BmBKTx1, a novel Ca2+-activated K+ channel blocker purified from the Asian scorpion Buthus martensi Karsch

BmBKTx1 is a novel short chain toxin purified from the venom of the Asian scorpion Buthus martensi Karsch. It is composed of 31 residues and is structurally related to SK toxins. However, when tested on the cloned rat SK2 channel, it only partially inhibited rSK2 currents, even at a concentration of 1 microm. To screen for other possible targets, BmBKTx1 was then tested on isolated metathoracic dorsal unpaired median neurons of Locusta migratoria, in which a wide variety of ion channels are expressed. The results suggested that BmBKTx1 could specifically block voltage-gated Ca(2+)-activated K(+) currents (BK-type). This was confirmed by testing the BmBKTx1 effect on the alpha subunits of BK channels of the cockroach (pSlo), fruit fly (dSlo), and human (hSlo), heterologously expressed in HEK293 cells. The IC(50) for channel blocking by BmBKTx1 was 82 nm for pSlo and 194 nm for dSlo. Interestingly, BmBKTx1 hardly affected hSlo currents, even at concentrations as high as 10 microm, suggesting that the toxin might be insect specific. In contrast to most other scorpion BK blockers that also act on the Kv1.3 channel, BmBKTx1 did not affect this channel as well as other Kv channels. These results show that BmBKTx1 is a novel kind of blocker of BK-type Ca(2+)-activated K(+) channels. As the first reported toxin active on the Drosophila Slo channel dSlo, it will also greatly facilitate studying the physiological role of BK channels in this model organism.     

Kbot1, a three disulfide bridges toxin from Buthus occitanus tunetanus venom highly active on both SK and Kv channels

On attempts to identify toxins showing original profile of activity among K+ channels, we purified Kbot1, a scorpion toxin that blocks Kv1 and SK potassium channels. With 28 amino-acid residues, Kbot1 is the shortest toxin sequenced in Buthus occitanus scorpion. It is linked by three disulfide bridges and its primary structure is 93% identical to that of BmP02 isolated from the venom of the Chinese scorpion Buthus martensi Karsch [Eur. J. Biochem. 245 (1996) 457]. Kbot1 exhibited a low neurotoxicity in mice after intracerebroventricular injection (LD50 approximately or = 0.8 microg per mouse). It competes with iodinated apamin for its rat brain synaptosomal membrane-binding site (IC50 of 20 nM). Despite 30% sequence identity between Kbot1 and ChTX, competitive experiments on the [125I] charybdotoxin, show that Kbot1 inhibits its binding to its rat brain synaptosomes with IC50 of 10 nM. This result was supported by electrophysiological experiments on cloned voltage-dependent K+ channels from rat brain, expressed in Xenopus oocytes. Kbot1 blocks Kv1.1, Kv1.2 and Kv1.3 currents with IC50 of 145, 2.5 and 15 nM, respectively. Based on these data, Kbot1 may be considered as the first member of subfamily 9 of scorpion toxins [Trends Pharmacol. Sci. 20 (1999) 444], highly active on both Kv and SK channels.     

Kbot55, purified from Buthus occitanus tunetanus venom,represents the first member of a novel α-KTx subfamily

Kbot55 is a 39 amino acid peptide isolated from the venom of the Tunisian scorpion Buthus occitanus tunetanus. This peptide is cross-linked by 3 disulfide bridges and has a molecular mass of 4128.65 Da. Kbot55 is very low represented in the venom and thus represents a challenge for biochemical characterization. In this study, Kbot55 has been subjected to a screening on ion channels expressed in Xenopus laevis oocytes. It was found that Kbot55 targets voltage-gated potassium channels with high affinity. Kbot55 shows very low amino acid identity with other scorpion potassium toxins and therefore was considered a bona fide novel type of scorpion toxin. Sequence alignment analysis indicated that Kbot55 is the first representative of the new α-Ktx31 subfamily and therefore was classified as α-Ktx31.1.     

Solution structure of BmBKTx1, a new BKCa1 channel blocker from the Chinese scorpion Buthus martensi Karsch

BmBKTx1 is a 31-amino acid peptide identified from the venom of the Chinese scorpion Buthus martensi Karsch, blocking high-conductance calcium-activated potassium channels. Sequence homology analysis indicates that BmBKTx1 is a new subfamily of short-chain alpha-KTx toxins of the potassium channel, which we term alpha-KTx19. Synthetic BmBKTx1 was prepared by using solid-phase peptide synthesis. Two-dimensional NMR spectroscopy techniques were used to determine the solution structure of BmBKTx1. The results show that the BmBKTx1 forms a typical cysteine-stabilized alpha/beta scaffold adopted by most short-chain scorpion toxins. The structure of BmBKTx1 consists of a two-stranded antiparallel beta-sheet (residues 20-29) and an alpha-helix (residues 5-15). The three-dimensional structure of BmBKTx1 was also compared with those of two function-related scorpion toxins, charybdotoxin (ChTx) and BmTx1, and their structural and functional implications are discussed.     

Structure–Activity Relationship of a Highly Selective Peptidyl Inhibitor of Kv1.3 Voltage-Gated K+-Channel from Scorpion (B. sindicus) Venom

Venomous fauna of the world is a unique source of protein and peptide toxins with a wide range of pharmacological and physiological activities targeting in particular the bio-signaling system. Thus the most widely known source of peptidyl neurotoxins used for callipering different ion channels (e.g. Na⁺, K⁺, Ca⁺⁺ or Cl^? etc.) as well as many medicinally important peptides which have been accepted as drugs or are in clinical trials originate from the venom of scorpions of Buthidae family. In the present study, structure–activity relationship of highly selective short-chain neurotoxin from scorpion Buthus sindicus (a common yellow scorpion of Sindh, Pakistan) has been established. The toxin named as Bs-KTx6 (4,115.4 Da) has been isolated, synthesized and found to be a potent as well as selective inhibitor of voltage gated potassium channel Kv1.3 (IC₅₀ = 7.7 pM). The structural studies on channel selective modulators like Bs-KTx6 may serve as a possible template in establishing libraries of peptidyl toxins for treating diseases such as multiple sclerosis, type-1 diabetes mellitus, rheumatoid arthritis, allergic contact dermatitis, bone resorption due to periodontitis and delayed type hypersensitivity. It is also worth mentioning that the peptide based drugs have excellent safety profiles as well as selectivity compared to other non-protein molecules. In conclusion, identified peptide Bs-KTx6 is a specific as well as very selective blocker of Kv1.3 and provides a starting template for the synthesis of peptidyl drugs to treat autoimmune diseases.     

A positive charge at the N-terminal segment of Discrepin increases the blocking effect of K channels responsible for the IA currents in cerebellum granular cells

Discrepin is a scorpion peptide that blocks preferentially the I_A currents of the voltage-dependent K⁺ channel of rat cerebellum granular cells. It was isolated from the venom of the buthid scorpion Tityus discrepans and contains 38 amino acid residues with a pyroglutamic acid at the N-terminal site. Discrepin has the lowest sequence identity (approx. 50%) among the six members of the α-KTx15 sub-family of scorpion toxins. In order to find out which residues are important for the blocking effects of Discrepin, six mutants were chemically synthesized (V6K, I19R, D20K, T35V, I19R-D20K, I19R-D20K-R21V), correctly folded and their physiological properties were examined. Substitution of residues V6 and D20 for basically charged amino acids increases the blocking activity of Discrepin, specially the mutation V6K at the N-terminal segment of the toxin. Analysis of 3D-structure models of the mutants V6K and D20K supports the idea that basic residues improve their blocking activities, similarly to what happens with BmTx3, a toxic peptide obtained from Buthus martensi scorpion, which has the highest known blocking effects of I_A currents in K⁺ channels of rat cerebellum granular cells.     

Martentoxin:一种大电导钙激活钾离子通道的独有配体(英文)

大电导钙激活钾离子(BK)通道广泛分布于可兴奋细胞与非兴奋细胞中,行使着一系列重要的生理功能。以源于蝎粗毒的高亲和性毒素作为研究工具,使BK通道的药理学和结构性质正逐步被揭示。Martentoxin是一种分离提取自东亚短钳蝎(Buthus martensi Karsch)粗毒的短链多肽,由37个氨基酸残基构成。研究表明,其对BK通道的特异性远高于其它各类型的电压门控钾通道(Kv)。迄今为止,由于用以探明BK通道亚型结构与功能及相关病理的特异性药物工具仍然稀缺,因此阐明martentoxin与BK通道间的相互作用模式就显得至关重要了。鉴于此原因,本综述将针对martentoxin的药理性质和其与BK通道相互作用的分子机制做进一步阐明。     

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.     

Isolation and characterization of SsmTx‐I,a Specific Kv2.1 blocker from the venom of the centipede Scolopendra Subspinipes Mutilans L. Koch

Scolopendra subspinipes mutilans, also known as Chinese red‐headed centipede, is a venomous centipede from East Asia and Australasia. Venom from this animal has not been researched as thoroughly as venom from snakes, snails, scorpions, and spiders. In this study, we isolated and characterized SsmTx‐I, a novel neurotoxin from the venom of S. subspinipes mutilans. SsmTx‐I contains 36 residues with four cysteines forming two disulfide bonds. It had low sequence similarity (<10%) with other identified peptide toxins. By whole‐cell recording, SsmTx‐I significantly blocked voltage‐gated K⁺ channels in dorsal root ganglion neurons with an IC₅₀ value of 200 nM, but it had no effect on voltage‐gated Na⁺ channels. Among the nine K⁺ channel subtypes expressed in human embryonic kidney 293 cells, SsmTx‐I selectively blocked the Kv2.1 current with an IC₅₀ value of 41.7 nM, but it had little effect on currents mediated by other K⁺ channel subtypes. Blockage of Kv2.1 by SsmTx‐I was not associated with significant alteration of steady‐state activation, suggesting that SsmTx‐I might act as a simple inhibitor or channel blocker rather than a gating modifier. Our study reported a specific Kv2.1‐blocker from centipede venom and provided a basis for future investigations of SsmTx‐I, for example on structure–function relationships, mechanism of action, and pharmacological potential. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Use of toxins to study potassium channels

Potassium channels comprise groups of diverse proteins which can be distinguished according to each member's biophysical properties. Some types of K⁺ channels are blocked with high affinity by specific peptidyl toxins. Three toxins, charybdotoxin, iberiotoxin, and noxiustoxin, which display a high degree of homology in their primary amino acid sequences, have been purified to homogeneity from scorpion venom. While charybdotoxin and noxiustoxin are known to inhibit more than one class of channel (i.e., several Ca²⁺-activated and voltage-dependent K⁺ channels), iberiotoxin appears to be a selective blocker of the high-conductance, Ca²⁺-activated K⁺ channel that is present in muscle and neuroendocrine tissue. A distinct class of small-conductance Ca²⁺-activated K⁺ channel is blocked by two other toxins, apamin and leiurotoxin-1, that share no sequence homology with each other. A family of homologous toxins, the dendrotoxins, have been purified from venom of various related species of snakes. These toxins inhibit several inactivating voltage-dependent K⁺ channels. Although molecular biology approaches have been employed to identify and characterize several species of voltagegated K⁺ channels, toxins directed against a particular channel can still be useful in defining the physiological role of that channel in a particular tissue. In addition, for those K⁺ channels which are not yet successfully probed by molecular biology techniques, toxins can be used as biochemical tools with which to purify the target protein of interest.     

Potassium Channel Modulation by a Toxin Domain in Matrix Metalloprotease 23

Peptide toxins found in a wide array of venoms block K⁺ channels, causing profound physiological and pathological effects. Here we describe the first functional K⁺ channel-blocking toxin domain in a mammalian protein. MMP23 (matrix metalloprotease 23) contains a domain (MMP23_TxD) that is evolutionarily related to peptide toxins from sea anemones. MMP23_TxD shows close structural similarity to the sea anemone toxins BgK and ShK. Moreover, this domain blocks K⁺ channels in the nanomolar to low micromolar range (Kv1.6 > Kv1.3 > Kv1.1 = Kv3.2 > Kv1.4, in decreasing order of potency) while sparing other K⁺ channels (Kv1.2, Kv1.5, Kv1.7, and KCa3.1). Full-length MMP23 suppresses K⁺ channels by co-localizing with and trapping MMP23_TxD-sensitive channels in the ER. Our results provide clues to the structure and function of the vast family of proteins that contain domains related to sea anemone toxins. Evolutionary pressure to maintain a channel-modulatory function may contribute to the conservation of this domain throughout the plant and animal kingdoms.     

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.     

Electrophysiological characterization of Tityus obscurus β toxin 1 (To1) on Na+-channel isoforms

To1, previously named Tc49b, is a peptide neurotoxin isolated from venom of the scorpion Tityus obscurus that is responsible for lethal human poisoning cases in the Brazilian Amazonian region. Previously, To1 was shown to be lethal to mice and to change Na⁺ permeation in cerebellum granular neurons from rat brain. In addition, To1 did not affect Shaker B K⁺ channels. Based on sequence similarities, To1 was described as a β-toxin. In the present work, To1 was purified from T. obscurus venom and submitted to an electrophysiological characterization in human and invertebrate Na_V channels. The analysis of the electrophysiological experiments reveal that To1 enhances the open probability at more negative potentials of human Na_V 1.3 and 1.6, of the insect channel BgNa_V1 and of arachnid VdNa_V1 channel. In addition, To1 reduces the peak of Na⁺ currents in some of the Na_Vs tested. These results support the classification of the To1 as a β-toxin. A structure and functional comparison to other β-toxins that share sequence similarity to To1 is also presented.     

Functional evolution of scorpion venom peptides with an inhibitor cystine knot fold

The ICK (inhibitor cystine knot) defines a large superfamily of polypeptides with high structural stability and functional diversity. Here, we describe a new scorpion venom-derived K⁺ channel toxin (named λ-MeuKTx-1) with an ICK fold through gene cloning, chemical synthesis, nuclear magnetic resonance spectroscopy, Ca²⁺ release measurements and electrophysiological recordings. λ-MeuKTx-1 was found to adopt an ICK fold that contains a three-strand anti-parallel β-sheet and a 3₁₀-helix. Functionally, this peptide selectively inhibits the Drosophila Shaker K⁺ channel but is not capable of activating skeletal-type Ca²⁺ release channels/ryanodine receptors, which is remarkably different from the previously known scorpion venom ICK peptides. The removal of two C-terminal residues of λ-MeuKTx-1 led to the loss of the inhibitory activity on the channel, whereas the C-terminal amidation resulted in the emergence of activity on four mammalian K⁺ channels accompanied by the loss of activity on the Shaker channel. A combination of structural and pharmacological data allows the recognition of three putative functional sites involved in channel blockade of λ-MeuKTx-1. The presence of a functional dyad in λ-MeuKTx-1 supports functional convergence among scorpion venom peptides with different folds. Furthermore, similarities in precursor organization, exon–intron structure, 3D-fold and function suggest that scorpion venom ICK-type K⁺ channel inhibitors and Ca²⁺ release channel activators share a common ancestor and their divergence occurs after speciation between buthidae and non-buthids. The structural and functional characterizations of the first scorpion venom ICK toxin with K⁺ channel-blocking activity sheds light on functionally divergent and convergent evolution of this conserved scaffold of ancient origin.     

Gating currents from neuronal KV7.4 Channels: General features and correlation with the ionic conductance

The K_V7 (KCNQ) subfamily of voltage-gated K⁺ channels consists of five members (K_V7.1- K_V7.5) giving rise to non-inactivating, and slowly activating/deactivating currents mainly expressed in cardiac (K_V7.1) and neuronal (K_V7.2- K_V7.5) tissue. In the present study, using the cut-open oocyte voltage clamp, we studied the relation of the ionic currents from homomeric neuronal Kv7 channels (K_V7.2-K_V7.5) with the gating currents recorded after K⁺ conductance blockade from the same channels. Increasing the recording temperature from 18{degree sign}C to 28{degree sign}C accelerated activation/deactivation kinetics of the ionic currents in all homomeric K_V7 channels (activation Q10s at 0 mV were 3.8, 4.1, 8.3, and 2.8 for Kv7.2, Kv7.3, Kv7.4 and Kv7.5 channels, respectively), without large changes in currents voltage-dependence; moreover, at 28{degree sign}C, ionic currents carried by K_V7.4 channels also showed a significant increase in their maximal value. Gating currents were only resolved in K_V7.4 and K_V7.5 channels; the size of the ON gating charges at +40 mV was 1.34 ± 0.34 nC for K_V7.4, and 0.79 ± 0.20 nC for K_V7.5. At 28{degree sign}C, K_V7.4 gating currents had the following salient properties: 1) similar time integral of QON and QOFF, indicating no charge immobilization; 2) a left-shift in the V1/2 of the QON/V when compared to the G/V (≈ 50 mV in the presence of 2 mM extracellular Ba²⁺); 3) a QON decay faster than ionic current activation; and 4) a rising phase in the OFF gating charge after depolarizations larger than 0 mV. These observations suggest that, in K_V7.4 channels, VSD movement is followed by a slow and/or low bearing charge step linking to pore opening, a result which may help to clarify the molecular consequence of disease-causing mutations and drugs affecting channel gating.     

Kv1.3 potassium channel-blocking toxin Ctri9577, novel gating modifier of Kv4.3 potassium channel from the scorpion toxin family

Scorpion toxin Ctri9577, as a potent Kv1.3 channel blocker, is a new member of the α-KTx15 subfamily which are a group of blockers for Kv4.x potassium channels. However, the pharmacological function of Ctri9577 for Kv4.x channels remains unknown. Scorpion toxin Ctri9577 was found to effectively inhibit Kv4.3 channel currents with IC₅₀ value of 1.34 ± 0.03 μM. Different from the mechanism of scorpion toxins as the blocker recognizing channel extracellular pore entryways, Ctri9577 was a novel gating modifier affecting voltage dependence of activation, steady-state inactivation, and the recovery process from the inactivation of Kv4.3 channel. However, Ctri9755, as a potent Kv1.3 channel blocker, was found not to affect voltage dependence of activation of Kv1.3 channel. Interestingly, pharmacological experiments indicated that 1 μM Ctri9755 showed less inhibition on Kv4.1 and Kv4.2 channel currents. Similar to the classical gating modifier of spider toxins, Ctri9577 was shown to interact with the linker between the transmembrane S3 and S4 helical domains through the mutagenesis experiments. To the best of our knowledge, Ctri9577 was the first gating modifier of potassium channels among scorpion toxin family, and the first scorpion toxin as both gating modifier and blocker for different potassium channels. These findings further highlighted the structural and functional diversity of scorpion toxins specific for the potassium channels.     

To4, the first Tityus obscurus β-toxin fully electrophysiologically characterized on human sodium channel isoforms

Many scorpion toxins that act on sodium channels (NaScTxs) have been characterized till date. These toxins may act modulating the inactivation or the activation of sodium channels and are named α- or β-types, respectively. Some venom toxins from Tityus obscurus (Buthidae), a scorpion widely distributed in the Brazilian Amazon, have been partially characterized in previous studies; however, little information about their electrophysiological role on sodium ion channels has been published. In the present study, we describe the purification, identification and electrophysiological characterization of a NaScTx, which was first described as Tc54 and further fully sequenced and renamed To4. This toxin shows a marked β-type effect on different sodium channel subtypes (hNa_v1.1–hNa_v1.7) at low concentrations, and has more pronounced activity on hNa_v1.1, hNa_v1.2 and hNa_v1.4. By comparing To4 primary structure with other Tityus β-toxins which have already been electrophysiologically tested, it is possible to establish some key amino acid residues for the sodium channel activity. Thus, To4 is the first toxin from T. obscurus fully electrophysiologically characterized on different human sodium channel isoforms.     

Peptidomimetic Star Polymers for Targeting Biological Ion Channels

Four end-functionalized star polymers that could attenuate the flow of ionic currents across biological ion channels were first de novo designed computationally, then synthesized and tested experimentally on mammalian K⁺ channels. The 4-arm ethylene glycol conjugate star polymers with lysine or a tripeptide attached to the end of each arm were specifically designed to mimic the action of scorpion toxins on K⁺ channels. Molecular dynamics simulations showed that the lysine side chain of the polymers physically occludes the pore of Kv1.3, a target for immuno-suppression therapy. Two of the compounds tested were potent inhibitors of Kv1.3. The dissociation constants of these two compounds were computed to be 0.1 μM and 0.7 μM, respectively, within 3-fold to the values derived from subsequent experiments. These results demonstrate the power of computational methods in molecular design and the potential of star polymers as a new infinitely modifiable platform for ion channel drug discovery.     

Ts17, a Tityus serrulatus β-toxin structurally related to α-scorpion toxins

Ts17 was purified from the venom of the scorpion Tityus serrulatus, the most dangerous scorpion species in Brazil. The activity on Na_v1.1-Na_v1.7 channels was electrophysiologically characterized by patch-clamp technique. Ts17 amino acid sequence indicated high similarity to alpha-scorpion toxins; however, it presented beta-toxin activity, altering the kinetics of the Na⁺-channels. The most affected subtypes during activation (with and without prepulse) and inactivation phases were Na_v1.2 and Na_v1.5, respectively. For recovery from inactivation, the most affected voltage-gated sodium channel was Na_v1.5. Circular dichroism spectra showed that Ts17 presents mainly β-sheet and unordered structures at all analyzed pHs, and the maximum value of α-helix was found at pH 4.0 (13.3 %). Based on the results, Ts17 might be used as a template to develop a new cardiac drug.Key contributionPurification of Ts17 from Tityus serrulatus, electrophysiological characterization of Ts17 on voltage-gated sodium channel subtypes, β-toxin classification.