additionally, only the lobster hepatopancreas were found to contain PSP, and the toxin profiles were the same with those in the viscera of the scallop, including carbamate toxins (GTX₁₋₃), N-sulfocarbamoyl toxins (C₁₊₂ and B₁) and decarbamoyl toxins (dcGTX₂₊₃). Unlike the lobster, the scallop contained more than β toxins. After being fed with toxic Chlamys nobili for 6 d, Panulirus stimpsoni selectively accumulated N-sulfocarbamoyl toxins with low toxicity. However, when they were depurated with non-toxic squid, N-sulfocarbamoyl toxins tended to transform into carbamate toxins with higher toxicity. The concentration of dcGTX₂₊₃ in Panulirus stimpsoni decreased significantly and wasn’t detectable after depuration for 6 d, which was likely due to their initial low accumulation of toxins. These results reveal that PSP could be transferred and transformed in Crustaceans along the given food chain under the conditions of laboratory, but there are many questions remained to be solved, and the further studies should be carried out.     

Isolation and characterization of four toxins from the blue-green alga, Microcystis aeruginosa

—Two alternative procedures for the isolation of toxins from the blue-green alga, Microcystis aeruginosa forma aeruginosa, are described. A novel approach is reported, whereby contaminating impurities are succinylated, exploiting the absence of free amino groups in toxin variants. All toxin variants comprise a hydrocarbon blocking group, five amino acid residues detectable by conventional means, while methylamine is liberated upon acid hydrolysis. Possible structural features are discussed relating to the observed chemical and physical properties of the toxins.     

Amino acid sequence and physiological characterization of toxins from the venom of the scorpion Centruroides limpidus tecomanus Hoffmann

The complete amino acid sequence of the major toxic component (II.20.3.4), named toxin 1, from the venom of the Mexican scorpion C. l. tecomanus is reported. The sequence (66 amino acids) was obtained by direct Edman degradation of reduced and alkylated toxin, followed by sequence determination of selected peptides separated after enzymatic cleavage with S. aureus V8 protease. In cultured chick dorsal root ganglion cells, 0.5 microM toxin 1 slowed down specifically the time course of Na+ current inactivation, while Ca2+ currents from the same preparation were little affected. In neonatal rat ventricular heart cells, toxin 1, at concentrations between 0.1 and 0.5 microM, reduced Na+ currents without changing the kinetics and Ca2+ currents were unaffected. Comparative analysis of the primary structure of this toxin with other scorpion toxins shows a high degree of similarity with the north American scorpion toxins. This analysis suggests that the 'fine tuning' of the molecular mechanism of action of these toxins is related to variations in the primary structure as well as to the type of membrane under study (tissue specificity).     

Overview of scorpion toxins specific for Na channels and related peptides: biodiversity, structure–function relationships and evolution

Scorpion venoms contain a large number of bioactive components. Several of the long-chain peptides were shown to be responsible for neurotoxic effects, due to their ability to recognize Na⁺ channels and to cause impairment of channel functions. Here, we revisited the basic paradigms in the study of these peptides in the light of recent data concerning their structure–function relationships, their functional divergence and extant biodiversity. The reviewed topics include: the criteria for classification of long-chain peptides according to their function, and a revision of the state-of-the-art knowledge concerning the surface areas of contact of these peptides with known Na⁺ channels. Additionally, we compiled a comprehensive list encompassing 191 different amino acid sequences from long-chain peptides purified from scorpion venoms. With this dataset, a phylogenetic tree was constructed and discussed taking into consideration their documented functional divergence. A critical view on problems associated with the study of these scorpion peptides is presented, drawing special attention to the points that need revision and to the subjects under intensive research at this moment, regarding scorpion toxins specific for Na⁺ channels and the other related long-chain peptides recently described.     

Binding domain of oligomycin on Na(+),K(+)-ATPase

Oligomycin inhibits Na⁺,K⁺-ATPase activity by stabilizing the Na⁺ occlusion but not the K⁺ occlusion. To locate the binding domain of oligomycin on Na⁺,K⁺-ATPase, the tryptic-digestion profile of Na⁺,K⁺-ATPase was compared with the profile of Na⁺ occlusion within the digested Na⁺,K⁺-ATPase in the presence of oligomycin. The Na⁺ occlusion profile is responsible for the digestion profile of the -subunit, which is the catalytic subunit of the ATPase. The effect of oligomycin on chimeric Ca²⁺-ATPase activity was examined. The chimera used, in which the 163 N-terminal amino acids of chicken sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase 1 were replaced with the 200 N-terminal amino acids of the chicken Na⁺,K⁺-ATPase 1-subunit, partially retains the Na⁺-dependent characteristics of Na⁺,K⁺-ATPase, because the chimeric Ca²⁺-ATPase activity is activated by Na⁺ but inhibited by ouabain, a specific inhibitor of Na⁺,K⁺-ATPase (Ishii, T., Lemas, M.V., Takeyasu, K., 1994, Proc. Natl. Acad. Sci. U. S. A., 91, 6103–6107). Oligomycin depressed the activation by Na⁺ of the chimeric Ca²⁺-ATPase activity. These findings suggest that the 200 N-terminal amino acids of the Na⁺,K⁺-ATPase -subunit include a binding domain for oligomycin.     

The effect of the myotoxic Lys49 phospholipase A2 from Agkistrodon contortrix laticinctus snake venom on Na/K-ATPase activity of toad bladders

ACLMT is a myotoxic Lys49 phospholipase A₂ isolated from the venom of the snakeAgkistrodon contortrix laticinctus. We have previously shown that ACLMT increases baseline water transport and partially inhibits vasopressin-stimulated water transport across toad bladders due to an increase in cytosolic calcium. However, these evidences provide insufficient insight into the mechanisms involved in the effects of ACLMT on membrane permeability. In an attempt to better understand such mechanisms, the current study aimed to investigate whether the Na⁺/K⁺-ATPase activity of isolated toad bladders can be affected by the ACLMT and the synthetic peptide from its C-terminal region. The toxin significantly decreased the Na⁺/K⁺-ATPase, while the peptide did not alter it. These findings suggest that the effects of ACLMT on membrane permeability may be due to the inhibition of the Na⁺/K⁺-ATPase activity, and that the C-terminal region may not play a relevant role in this effect. This study contributes toward a better understanding of the mechanisms involved in the toxicity of the snake venom Lys49 PLA₂ myotoxins on biological tissues.     

Development of an in vitro drug screening system for Mycobacterium leprae based on the determination of the intrabacterial sodium to potassium ratio of individual bacterial organisms

In vitro drug effects on Mycobacterium leprae (M. leprae) in a cell-free system have been monitored by mass spectrometric determination of the ratio of the intrabacterial concentrations of the sodium and potassium ions (Na⁺, K⁺ ratio) of a limited number of individual bacteria per sample. From the drug-induced increase of the median values of the distributions of the Na⁺, K⁺ ratio, information on the concentration and time dependence of drug effects as well as on antagonistic or synergistic interactions of drugs has been obtained. Moreover, absolute values for the percentage of killed bacteria (% kill) have been derived from the distribution of the Na⁺, K⁺ ratios within a bacterial population. For this, the limiting value of the Na⁺, K⁺ ratio (up to which bacteria are viable) —which had been determined as 0.45 for cultivable bacteria — has been presumed to be valid also for M. leprae. Highest killing rates have been observed for fusidic acid and clarithromycin, followed by rifabutine, rifampin, and clofazimine. Minocycline and dapsone have shown only moderate killing effects and isoniazid and — probably due to the restricted metabolism of M. leprae in a cell-free medium — ofloxacin have been completely inactive. Strong ofloxacin effects, however, have been observed for cultivable mycobacteria and intracellular M. leprae phagocytized by a murine macrophage cell line.     

TsTX-IV, a short chain four-disulfide-bridged neurotoxin from Tityus serrulatus venom which acts on Ca2+-activated K+ channels.

The primary structure of TsTX-IV, a neurotoxin isolated from Tityrus serrulatus scorpion venom, is reported. Its amino acid sequence was determined by automated Edman sequential degradation of the reduced and carboxymethylated toxin and of relevant peptides obtained by digestion with Staphylococcus aureus strain V8 protease or trypsin and cleavage by CNBr. The complete sequence showed 41 amino acid residues, which account for an estimated molecular weight of 4520, and eight half-cystine residues which cross-link the toxin molecule with four disulfide bonds. The molecular weight determined by mass spectrometry was 4518. Comparison of this sequence with those from other scorpion toxins showed a resemblance with toxins which act on different types of K+ channels. TsTx-IV was able to block Ca2+-activated K+ channels of high conductance. TsTX-IV is the first four-disulfide-bridged short toxin from T. serrulatus so far completely sequenced.     

Characterization of PsTX-60B, a new membrane-attack complex/perforin (MACPF) family toxin, from the venomous sea anemone Phyllodiscus semoni

The sea anemone Phyllodiscus semoni has been known as one of the most venomous sea anemones. Previously, we reported the isolation of the major lethal protein toxins, PsTX-60A and PsTX-60B, from P. semoni and the primary structure of PsTX-60A. In this paper, we report the complete sequence of cDNA (1600 base pairs) encoding PsTX-60B and the deduced primary structure (488 amino acids) of PsTX-60B. The amino acid sequence of PsTX-60B showed the homology with those of PsTX-60A and Actineria villosa toxin, AvTX-60A. The results showed that PsTX-60B is a new member of the membrane-attack complex/perforin (MACPF) family toxin.     

Amylosin from Bacillus amyloliquefaciens, a K and Na channel-forming toxic peptide containing a polyene structure

Bacillus amyloliquefaciens strains isolated from the indoor environment of moisture-damaged buildings produce a 1197 Da toxin, named amylosin. Nuclear magnetic resonance (NMR) data showed that amylosin contains a chromophoric polyene structure and the amino acids leucine/isoleucine, proline, aspartic acid/asparagine, glutamic acid/glutamine and tyrosine. A quantitation method for amylosin was developed using commercially available amphotericin B as a reference compound and a known concentration of amylosin determined by NMR with the electronic reference to access in vivo concentration (ERETIC) method. Purified amylosin inhibited motility of boar sperm cells at an exposure concentration of 135 nM and hyperpolarized their cell membrane and depolarized their mitochondria at exposure to concentration of 33–67 nM for 10 min. In a 3-d exposure time only 27 nM of amylosin was needed to provoke the same toxicity functions. Amylosin was cytotoxic to feline lung cells at concentrations of . Purified amylosin provoked adenosine 5′-triphosphate (ATP)-independent cation influx into isolated rat liver mitochondria (RLM), inducing swelling of the mitochondria at concentrations of 200 nM K⁺ or >250 nM Na⁺ medium. In the K⁺- or Na⁺-containing medium, amylosin uncoupled RLM, causing oxidation of pyridine nucleotides (PN), loss of the mitochondrial membrane potential, and suppressed ATP synthesis. Purified amylosin produced cation channels in black-lipid membranes (BLMs) with a selectivity K⁺>Na⁺ at a concentration of 26 nM, i.e. the same concentration at which amylosin was toxic to boar sperm cells. The amylosin cation channels were cholesterol- and ATP-independent and more effective with K⁺ than with Na⁺. We propose that the toxicity of amylosin may be due its ionophoric properties, representing the first K⁺/Na⁺ channel-forming substance reported from B. amyloliquefaciens.     

Non-selective retention of PSP toxins by the mussel Mytilus galloprovincialis fed with the toxic dinoflagellate Alexandrium tamarense.

Mussels, Mytilus galloprovincialis, were contaminated by paralytic shellfish poisoning (PSP) toxins by being fed with the toxic dinoflagellate Alexandrium tamarense. Temporal variations in the toxin content and the profile of mussels during the feeding experiment were monitored by high-performance liquid chromatography (HPLC). The toxin profile of mussels was compared with that of A. tamarense to clarify the mechanism of uptake of toxins in mussels. The prominent toxins in mussels and A. tamarense were N-sulfocarbamoyl toxins (C1,2) and carbamate toxins, gonyautoxin-1,4 (GTX1,4). The toxin profiles of both mussels and A. tamarense were almost constant throughout the experimental period. There were no remarkable differences in the toxin proportion between mussel and A. tamarense. These results indicate that mussels do not selectively accumulate particular toxins.     

Effect of oral Saccharomyces boulardii treatment on the activity of Clostridium difficile toxins in mouse digestive tract.

Human antibiotic-associated diarrhoea and pseudomembranous colitis are partly due to toxin production by Clostridium difficile. It is now well documented that Saccharomyces boulardii protects against C. difficile induced diseases. In an attempt to understand better the mechanism of this protective effect, the action of S. boulardii on a crude toxin preparation was studied in vitro and in vivo. The results showed that the yeast had no effect on the toxins in vitro but was able to protect mice inoculated with these toxins. Furthermore, the observation by scanning electron microscopy that the mucosa of S. boulardii protected mice was not damaged suggests that the yeast mainly acts on the intestinal mucosa.     

Partial purification of Lophozozymus pictor toxin

An aqueous extract of a coral reef crab Lophozozymus pictor was subjected to a 6-stage purification and chemical testing procedure. The semi-purified toxin had a molecular weight between 1000 and 5000 and gave reactions that suggested it contained free amino and phenolic groups. The ₅₀ for mice was 377 μg/kg. The dose-death time relationships for the crab toxin, saxitoxin and tetrodotoxin were determined. The relationship for the crab toxin differed markedly from those for the other two toxins, the crab toxin being slower in its action.