Molecular cloning and characterization of four scorpion K(+)-toxin-like peptides: a new subfamily of venom peptides (alpha-KTx14) and genomic analysis of a member.

Four full-length cDNAs encoding the precursors of four K(+)-toxin-like peptides (named BmKK(1), BmKK(2), BmKK(3) and BmmKK(4), respectively) were first isolated from a venom gland cDNA library of the Chinese scorpion Buthus martensii Karsch. The deduced precursors of BmKK(1), BmKK(2) and BmKK(3) are all made of 54 amino acid residues including a signal peptide of 23 residues, and a mature toxin of 31 residues with three disulfide bridges. The precursor of BmKK(4) is composed of 55 amino acid residues including a signal peptide of 23 residues, a mature toxin of 30 residues cross-linked by three disulfide bridges, and an extra Gly-Lys tail which should be removed in the processing step. The four peptides displayed 24-97% sequence identity with each other, and less than 27% homology with any other scorpion toxins described. However, they shared a common disulfide bridge pattern, which was consistent with that of most short-chain K(+)-toxins, suggesting they represent a new class of scorpion toxins and their target receptors may be a subfamily of K(+) channels. We classified the BmKK toxin subfamily as alpha-KTx14 according to the classification rules. The genomic sequence of BmKK(2) was also cloned and sequenced. It consisted of two exons, disrupted by an intron of 79 bp inserted in the region encoding the C-terminal part of the signal peptide. This structure was very similar to that of other K(+)-toxins described previously.     

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 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.     

Scorpion toxins specific for Na+-channels.

Na+-channel specific scorpion toxins are peptides of 60-76 amino acid residues in length, tightly bound by four disulfide bridges. The complete amino acid sequence of 85 distinct peptides are presently known. For some toxins, the three-dimensional structure has been solved by X-ray diffraction and NMR spectroscopy. A constant structural motif has been found in all of them, consisting of one or two short segments of alpha-helix plus a triple-stranded beta-sheet, connected by variable regions forming loops (turns). Physiological experiments have shown that these toxins are modifiers of the gating mechanism of the Na+-channel function, affecting either the inactivation (alpha-toxins) or the activation (beta-toxins) kinetics of the channels. Many functional variations of these peptides have been demonstrated, which include not only the classical alpha- and beta-types, but also the species specificity of their action. There are peptides that bind or affect the function of Na+-channels from different species (mammals, insects or crustaceans) or are toxic to more than one group of animals. Based on functional and structural features of the known toxins, a classification containing 10 different groups of toxins is proposed in this review. Attempts have been made to correlate the presence of certain amino acid residues or 'active sites' of these peptides with Na+-channel functions. Segments containing positively charged residues in special locations, such as the five-residue turn, the turn between the second and the third beta-strands, the C-terminal residues and a segment of the N-terminal region from residues 2-11, seems to be implicated in the activity of these toxins. However, the uncertainty, and the limited success obtained in the search for the site through which these peptides bind to the channels, are mainly due to the lack of an easy method for expression of cloned genes to produce a well-folded, active peptide. Many scorpion toxin coding genes have been obtained from cDNA libraries and from polymerase chain reactions using fragments of scorpion DNAs, as templates. The presence of an intron at the DNA level, situated in the middle of the signal peptide, has been demonstrated.     

BmP09, a "long chain" scorpion peptide blocker of BK channels

A novel "long chain" toxin BmP09 has been purified and characterized from the venom of the Chinese scorpion Buthus martensi Karsch. The toxin BmP09 is composed of 66 amino acid residues, including eight cysteines, with a mass of 7721.0 Da. Compared with the B. martensi Karsch AS-1 as a Na(+) channel blocker (7704.8 Da), the BmP09 has an exclusive difference in sequence by an oxidative modification at the C terminus. The sulfoxide Met-66 at the C terminus brought the peptide a dramatic switch from a Na(+) channel blocker toaK(+) channel blocker. Upon probing the targets of the toxin BmP09 on the isolated mouse adrenal medulla chromaffin cells, where a variety of ion channels coexists, we found that the toxin BmP09 specifically blocked large conductance Ca(2+)- and voltage-dependent K(+) channels (BK) but not Na(+) channels at a range of 100 nm concentration. This was further confirmed by blocking directly the BK channels encoded with mSlo1 alpha-subunits in Xenopus oocytes. The half-maximum concentration EC(50) of BmP09 was 27 nm, and the Hill coefficient was 1.8. In outside-out patches, the 100 nm BmP09 reduced approximately 70% currents of BK channels without affecting the single-channel conductance. In comparison with the "short chain" scorpion peptide toxins such as Charybdotoxin, the toxin BmP09 behaves much better in specificity and reversibility, and thus it will be a more efficient tool for studying BK channels. A three-dimensional simulation between a BmP09 toxin and an mSlo channel shows that the Lys-41 in BmP09 lies at the center of the interface and plugs into the entrance of the channel pore. The stable binding between the toxin BmP09 and the BK channel is favored by aromatic pi -pi interactions around the center.     

ImKTx1, a new Kv1.3 channel blocker with a unique primary structure

Toxins from the venoms of scorpion, snake, and spider are valuable tools to probe the structure-function relationship of ion channels. In this investigation, a new toxin gene encoding the peptide ImKTx1 was isolated from the venom gland of the scorpion Isometrus maculates by constructing cDNA library method, and the recombinant ImKTx1 peptide was characterized physiologically. The mature peptide of ImKTx1 has 39 amino acid residues including six cross-linked cysteines. The electrophysiological experiments showed that the recombinant ImKTx1 peptide had a pharmacological profile where it inhibited Kv1.3 channel currents with IC(50) of 1.70 n± 1.35 μM, whereas 10 μM rImKTx1 peptide inhibited about 40% Kv1.1 and 42% Kv1.2 channel currents, respectively. In addition, 10 μM rImKTx1 had no effect on the Nav1.2 and Nav1.4 channel currents. Multiple sequence alignments showed that ImKTx1 had no homologous toxin peptide, but it was similar with Ca(2+) channel toxins from scorpion and spider in the arrangement of cysteine residues. These results indicate that ImKTx1 is a new Kv1.3 channel blocker with a unique primary structure. Our results indicate the diversity of K(+) channel toxins from scorpion venoms and also provide a new molecular template targeting Kv1.3 channel.     

Phaiodotoxin, a novel structural class of insect-toxin isolated from the venom of the Mexican scorpion Anuroctonus phaiodactylus.

A peptide called phaiodotoxin was isolated from the venom of the scorpion Anuroctonus phaiodactylus. It is lethal to crickets, but non toxic to mice at the doses assayed. It has 72 amino acid residues, with a molecular mass of 7971 atomic mass units. Its covalent structure was determined by Edman degradation and mass spectrometry; it contains four disulfide-bridges, of which one of the pairs is formed between cysteine-7 and cysteine-8 (positions Cys63-Cys71). The other three pairs are formed between Cys13-Cys38, Cys23-Cys50 and Cys27-Cys52. Comparative sequence analysis shows that phaiodotoxin belongs to the long-chain subfamily of scorpion peptides. Several genes coding for this peptide and similar ones were cloned by PCR, using cDNA prepared from the RNA of venomous glands of this scorpion. Electrophysiological assays conducted with this toxin in several mammalian cell lines (TE671, COS7, rat GH3 and cerebellum granular cells), showed no effect on Na+ currents. However, it shifts the voltage dependence of activation and inactivation of insect Na+ channels (para/tipE) to more negative and positive potentials, respectively. Therefore, the 'window' current is increased by 225%, which is thought to be the cause of its toxicity toward insects. Phaiodotoxin is the first toxic peptide ever purified from a scorpion of the family Iuridae.     

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.     

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.     

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.     

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.     

An ERG channel inhibitor from the scorpion Buthus eupeus

The isolation of the peptide inhibitor of M-type K(+) current, BeKm-1, from the venom of the Central Asian scorpion Buthus eupeus has been described previously (Fillipov A. K., Kozlov, S. A., Pluzhnikov, K. A., Grishin, E. V., and Brown, D. A. (1996) FEBS Lett. 384, 277-280). Here we report the cloning, expression, and selectivity of BeKm-1. A full-length cDNA of 365 nucleotides encoding the precursor of BeKm-1 was isolated using the rapid amplification of cDNA ends polymerase chain reaction technique from mRNA obtained from scorpion telsons. Sequence analysis of the cDNA revealed that the precursor contains a signal peptide of 21 amino acid residues. The mature toxin consists of 36 amino acid residues. BeKm-1 belongs to the family of scorpion venom potassium channel blockers and represents a new subgroup of these toxins. The recombinant BeKm-1 was produced as a Protein A fusion product in the periplasm of Escherichia coli. After cleavage and high performance liquid chromatography purification, recombinant BeKm-1 displayed the same properties as the native toxin. Three BeKm-1 mutants (R27K, F32K, and R27K/F32K) were generated, purified, and characterized. Recombinant wild-type BeKm-1 and the three mutants partly inhibited the native M-like current in NG108-15 at 100 nm. The effect of the recombinant BeKm-1 on different K(+) channels was also studied. BeKm-1 inhibited hERG1 channels with an IC(50) of 3.3 nm, but had no effect at 100 nm on hEAG, hSK1, rSK2, hIK, hBK, KCNQ1/KCNE1, KCNQ2/KCNQ3, KCNQ4 channels, and minimal effect on rELK1. Thus, BeKm-1 was shown to be a novel specific blocker of hERG1 potassium channels.     

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.     

New "Birtoxin analogs" from Androctonus australis venom

From the venom of the scorpion Androctonus australis, we have isolated a new bioactive polypeptide termed AaBTX-L1. When tested on the insect voltage-gated Na(+) channel (para) of the fruit fly, this toxin was able to induce a clear shift in activation (V(1/2)), resulting in the opening of the channel at more negative membrane potentials. Furthermore, AaBTX-L1 was totally devoid of toxicity when injected into mice intracerebroventricularly and did not compete with radiolabeled voltage-gated K(+) and Na(+) channel toxins in binding experiments on rat brain synaptosomes. Using its N-terminal amino acid sequence to design degenerate primers, several clones were amplified by PCR from the A. australis venom gland cDNA library. As a consequence, seven full oligonucleotide sequences encoding "long-chain" polypeptides with only three disulfide bridges have been cloned for the first time and are described here. Remarkably, they share high similarity with the anti-insect toxin Birtoxin from Parabuthus transvaalicus.     

A novel conus peptide ligand for K+ channels

Voltage-gated ion channels determine the membrane excitability of cells. Although many Conus peptides that interact with voltage-gated Na(+) and Ca(2+) channels have been characterized, relatively few have been identified that interact with K(+) channels. We describe a novel Conus peptide that interacts with the Shaker K(+) channel, kappaM-conotoxin RIIIK from Conus radiatus. The peptide was chemically synthesized. Although kappaM-conotoxin RIIIK is structurally similar to the mu-conotoxins that are sodium channel blockers, it does not affect any of the sodium channels tested, but blocks Shaker K(+) channels. Studies using Shaker K(+) channel mutants with single residue substitutions reveal that the peptide interacts with the pore region of the channel. Introduction of a negative charge at residue 427 (K427D) greatly increases the affinity of the toxin, whereas the substitutions at two other residues, Phe(425) and Thr(449), drastically reduced toxin affinity. Based on the Shaker results, a teleost homolog of the Shaker K(+) channel, TSha1 was identified as a kappaM-conotoxin RIIIK target. Binding of kappaM-conotoxin RIIIK is state-dependent, with an IC(50) of 20 nm for the closed state and 60 nm at 0 mV for the open state of TSha1 channels.     

Amino acid sequence and immunological characterization with monoclonal antibodies of two toxins from the venom of the scorpion Centruroides noxius Hoffmann.

Two toxins, which we propose to call toxins 2 and 3, were purified to homogeneity from the venom of the scorpion Centruroides noxius Hoffmann. The full primary structures of both peptides (66 amino acid residues each) was determined. Sequence comparison indicates that the two new toxins display 79% identity and present a high similarity to previously characterized Centruroides toxins, the most similar toxins being Centruroides suffusus toxin 2 and Centruroides limpidus tecomanus toxin 1. Six monoclonal antibodies (mAb) directed against purified fraction II-9.2 (which contains toxins 2 and 3) were isolated in order to carry out the immunochemical characterization of these toxins. mAb BCF2, BCF3, BCF7 and BCF9 reacted only with toxin 2, whereas BCF1 and BCF8 reacted with both toxins 2 and 3 with the same affinity. Simultaneous binding of mAb pairs to the toxin and cross-reactivity of the venoms of different scorpions with the mAb were examined. The results of these experiments showed that the mAb define four different epitopes (A-D). Epitope A (BCF8) is topographically unrelated to epitopes B (BCF2 and BCF7), C (BCF3) and D (BCF9) but the latter three appear to be more closely related or in close proximity to each other. Epitope A was found in all Centruroides venoms tested as well as on four different purified toxins of C. noxius, and thus seems to correspond to a highly conserved structure. Based on the cross-reactivity of their venoms with the mAb, Centruroides species could be classified in the following order: Centruroides elegans, Centruroides suffusus suffusus = Centruroides infamatus infamatus, Centruroides limpidus tecomanus, Centruroides limpidus limpidus, and Centruroides limpidus acatlanensis, according to increasing immunochemical relatedness of their toxins to those of Centruroides noxius. All six mAb inhibited the binding of toxin 2 to rat brain synaptosomal membranes, but only mAb BCF2, which belongs to the IgG2a subclass, displayed a clear neutralizing activity in vivo.     

Proteomic analysis of Tityus discrepans scorpion venom and amino acid sequence of novel toxins

The Venezuelan scorpion Tityus discrepans is known to cause human fatalities. We describe the first complete proteomic analysis of its venom. By HPLC 58 different fractions were obtained and 205 different components were identified by MS analysis. Components having molecular masses from 272 to 57 908 amu were found. Forty homogeneous components had their N-terminal amino acid sequence determined by Edman degradation, from which two new peptides named TdK2 and TdK3 (meaning T. discrepans (Td) K(+) channel toxins 2 and 3) were fully characterized. The first contains 34 amino acid residues with a molecular mass of 3451 amu, and the second has 36 amino acids with 3832 amu. Both peptides are tightly bound by three disulfide bridges. TdK2 was shown to block reversibly the Shaker B K(+)-channel expressed heterologously in Sf9 cells. The systematic number assigned to TdK2 is alpha-KTx-18.2 and that of TdK3 is alpha-KTx-18.3. Comparative analysis of the amino acid sequences found suggests that this venom contains peptides highly similar to those that block K(+) channels, as well as those that modify the gating mechanisms of Na(+) channels, found in other scorpions. Additionally, peptides similar to defensins were also identified.     

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.     

Gene expression, mutation, and structure-function relationship of scorpion toxin BmP05 active on SK(Ca) channels

Four peptide inhibitors of small-conductance Ca(2+)-activated, apamin-sensitive K(+) channels (SK(Ca)) have been isolated from the venom of the Chinese scorpion Buthus martensi, named BmP01, BmP02, BmP03, and BmP05, respectively [Romi-Lebrun, R. (1997) Eur. J. Biochem. 245, 457-464]. Among them BmP05 with 31 amino acid residues has been intensively studied due to its most potent toxicity. To investigate the structure-function relationship of BmP05, its wild type and seven mutants (their C-termini unamidated) were successfully expressed in the yeast secretion system and purified with a high yield over 8 mg/L. Their toxicity to mice and electrophysiological activity on the K(+) currents (SK(Ca) and Kv) in rat adrenal chromaffin cells were measured and compared. The results indicated the following: (1) As a selective antagonist against SK(Ca), 1 microM rBmP05 is equivalent to 0.2 microM apamin, and its IC(50) is 0.92 microM. (2) The basic residues Lys and Arg located at positions 6 and 13 in the N-terminal alpha-helix region are essential and synergetic in the interaction of the toxin with SK(Ca). (3) Disruption of the alpha-helix by mutation of Gln at position 9 with Pro results in almost total loss of toxicity. (4) The C-terminal residue His31 plays an auxiliary role in the interaction of the toxin with SK(Ca). (5) The beta-turn connecting two beta-sheets near the C-terminal part is responsible for the specificity of the toxin to the different subtypes of K(+) channels.