Venom characterization of the Amazonian scorpion Tityus metuendus.

The soluble venom from the scorpion Tityus metuendus was characterized by various methods. In vivo experiments with mice showed that it is lethal. Extended electrophysiological recordings using seven sub-types of human voltage gated sodium channels (hNav1.1 to 1.7) showed that it contains both α- and ß-scorpion toxin types. Fingerprint analysis by mass spectrometry identified over 200 distinct molecular mass components. At least 60 sub-fractions were recovered from HPLC separation. Five purified peptides were sequenced by Edman degradation, and their complete primary structures were determined. Additionally, three other peptides have had their N-terminal amino acid sequences determined by Edman degradation and reported. Mass spectrometry analysis of tryptic digestion of the soluble venom permitted the identification of the amino acid sequence of 111 different peptides. Search for similarities of the sequences found indicated that they probably are: sodium and potassium channel toxins, metalloproteinases, hyaluronidases, endothelin and angiotensin-converting enzymes, bradykinin-potentiating peptide, hypothetical proteins, allergens, other enzymes, other proteins and peptides.

Toxin modulators and blockers of hERG K(+) channels.

The K(+) channel encoded by the Ether-á-go-go-Related Gene (ERG) is expressed in different tissues of different animal species. There are at least three subtypes of this channel, being the sub-type 1 (ERG1) crucial in the repolarization phase of the cardiac action potential. Mutations in this gene can affect the properties of the channel producing the type II long QT syndrome (LQTS2) and many drugs are also known to affect this channel with a similar side effect. Various scorpion, spider and sea anemone toxins affect the ERG currents by blocking the ion-conducting pore from the external side or by modulating channel gating through binding to the voltage-sensor domain. By doing so, these toxins become very useful tools for better understanding the structural and functional characteristics of these ion channels. This review discusses the interaction between the ERG channels and the peptides isolated from venoms of these animals. Special emphasis is placed on scorpion toxins, although the effects of several spider venom toxins and anemone toxins will be also revised.

Interacting sites of scorpion toxin ErgTx1 with hERG1 K+ channels.

Peptides purified from scorpion venoms were shown to interact with specific amino acid residues present in the outer vestibule of various sub-types of potassium channels, occluding the pore and causing a decrement of K(+) permeability through the membrane of excitable and non excitable cells. This communication describes the identification of several interacting sites of toxin ErgTx1, a toxin purified from the venom of the scorpion Centruroides noxius, with the human ERG1 K(+) channels, by means of site-directed mutagenesis of specific residues of the toxin. Recombinant mutants of the gene coding for ErgTx1 were expressed heterologously in Escherichia coli, properly folded and their affinities and interactions with hERG1 channels were determined by patch-clamp techniques. Residues in position Y14, Y17 and F37 of the solvent exposed hydrophobic surface, and charged residues at the position K13 and K38 of ErgTx1 were shown to cause a decrement of the affinity from 20 folds to 3 orders of magnitude, thus suggesting that they are certainly participating on the binding surface of this toxin towards the hERG1 channels. Double mutants at positions K13 and F37, Y14 and F37, Y17 and F37 and K13 and K38 were also prepared and assayed, but the results obtained are not much different from the single point mutants of ErgTx1. The results of the present work indicate the most probable surface area of ErgTx1 that makes contact with the hERG channels.

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.

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.

Recombinant expression of the toxic peptide ErgTx1 and role of Met35 on its stability and function.

Ergtoxin 1 (ErgTx1) is a 42 amino acid peptide purified from the venom of the Mexican scorpion Centruroides noxius Hoffmann, capable of blocking specifically human potassium channels of the ether-á-go-go-related gene family (hERG). This peptide binds to a partially overlapping site on the channel outer mouth, in which residues of the S5-P linker are critically involved. Here we describe results of site directed mutagenesis of the ErgTx1 gene and its heterologous expression in Escherichia coli. The recombinant products show the fundamental role played by methionine in position 35 (Met35) of the primary structure. Naturally oxidized Met35 decreases by three orders of magnitude the affinity of the peptide for the hERG1 channels. This result is quite relevant, because it shows two possible situations: either Met35 is involved in the proper folding of the molecule or it plays a direct role in the interaction with the channel, i.e., constitutes part of the interacting surfaces. These two situations were evaluated by preparing heterologously expressed ErgTx1 gene and a mutant containing alanine in position 35. Additionally circular dichroism measurements of both native and recombinant peptides were performed. The electrophysiological recordings and the structural values obtained by optical measurements, strongly support the idea that Met35 is indeed a key residue on the interacting surfaces of the toxin with the channels.

hERG1 channels are overexpressed in glioblastoma multiforme and modulate VEGF secretion in glioblastoma cell lines.

Recent studies have led to considerable advancement in our understanding of the molecular mechanisms that underlie the relentless cell growth and invasiveness of human gliomas. Partial understanding of these mechanisms has (1) improved the classification for gliomas, by identifying prognostic subgroups, and (2) pointed to novel potential therapeutic targets. Some classes of ion channels have turned out to be involved in the pathogenesis and malignancy of gliomas. We studied the expression and properties of K(+) channels in primary cultures obtained from surgical specimens: human ether a gò-gò related (hERG)1 voltage-dependent K(+) channels, which have been found to be overexpressed in various human cancers, and human ether a gò-gò-like 2 channels, that share many of hERG1's biophysical features. The expression pattern of these two channels was compared to that of the classical inward rectifying K(+) channels, IRK, that are widely expressed in astrocytic cells and classically considered a marker of astrocytic differentiation. In our study, hERG1 was found to be specifically overexpressed in high-grade astrocytomas, that is, glioblastoma multiforme (GBM). In addition, we present evidence that, in GBM cell lines, hERG1 channel activity actively contributes to malignancy by promoting vascular endothelial growth factor secretion, thus stimulating the neoangiogenesis typical of high-grade gliomas. Our data provide important confirmation for studies proposing the hERG1 channel as a molecular marker of tumour progression and a possible target for novel anticancer therapies.