A potent potassium channel blocker from Mesobuthus eupeus scorpion venom

Scorpion venom-derived peptidyl toxins are valuable pharmacological tools for investigating the structure–function relationship of ion channels. Here, we report the purification, sequencing and functional characterization of a new K⁺ channel blocker (MeuKTX) from the venom of the scorpion Mesobuthus eupeus. Effects of MeuKTX on ten cloned potassium channels in Xenopus oocytes were evaluated using two-electrode voltage-clamp recordings. MeuKTX is the orthologue of BmKTX (α-KTx3.6), a known Kv1.3 blocker from the scorpion Mesobuthus martensii, and classified as α-KTx3.13. MeuKTX potently blocks rKv1.1, rKv1.2 and hKv1.3 channels with 50% inhibitory concentration (IC₅₀) of 203.15 ± 4.06 pM, 8.92 ± 2.3 nM and 171 ± 8.56 pM, respectively, but does not affect rKv1.4, rKv1.5, hKv3.1, rKv4.3, and hERG channels even at 2 μM concentration. At this high concentration, MeuKTX is also active on rKv1.6 and Shaker IR. Our results also demonstrate that MeuKTX and BmKTX have the same channel spectrum and similar pharmacological potency. Analysis of the structure–function relationships of α-KTx3 subfamily toxins allows us to recognize several key sites which may be useful for designing toxins with improved activity on hKv1.3, an attractive target for T-cell mediated autoimmune diseases.     

Solution structure of BTK-2, a novel hK(v)1.1 inhibiting scorpion toxin, from the eastern Indian scorpion Mesobuthus tamulus

The three dimensional structure of a 32 residue three disulfide scorpion toxin, BTK-2, from the Indian red scorpion Mesobuthus tamulus has been determined using isotope edited solution NMR methods. Samples for structural and electrophysiological studies were prepared using recombinant DNA methods. Electrophysiological studies show that the peptide is active against hK(v)1.1 channels. The structure of BTK-2 was determined using 373 distance restraints from NOE data, 66 dihedral angle restraints from NOE, chemical shift and scalar coupling data, 6 constraints based on disulfide linkages and 8 constraints based on hydrogen bonds. The root mean square deviation (r.m.s.d) about the averaged co-ordinates of the backbone (N, C(α), C') and all heavy atoms are 0.81 ± 0.23? and 1.51 ± 0.29? respectively. The backbone dihedral angles (? and ψ) for all residues occupy the favorable and allowed regions of the Ramachandran map. The three dimensional structure of BTK-2 is composed of three well defined secondary structural regions that constitute the α-β-β structural motif. Comparisons between the structure of BTK-2 and other closely related scorpion toxins pointed towards distinct differences in surface properties that provide insights into the structure-function relationships among this important class of voltage-gated potassium channel inhibiting peptides.     

Bradycardia induced by Mesobuthus tamulus scorpion venom involves muscarinic receptor-G-protein-coupled cell signaling pathways

Indian red scorpion (Mesobuthus tamulus; MBT) envenomation produces various cardio-respiratory abnormalities including cardiac dysrhythmias. The underlying cell signaling pathways for the cardiac dysrhythmias produced by MBT venom are not known. The present study was therefore conducted to delineate the second messenger signaling pathways involved in MBT venom-induced atrial rhythm changes. The effects of venom and various antagonists were examined on spontaneously beating rat right atrial preparations in vitro. The MBT-venom produced an increase (35%), a decrease (45%) and again an increase (50%) in rate at 0.03, 0.3 and 3.0 microg/ml of venom, respectively. On the other hand, force of contraction exhibited a concentration-dependent rise (up to 40%) at all concentrations of venom. Pretreatment with atropine (0.3 microM) blocked the decrease in atrial rate at 0.3 microg/ml concentration of venom while no such blockade was seen in force of contraction. Submaximal concentration of ACh (0.1 nM) decreased the atrial rate by 25%. In the presence of MBT venom (0.3 microg/ml), ACh-induced fall in atrial rate was enhanced. The venom-induced fall in atrial rate and augmentation of ACh response were blocked by pertussis toxin (PTx; a Gi-inhibitor) or methylene blue (a G-cyclase inhibitor). The results indicate that the decrease in atrial rate produced by venom is mediated muscarinic by receptors via Gi-guanylyl cyclase mediated cell signaling pathways.     

BTK-2, a new inhibitor of the Kv1.1 potassium channel purified from Indian scorpion Buthus tamulus

A novel inhibitor of voltage-gated potassium channel was isolated and purified to homogeneity from the venom of the red scorpion Buthus tamulus. The primary sequence of this toxin, named BTK-2, as determined by peptide sequencing shows that it has 32 amino acid residues with six conserved cysteines. The molecular weight of the toxin was found to be 3452 Da. It was found to block the human potassium channel hKv1.1 (IC(50)=4.6 microM). BTK-2 shows 40-70% sequence similarity to the family of the short-chain toxins that specifically block potassium channels. Multiple sequence alignment helps to categorize the toxin in the ninth subfamily of the K+ channel blockers. The modeled structure of BTK-2 shows an alpha/beta scaffold similar to those of the other short scorpion toxins. Comparative analysis of the structure with those of the other toxins helps to identify the possible structure-function relationship that leads to the difference in the specificity of BTK-2 from that of the other scorpion toxins. The toxin can also be used to study the assembly of the hKv1.1 channel.     

Tamapin,a venom peptide from the Indian red scorpion (Mesobuthus tamulus) that targets small conductance Ca2+-activated K+ channels and afterhyperpolarization currents in central neurons

The biophysical properties of small conductance Ca(2+)-activated K(+) (SK) channels are well suited to underlie afterhyperpolarizations (AHPs) shaping the firing patterns of a conspicuous number of central and peripheral neurons. We have identified a new scorpion toxin (tamapin) that binds to SK channels with high affinity and inhibits SK channel-mediated currents in pyramidal neurons of the hippocampus as well as in cell lines expressing distinct SK channel subunits. This toxin distinguished between the SK channels underlying the apamin-sensitive I(AHP) and the Ca(2+)-activated K(+) channels mediating the slow I(AHP) (sI(AHP)) in hippocampal neurons. Compared with related scorpion toxins, tamapin displayed a unique, remarkable selectivity for SK2 versus SK1 ( approximately 1750-fold) and SK3 ( approximately 70-fold) channels and is the most potent SK2 channel blocker characterized so far (IC(50) for SK2 channels = 24 pm). Tamapin will facilitate the characterization of the subunit composition of native SK channels and help determine their involvement in electrical and biochemical signaling.     

A fusion protein containing a lepidopteran-specific toxin from the South Indian red scorpion (Mesobuthus tamulus) and snowdrop lectin shows oral toxicity to target insects

Background  

Despite evidence suggesting a role in plant defence, the use of plant lectins in crop protection has been hindered by their low and species-specific insecticidal activity. Snowdrop lectin (Galanthus nivalis agglutinin; GNA) is transported to the haemolymph of insects after oral ingestion, and can be used as a basis for novel insecticides. Recombinant proteins containing GNA expressed as a fusion with a peptide or protein, normally only toxic when injected into the insect haemolymph, have the potential to show oral toxicity as a result of GNA-mediated uptake.     

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 BmP02, a new potassium channel blocker from the venom of the Chinese scorpion Buthus martensi Karsch

BmP02 is a 28-amino acid residue peptide purified from the venom of the Chinese scorpion Buthus martensi Karsch, which had been demonstrated to be a weak blocker of apamin-sensitive calcium-activated potassium channels. Two-dimensional NMR spectroscopy techniques were used to determine the solution structure of BmP02. The results show that BmP02 formed a alpha/beta scorpion fold, the typical three-dimensional structure adopted by most short chain scorpion toxins whose structures have been determined. However, in BmP02 this alpha/beta fold was largely distorted. The alpha-helix was shortened to only one turn, and the loop connecting the helix to the first beta-strand exhibited conformational heterogeneity. The instability of BmP02 could be attributed to a proline at position 17, which is usually a glycine. Because the residue at this position makes intense contact with the alpha-helix, it was supposed that the bulky side chain of proline had pushed the helix away from the beta-sheet. This had a significant influence on the structure and function of BmP02. The alpha-helix rotated by about 40 degrees to avoid Pro17 while forming two disulfides with the second beta-strand. The rotation further caused both ends of the helix to be unwound due to covalent restrictions. According to its structure, BmP02 was supposed to interact with its target via the side chains of Lys11 and Lys13.     

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.     

Indian red scorpion (Mesobuthus tamulus concanesis, Pocock) venom prolongs repolarization time and refractoriness of the compound action potential of frog sciatic nerve in vitro through calcium dependent mechanism

Effects of Indian red scorpion (M. tamulus concanesis; MBT) venom on the compound action potential (CAP) of sciatic nerve in vitro were examined. MBT venom (0.1-6.0 micrograms/ml) prolonged the repolarization time and refractory period of the CAP in a concentration-dependent manner with maximal potentiation occurring at 6 micrograms/ml (about 100-200 times of the initial). At 1 microgram/ml of venom the prolongations were 40-50 times the initial durations and this concentration was used for subsequent experiments. Rise time, threshold, and conduction velocity of CAP were not altered by MBT venom (1 microgram/ml). In Ca(2+)-free medium, the venom-induced prolongations were only 2-6 times the initial response but addition of Ca2+ in the same medium then prolonged than by 50-70 times. The Ca2+ channel antagonists (nifedipine, 10 microM or Mg2+ ions, 5 mM) attenuated the venom (1 microgram/ml)-induced prolongation of repolarization time and refractory period. However, venom-induced prolongation of CAP responses were still significantly greater than the control in presence of these antagonists. The results indicate that MBT venom-induced increases in repolarization time and refractory period of the action potential greatly depend upon the presence of Ca2+ ions in the medium. The Ca2+ influx was through the L-type of Ca2+ channels.     

Purification and characterization of a unique, potent, peptidyl probe for the high conductance calcium-activated potassium channel from venom of the scorpion Buthus tamulus.

An inhibitor of the high conductance, Ca2(+)-activated K+ channel (PK,Ca) has been purified to homogeneity from venom of the scorpion Buthus tamulus by a combination of ion exchange and reversed-phase chromatography. This peptide, which has been named iberiotoxin (IbTX), is one of two minor components of the crude venom which blocks PK,Ca. IbTX consists of a single 4.3-kDa polypeptide chain, as determined by polyacrylamide gel electrophoresis, analysis of amino acid composition, and Edman degradation. Its complete amino acid sequence has been defined. IbTX displays 68% sequence homology with charybdotoxin (ChTX), another scorpion-derived peptidyl inhibitor of PK,Ca, and, like this latter toxin, its amino terminus contains a pyroglutamic acid residue. However, IbTX possesses 4 more acidic and 1 less basic amino acid residue than does ChTX, making this toxin much less positively charged than the other peptide. In single channel recordings, IbTX reversibly blocks PK,Ca in excised membrane patches from bovine aortic smooth muscle. It acts exclusively at the outer face of the channel and functions with an IC50 of about 250 pM. Block of channel activity appears distinct from that of ChTX since IbTX decreases both the probability of channel opening as well as the channel mean open time. IbTX is a selective inhibitor of PK,Ca; it does not block other types of voltage-dependent ion channels, especially other types of K+ channels that are sensitive to inhibition by ChTX. IbTX is a partial inhibitor of 125I-ChTX binding in bovine aortic sarcolemmal membrane vesicles (Ki = 250 pM). The maximal extent of inhibition that occurs is modulated by K+, decreasing as K+ concentration is raised, but K+ does not affect the absolute inhibitory potency of IbTX. A Scatchard analysis indicates that IbTX functions as a noncompetitive inhibitor of ChTX binding. Taken together, these data suggest that IbTX interacts at a distinct site on the channel and modulates ChTX binding by an allosteric mechanism. Therefore, IbTX defines a new class of peptidyl inhibitor of PK,Ca with unique properties that make it useful for investigating the characteristics of this channel in target tissues.     

The first potassium channel toxin from the venom of the Iranian scorpion Odonthobuthus doriae

The very first member of K(+) channels toxins from the venom of the Iranian scorpion Odonthobuthus doriae (OdK1) was purified, sequenced and characterized physiologically. OdK1 has 29 amino acids, six conserved cysteines and a pI value of 4.95. Based on multiple sequence alignments, OdK1 was classified as alpha-KTx 8.5. The pharmacological effects of OdK1 were studied on six different cloned K(+) channels (vertebrate Kv1.1-Kv1.5 and Shaker IR) expressed in Xenopus laevis oocytes. Interestingly, OdK1 selectively inhibited the currents through Kv1.2 channels with an IC50 value of 183+/-3 nM but did not affect any of the other channels.     

Cloning and characterization of a novel calcium channel toxin-like gene BmCa1 from Chinese scorpion Mesobuthus martensii Karsch

Many studies have been carried on peptides and genes encoding scorpion toxins from the venom of Mesobuthus martensii Karsch (synonym: Buthus martensii Karsch, BmK), such as Na+, K+ and Cl- channel modulators. In this study, a novel calcium channel toxin-like gene BmCa1 was isolated and characterized from the venom of Mesobuthus martensii Karsch. First, a partial cDNA sequence of the Ca2+ channel toxin-like gene was identified by random sequencing method from a venomous gland cDNA library of Mesobuthus martensii Karsch. The full-length sequence of BmCa1 was then obtained by 5'RACE technique. The peptide deduced from BmCa1 precursor nucleotide sequence contains a 27-residue signal peptide and a 37-residue mature peptide. Although BmCa1 and other scorpion toxins are different at the gene and protein primary structure levels, BmCa1 has the same precursor nucleotide organization and cysteine arrangement as that of the first subfamily members of calcium channel scorpion toxins. Genomic DNA sequence of BmCa1 was also cloned by PCR. Sequence analysis showed that BmCa1 gene consists of three exons separated by two introns of 72 bp and 1076 bp in length, respectively. BmCa1 is the first calcium channel toxin-like gene cloned from the venom of Mesobuthus martensii Karsch and potentially represents a novel class of calcium channel toxins in scorpion venoms.     

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.     

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.     

Probing the pH-dependent structural features of alpha-KTx12.1, a potassium channel blocker from the scorpion Tityus serrulatus

Potassium channels are widespread in living cells and are involved in many diseases. The scorpion toxin alpha-KTx(12.1) interacts with various K(+) channels, suggesting its capacity to match diverse channel pores. It is recognized that tissue injuries may affect the pH at toxins site of action, thereby modulating both protein conformation and activity. To better understand its molecular mechanism of action, we studied alpha-KTx(12.1) using pH as a tool to explore its plasticity and NMR in combination with MD calculations to detect it. The toxin solution structure consists of an alpha-helix and a triple-stranded beta-sheet stabilized by four disulfide bridges. The NMR results show, in addition, that His28 possesses an unusually low pK(a) of 5.2. The best set of protein conformers is obtained at pH 4.5, while at pH 7.0, the reduced number of NOEs resulting from a faster hydrogen exchange does not allow to reach a good structural convergence. Nonetheless, MD calculations show that the toxin structure does not vary significantly in that pH range, while conformational changes and modifications of the surface charge distribution occur when His28 is fully protonated. Moreover, essential dynamics analysis reveals variations in the toxin's coherent motions. In conclusion, His28, with its low pK(a) value, provides alpha-KTx(12.1) with the ability to preserve its active conformation over a wide pH interval, thus expanding the range of cellular conditions where the toxin can fully exhibit its activity. Overall, the results further underline the role of histidine as a natural controller of proteins' functionality.     

Hemitoxin, the first potassium channel toxin from the venom of the Iranian scorpion Hemiscorpius lepturus

Hemitoxin (HTX) is a new K+ channel blocker isolated from the venom of the Iranian scorpion Hemiscorpius lepturus. It represents only 0.1% of the venom proteins, and displaces [125 I]alpha-dendrotoxin from its site on rat brain synaptosomes with an IC50 value of 16 nm. The amino acid sequence of HTX shows that it is a 35-mer basic peptide with eight cysteine residues, sharing 29-69% sequence identity with other K+ channel toxins, especially with those of the alphaKTX6 family. A homology-based molecular model generated for HTX shows the characteristic alpha/beta-scaffold of scorpion toxins. The pairing of its disulfide bridges, deduced from MS of trypsin-digested peptide, is similar to that of classical four disulfide bridged scorpion toxins (Cys1-Cys5, Cys2-Cys6, Cys3-Cys7 and Cys4-Cys8). Although it shows the highest sequence similarity with maurotoxin, HTX displays different affinities for Kv1 channel subtypes. It blocks rat Kv1.1, Kv1.2 and Kv1.3 channels expressed in Xenopus oocytes with IC50 values of 13, 16 and 2 nM, respectively. As previous studies have shown the critical role played by the beta-sheet in Kv1.3 blockers, we suggest that Arg231 is also important for Kv1.3 versus Kv1.2 HTX positive discrimination. This article gives information on the structure-function relationships of Kv1.2 and Kv1.3 inhibitors targeting developing peptidic inhibitors for the rational design of new toxins targeting given K+ channels with high selectivity.     

Molecular characterization of a possible progenitor sodium channel toxin from the Old World scorpion Mesobuthus martensii

Toxins affecting sodium channels widely exist in the venoms of scorpions throughout the world. These molecules comprise an evolutionarily related peptide family with three shared features including conserved three-dimensional structure and gene organization, and similar function. Based on different pharmacological profiles and binding properties, scorpion sodium channel toxins are divided into alpha- and beta-groups. However, their evolutionary relationship is not yet established. Here, we report a gene isolated from the venom gland of scorpion Mesobuthus martensii which encodes a novel sodium channel toxin-like peptide of 64 amino acids, named Mesotoxin. The Mesotoxin gene is organized into three exons and two introns with the second intron location conserved across the family. This peptide is unusual in that it has only three disulfides and a long cysteine-free tail with loop size and structural characteristics close to beta-toxins. Evolutionary analysis favors its basal position in the origin of scorpion sodium channel toxins as a progenitor. The discovery of Mesotoxin will assist investigations into the key issue regarding the origin and evolution of scorpion toxins.     

A novel K+ channel blocking toxin from Tityus discrepans scorpion venom.

A novel toxin (TdK1) was purified from the venom of the scorpion Tityus discrepans, sequenced and functionally characterized. It contains 37 amino acid residues and blocks reversible the shakerB K+ channel expressed in SF9 cells with a Kd in the order of 280 nM. The proposed systematic nomenclature for this peptide is alpha-KTx4.3.