Molecular cloning, genomic organization and functional characterization of a new short-chain potassium channel toxin-like peptide BmTxKS4 from Buthus martensii Karsch(BmK)

Scorpion venom contains many small polypeptide toxins, which can modulate Na(+), K(+), Cl(-), and Ca(2+) ion-channel conductance in the cell membrane. A full-length cDNA sequence encoding a novel type of K(+)-channel toxin (named BmTxKS4) was first isolated and identified from a venom gland cDNA library of Buthus martensii Karsch (BmK). The encoded precursor contains 78 amino acid residues including a putative signal peptide of 21 residues, propeptide of 11 residues, and a mature peptide of 43 residues with three disulfide bridges. BmTxKS4 shares the identical organization of disulfide bridges with all the other short-chain K(+)-channel scorpion toxins. By PCR amplification of the genomic region encoding BmTxKS4, it was shown that BmTxKS4 composed of two exons is disrupted by an intron of 87 bp inserted between the first and the second codes of Phe (F) in the encoding signal peptide region, which is completely identical with that of the characterized scorpion K(+)-channel ligands in the size, position, consensus junctions, putative branch point, and A+T content. The GST-BmTxKS4 fusion protein was successfully expressed in BL21 (DE3) and purified with affinity chromatography. About 2.5 mg purified recombinant BmTxKS4 (rBmTxKS4) protein was obtained by treating GST-BmTxKS4 with enterokinase and sephadex chromatography from 1 L bacterial culture. The electrophysiological activity of 1.0 microM rBmTxKS4 was measured and compared by whole cell patch-clamp technique. The results indicated that rBmTxKS4 reversibly inhibited the transient outward K(+) current (I(to)), delayed inward rectifier K(+) current (I(k1)), and prolonged the action potential duration of ventricular myocyte, but it has no effect on the action potential amplitude. Taken together, BmTxKS4 is a novel subfamily member of short-strain K(+)-channel scorpion toxin.     

Molecular dissection of venom from Chinese scorpion Mesobuthus martensii: identification and characterization of four novel disulfide-bridged venom peptides

Scorpion venom is composed of a large repertoire of biologically active polypeptides. However, most of these peptides remain to be identified and characterized. In this paper, we report the identification and characterization of four novel disulfide-bridged venom peptides (named BmKBTx, BmKITx, BmKKx1 and BmKKx2, respectively) from the Chinese scorpion, Mesobuthus martensii (also named Buthus martensii Karsch). BmKBTx is composed of 58 amino acid residues and cross-linked by three disulfide bridges. The sequence of BmKBTx shows some similarities to that of the toxin, birtoxin, and its analogs. It is likely that BmKBTx is a beta-toxin active on Na+ channels, which is toxic to either insects or mammals. BmKITx is composed of 71 amino acid residues with four disulfide bridges. It is the longest venom peptide identified from M. martensii so far. BmKITx shows little sequence identity with scorpion alpha-toxins toxic to insects. It is likely that BmKITx is a new type of Na+ -channel specific toxin active on both insects and mammals. BmKKx1 contains 38 amino acid residues cross-linked by three disulfide bridges and shows 84% sequence identity with BmTx3, an inhibitor of A-type K+ channel and HERG currents. BmKKx1 has been classified as alpha-KTx-15.8. BmKKx2 is composed of 36 residues and stabilized by three disulfide bridges. BmKKx2 is a new member of the gamma-K+ -channel toxin subfamily (classified as gamma-KTx 2.2). The venoms of scorpions thus continue to provide novel toxins with potential novel actions on targets.     

A novel class of peptide found in scorpion venom with neurodepressant effects in peripheral and central nervous system of the rat

A novel toxin, named Cll9, was isolated from the venom of the scorpion Centruroides limpidus limpidus Karsch. It is composed of 63 amino acid residues closely packed by four disulfide bridges. It showed no apparent effect when injected to insects, crustaceans and i.p. to mice. However, when i.c.v. injected in the rat it immediately induced sleep, suggesting that it has a neurodepressant effect. We confirmed this by showing that it has a strong antiepileptic action, as assessed with the penicillin focus model. Its effectiveness in inhibiting Na(+) permeability in (cultured) rat peripheral ganglia further supports its neurodepressant actions. However, this peptide did not affect other Na(+) channels such as those from cerebellum granular cells in culture or the rSkM1 Na(+) channels expressed in HEK293. The cDNA and genomic regions encoding this peptide were cloned and sequenced. This peptide is synthesized as a precursor of 84 amino acid residues and processed by removing 19 amino acids (signal peptide) from the amino terminal region and a couple of lysine residues from the carboxyl end. The presence of an intron of 777 bases interrupting the region encoding the signal peptide was also revealed. A comparison of its primary sequence, with more than 100 scorpion toxins known, showed that together with toxin CsE9 they constitute a new subfamily of peptides considered to be one of the most divergent groups of scorpion toxin-like peptides discovered.     

Synthesis, 1H NMR structure, and activity of a three-disulfide-bridged maurotoxin analog designed to restore the consensus motif of scorpion toxins

Maurotoxin (MTX) is a 34-residue toxin that has been isolated from the venom of the chactidae scorpion Scorpio maurus palmatus. The toxin displays an exceptionally wide range of pharmacological activity since it binds onto small conductance Ca(2+)-activated K(+) channels and also blocks Kv channels (Shaker, Kv1.2 and Kv1.3). MTX possesses 53-68% sequence identity with HsTx1 and Pi1, two other K(+) channel short chain scorpion toxins cross-linked by four disulfide bridges. These three toxins differ from other K(+)/Cl(-)/Na(+) channel scorpion toxins cross-linked by either three or four disulfide bridges by the presence of an extra half-cystine residue in the middle of a consensus sequence generally associated with the formation of an alpha/beta scaffold (an alpha-helix connected to an antiparallel beta-sheet by two disulfide bridges). Because MTX exhibits an uncommon disulfide bridge organization among known scorpion toxins (C1-C5, C2-C6, C3-C4, and C7-C8 instead of C1-C4, C2-C5, and C3-C6 for three-disulfide-bridged toxins or C1-C5, C2-C6, C3-C7, and C4-C8 for four-disulfide-bridged toxins), we designed and chemically synthesized an MTX analog with three instead of four disulfide bridges ([Abu(19),Abu(34)]MTX) and in which the entire consensus motif of scorpion toxins was restored by the substitution of the two half-cystines in positions 19 and 34 (corresponding to C4 and C8) by two isosteric alpha-aminobutyrate (Abu) derivatives. The three-dimensional structure of [Abu(19), Abu(34)]MTX in solution was solved by (1)H NMR. This analog adopts the alpha/beta scaffold with now conventional half-cystine pairings connecting C1-C5, C2-C6, and C3-C7 (with C4 and C8 replaced by Abu derivatives). This novel arrangement in half-cystine pairings that concerns the last disulfide bridge results mainly in a reorientation of the alpha-helix regarding the beta-sheet structure. In vivo, [Abu(19),Abu(34)]MTX remains lethal in mice as assessed by intracerebroventricular injection of the peptide (LD(50) value of 0. 25 microg/mouse). The structural variations are also accompanied by changes in the pharmacological selectivity of the peptide, suggesting that the organization pattern of disulfide bridges should affect the three-dimensional presentation of certain key residues critical to the blockage of K(+) channel subtypes.     

Purification, characterization and sequence determination of BmKK4, a novel potassium channel blocker from Chinese scorpion Buthus martensi Karsch

The scorpion neurotoxin BmKK4 was purified from the venom of the Chinese scorpion Buthus martensi Karsch by a combination of gel-filtration, ion exchange and reversed phase chromatography. The primary sequence of BmKK4 was determined using the tandem MS/MS technique and the cDNA database searching as followings: ZTQCQ SVRDC QQYCL TPDRC SYGTC YCKTT (NH(2)). BmKK4 is the first isolated member of a new subfamily alpha-KTx17 of scorpion K(+) toxins.     

Molecular cloning and functional expression of a gene encoding an antiarrhythmia peptide derived from the scorpion toxin.

From a cDNA library of Chinese scorpion Buthus martensii Karsch, full-length cDNAs of 351 nucleotides encoding precursors (named BmKIM) that contain signal peptides of 21 amino acid residues, a mature toxin of 61 residues with four disulfide bridges, and an extra Gly-Lys-Lys tail, were isolated. The genomic sequence of BmKIM was cloned and sequenced; it consisted of two exons disrupted by an intron of 1622 bp, the largest known in scorpion toxin genomes, inserted in the region encoding the signal peptide. The cDNA was expressed in Escherichia coli. The recombinant BmKIM was toxic to both mammal and insects. This is the first report that a toxin with such high sequence homology with an insect-specific depressant toxin group exhibits toxicity to mammals. Using whole cell patch-clamp recording, it was discovered that the recombinant BmKIM inhibited the sodium current in rat dorsal root ganglion neurons and ventricular myocytes and protected against aconitine- induced cardiac arrhythmia.     

Cloning and characterization of BmK86, a novel K+ -channel blocker from scorpion venom

Scorpion venom represents a tremendous hitherto unexplored resource for understanding ion channels. BmK86 is a novel K+ -channel toxin gene isolated from a cDNA library of Mesobuthus martensii Karsch, which encodes a signal peptide of 22 amino acid residues and a mature toxin of 35 residues with three disulfide bridges. The genomic sequence of BmK86 consists of two exons disrupted by an intron of 72 bp. Comparison with the other scorpion toxins BmK86 shows low sequence similarity. The GST-BmK86 fusion protein was successfully expressed in Escherichia coli. The fusion protein was cleaved by enterokinase and the recombinant BmK86 was purified by HPLC. Using whole-cell patch-clamp recording, the recombinant BmK86 was found to inhibit the potassium current of mKv1.3 channel expressed in COS7 cells. These results indicated that BmK86 belongs to a representative member of a novel subfamily of alpha-KTxs. The systematic number assigned to BmK86 is alpha-KTx26.1.     

Solution structure of Pi4, a short four-disulfide-bridged scorpion toxin specific of potassium channels

Pi4 is a short toxin found at very low abundance in the venom of Pandinus imperator scorpions. It is a potent blocker of K(+) channels. Like the other members of the alpha-KTX6 subfamily to which it belongs, it is cross-linked by four disulfide bonds. The synthetic analog (sPi4) and the natural toxin (nPi4) have been obtained by solid-phase synthesis or from scorpion venom, respectively. Analysis of two-dimensional (1)H NMR spectra of nPi4 and sPi4 indicates that both peptides have the same structure. Moreover, electrophysiological recordings of the blocking of Shaker B K(+) channels by sPi4 (K(D) = 8.5 nM) indicate that sPi4 has the same blocking activity of nPi4 (K(D) = 8.0 nM), previously described. The disulfide bonds have been independently determined by NMR and structure calculations, and by Edman-degradation/mass-spectrometry identification of peptides obtained by proteolysis of nPi4. Both approaches indicate that the pairing of the half-cystines is (6)C-(27)C, (12)C-(32)C, (16)C-(34)C, and (22)C-(37)C. The structure of the toxin has been determined by using 705 constraints derived from NMR data on sPi4. The structure, which is well defined, shows the characteristic alpha/beta scaffold of scorpion toxins. It is compared to the structure of the other alpha-KTX6 subfamily members and, in particular, to the structure of maurotoxin, which shows a different pattern of disulfide bridges despite its high degree of sequence identity (76%) with Pi4. The structure of Pi4 and the high amounts of synthetic peptide available, will enable the detailed analysis of the interaction of Pi4 with K(+) channels.     

Characterisation of a novel toxin active on potassium apanaine channels, purified from Buthus occitanus tunetanus scorpion venom

A new peptidyl inhibitor of the small-conductance Ca(2+)-activated K+ channels (SKca) was purified to homogeneity from the venom of the Tunisian scorpion Buthus occitanus tunetanus. The molecular mass determined by SDS-PAGE, shows that it's a short peptide (3300 Da). The primary sequence of this toxin shows that it is a 31-residue polypeptide cross-linked by three disulfide bridges and structurally related to subfamily 5 of short scorpion toxins. This molecule shows similar pharmacological properties with this group of peptides inducing high toxicity in mice after intracerebro-ventricular injection, and competing with iodinated apamin for binding to its receptor site from rat brain synaptosomes (K0.5 = 4 nM).     

Solution structure of BmKK2, a new potassium channel blocker from the venom of chinese scorpion Buthus martensi Karsch

A natural K+ channel blocker, BmKK2 (a member of scorpion toxin subfamily alpha-KTx 14), which is composed of 31 amino acid residues and purified from the venom of the Chinese scorpion Buthus martensi Karsch, was characterized using whole-cell patch-clamp recording in rat hippocampal neurons. The three dimensional structure of BmKK2 was determined with two-dimensional NMR spectroscopy and molecular modelling techniques. In solution this toxin adopted a common alpha/beta-motif, but showed distinct local conformation in the loop between alpha-helix and beta-sheet in comparison with typical short-chain scorpion toxins (e.g., CTX and NTX). Also, the alpha helix is shorter and the beta-sheet element is smaller (each strand consisted only two residues). The unusual structural feature of BmKK2 was attributed to the shorter loop between the alpha-helix and beta-sheet and the presence of two consecutive Pro residues at position 21 and 22 in the loop. Moreover, two models of BmKK2/hKv1.3 channel and BmKK2/rSK2 channel complexes were simulated with docking calculations. The results demonstrated the existence of a alpha-mode binding between the toxin and the channels. The model of BmKK2/rSK2 channel complex exhibited favorable contacts both in electrostatic and hydrophobic, including a network of five hydrogen bonds and bigger interface containing seven pairs of inter-residue interactions. In contrast, the model of BmKK2/hKv1.3 channel complex, containing only three pairs of inter-residue interactions, exhibited poor contacts and smaller interface. The results well explained its lower activity towards Kv channel, and predicted that it may prefer a type of SK channel with a narrower entryway as its specific receptor.     

Solution structure of a K+-channel blocker from the scorpion Tityus cambridgei

A new K(+)-channel blocking peptide identified from the scorpion venom of Tityus cambridgei (Tc1) is composed of 23 amino acid residues linked with three disulfide bridges. Tc1 is the shortest known toxin from scorpion venom that recognizes the Shaker B K(+) channels and the voltage-dependent K(+) channels in the brain. Synthetic Tc1 was produced using solid-phase synthesis, and its activity was found to be the same as that of native Tc1. The pairings of three disulfide bridges in the synthetic Tc1 were identified by NMR experiments. The NMR solution structures of Tc1 were determined by simulated annealing and energy-minimization calculations using the X-PLOR program. The results showed that Tc1 contains an alpha-helix and a 3(10)-helix at N-terminal Gly(4)-Lys(10) and a double-stranded beta-sheet at Gly(13)-Ile(16) and Arg(19)-Tyr(23), with a type I' beta-turn at Asn(17)-Gly(18). Superposition of each structure with the best structure yielded an average root mean square deviation of 0.26 +/- 0.05 A for the backbone atoms and of 1.40 +/- 0.23 A for heavy atoms in residues 2 to 23. The three-dimensional structure of Tc1 was compared with two structurally and functionally related scorpion toxins, charybdotoxin (ChTx) and noxiustoxin (NTx). We concluded that the C-terminal structure is the most important region for the blocking activity of voltage-gated (Kv-type) channels for scorpion K(+)-channel blockers. We also found that some of the residues in the larger scorpion K(+)-channel blockers (31 to 40 amino acids) are not involved in K(+)-channel blocking activity.     

Proteomics of the venom from the Amazonian scorpion Tityus cambridgei and the role of prolines on mass spectrometry analysis of toxins

Scorpion venom are complex mixtures of peptides, known to cause impairment of ion-channel function in biological membranes. This report describes the separation of approximately 60 different components by high performance liquid chromatography and the characterization by Edman degradation and mass spectrometry of 26 peptides from the soluble venom of the Amazonian scorpion Tityus cambridgei. One of these peptides, named Tc48a, was fully characterized. It contains 65 amino acid residues, the C-terminal residue is amidated and it affects Na(+)-channels with a K(d) of about 82 nM. Furthermore, this report shows the thermo-instability of scorpion toxins subjected to electron spray ionization-mass spectrometry (ESI-MS). When a proline residue is located near the N-terminal region of the toxin, not stabilized by disulfide bridges, artificial components are generated by the mass spectrometer conditions, due to the cleavage of the peptide bond at the proline positions. This phenomenon was confirmed by using four model proteins (variable regions of immunoglobulins) studied by ESI-MS and matrix assisted laser desorption ionization-time of flight (MALDI-TOF)/MS.     

Mechanisms of maurotoxin action on Shaker potassium channels

Maurotoxin (alpha-KTx6.2) is a toxin derived from the Tunisian chactoid scorpion Scorpio maurus palmatus, and it is a member of a new family of toxins that contain four disulfide bridges (, Eur. J. Biochem. 254:468-479). We investigated the mechanism of the maurotoxin action on voltage-gated K(+) channels expressed in Xenopus oocytes. Maurotoxin blocks the channels in a voltage-dependent manner, with its efficacy increasing with greater hyperpolarization. We show that an amino acid residue in the external mouth of the channel pore segment that is known to be involved in the actions of other peptide toxins is also involved in maurotoxin's interaction with the channel. We conclude that, despite the unusual disulfide bridge pattern, the mechanism of the maurotoxin action is similar to those of other K(+) channel toxins with only three disulfide bridges.     

Maurotoxin versus Pi1/HsTx1 scorpion toxins. Toward new insights in the understanding of their distinct disulfide bridge patterns

Maurotoxin (MTX) is a scorpion toxin acting on several K(+) channel subtypes. It is a 34-residue peptide cross-linked by four disulfide bridges that are in an "uncommon" arrangement of the type C1-C5, C2-C6, C3-C4, and C7-C8 (versus C1-C5, C2-C6, C3-C7, and C4-C8 for Pi1 or HsTx1, two MTX-related scorpion toxins). We report here that a single mutation in MTX, in either position 15 or 33, resulted in a shift from the MTX toward the Pi1/HsTx1 disulfide bridge pattern. This shift is accompanied by structural and pharmacological changes of the peptide without altering the general alpha/beta scaffold of scorpion toxins.     

Molecular cloning and sequencing of two 'short chain' and two 'long chain' K(+) channel-blocking peptides from the Chinese scorpion Buthus martensii Karsch.

Five full-length cDNAs encoding the precursors of two 'short chain' scorpion non-toxic peptides active on Ca(2+)-activated K(+) channels (BmP02 and BmP03) and two novel putative long chain K(+) channel-blocking peptides (named BmTXKbeta and BmTXKbeta2) were first isolated from the venom gland cDNA library of the Chinese scorpion Buthus martensii Karsch (BmK). BmTXKbeta2 showed a high similarity with AaTXKbeta, while BmTXKbeta was completely different in the deduced primary structure from the long chain and short chain scorpion toxins already characterized. Thus, BmTXKbeta expands the scorpion long chain K(+) channel-blocking peptide family. Although little sequence similarity exists between the above two short and two long peptides, they are similar at the positions of six cysteines, suggesting that they should all share a similar scaffold composed of an alpha-helix and a three-stranded beta-sheet.     

NMR solution structure of Cn12, a novel peptide from the Mexican scorpion Centruroides noxius with a typical beta-toxin sequence but with alpha-like physiological activity.

Cn12 isolated from the venom of the scorpion Centruroides noxius has 67 amino-acid residues, closely packed with four disulfide bridges. Its primary structure and disulfide bridges were determined. Cn12 is not lethal to mammals and arthropods in vivo at doses up to 100 microg per animal. Its 3D structure was determined by proton NMR using 850 distance constraints, 36 phi angles derived from 36 coupling constants obtained by two different methods, and 22 hydrogen bonds. The overall structure has a two and half turn alpha-helix (residues 24-32), three strands of antiparallel beta-sheet (residues 2-4, 37-40 and 45-48), and a type II turn (residues 41-44). The amino-acid sequence of Cn12 resembles the beta scorpion toxin class, although patch-clamp experiments showed the induction of supplementary slow inactivation of Na(+) channels in F-11 cells (mouse neuroblastoma N18TG-2 x rat DRG2), which means that it behaves more like an alpha scorpion toxin. This behaviour prompted us to analyse Na(+) channel binding sites using information from 112 Na(+) channel gene clones available in the literature, focusing on the extracytoplasmic loops of the S5-S6 transmembrane segments of domain I and the S3-S4 segments of domain IV, sites considered to be responsible for binding alpha scorpion toxins.     

Proteomic analysis of the venom and characterization of toxins specific for Na+ - and K+ -channels from the Colombian scorpion Tityus pachyurus

The Colombian scorpion Tityus pachyurus is toxic to humans and is capable of producing fatal accidents, but nothing is known about its venom components. This communication reports the separation of at least 57 fractions from the venom by high performance liquid chromatography. From these, at least 104 distinct molecular weight compounds were identified by mass spectrometry analysis. The complete amino acid sequences of three peptides were determined and the partial sequences of three others were also identified. Electrophysiological experiments conducted with ion-channels expressed heterologously on Sf9 cells showed the presence of a potent Shaker B K(+)-channel blocker. This peptide (trivial name Tpa1) contains 23 amino acid residues closely packed by three disulfide bridges with a molecular mass of 2,457 atomic mass units. It is the third member of the sub-family 13, for which the systematic name is proposed to be alpha-KTx13.3. The mice assay showed clearly the presence of toxic peptides to mammals. One of them named Tpa2, containing 65 amino acid residues with molecular mass of 7,522.5 atomic mass units, is stabilized by four disulfide bridges. It was shown to modify the Na(+)-currents of F-11 and TE671 cells in culture, similar to the beta scorpion toxins. These results demonstrate the presence of toxic peptides in the venom of T. pachyurus and confirm that accidents with this species of scorpion should be considered an important human hazard in Colombia.     

Genomic and Structural Characterization of Kunitz-Type Peptide LmKTT-1a Highlights Diversity and Evolution of Scorpion Potassium Channel Toxins

Background

Recently, a new subfamily of long-chain toxins with a Kunitz-type fold was found in scorpion venom glands. Functionally, these toxins inhibit protease activity and block potassium channels. However, the genomic organization and three-dimensional (3-D) structure of this kind of scorpion toxin has not been reported.

Principal Findings

Here, we characterized the genomic organization and 3-D nuclear magnetic resonance structure of the scorpion Kunitz-type toxin, LmKTT-1a, which has a unique cysteine pattern. The LmKTT-1a gene contained three exons, which were interrupted by two introns located in the mature peptide region. Despite little similarity to other Kunitz-type toxins and a unique pattern of disulfide bridges, LmKTT-1a possessed a conserved Kunitz-type structural fold with one α-helix and two β-sheets. Comparison of the genomic organization, 3-D structure, and functional data of known toxins from the α-KTx, β-KTx, γ-KTx, and κ-KTx subfamily suggested that scorpion Kunitz-type potassium channel toxins might have evolved from a new ancestor that is completely different from the common ancestor of scorpion toxins with a CSα/β fold. Thus, these analyses provide evidence of a new scorpion potassium channel toxin subfamily, which we have named δ-KTx.

Conclusions/Significance

Our results highlight the genomic, structural, and evolutionary diversity of scorpion potassium channel toxins. These findings may accelerate the design and development of diagnostic and therapeutic peptide agents for human potassium channelopathies.     

OsK2, a new selective inhibitor of Kv1.2 potassium channels purified from the venom of the scorpion Orthochirus scrobiculosus

A novel inhibitor of voltage-gated K(+) channels has been purified to homogeneity from the venom of the black scorpion Orthochirus scrobiculosus. This toxin, named OsK2, has been characterized as a 28-residue peptide, containing six conserved cysteine residues and was shown to be a potent and selective blocker of Kv1.2 channels (K(d) = 97 nM). OsK2 is the second member of the 13th subfamily of short-chain K(+) channel-blocking peptides known thus far and is therefore called alpha-KTx 13.2.