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.     

Unique Mechanism of the Interaction between Honey Bee Toxin TPNQ and rKir1.1 Potassium Channel Explored by Computational Simulations: Insights into the Relative Insensitivity of Channel towards Animal Toxins

Background

The 21-residue compact tertiapin-Q (TPN_Q) toxin, a derivative of honey bee toxin tertiapin (TPN), is a potent blocker of inward-rectifier K⁺ channel subtype, rat Kir1.1 (rKir1.1) channel, and their interaction mechanism remains unclear.

Principal Findings

Based on the flexible feature of potassium channel turrets, a good starting rKir1.1 channel structure was modeled for the accessibility of rKir1.1 channel turrets to TPN_Q toxin. In combination with experimental alanine scanning mutagenesis data, computational approaches were further used to obtain a reasonable TPN_Q toxin-rKir1.1 channel complex structure, which was completely different from the known binding modes between animal toxins and potassium channels. TPN_Q toxin mainly adopted its helical domain as the channel-interacting surface together with His12 as the pore-blocking residue. The important Gln13 residue mainly contacted channel residues near the selectivity filter, and Lys20 residue was surrounded by a polar “groove” formed by Arg118, Thr119, Glu123, and Asn124 in the channel turret. On the other hand, four turrets of rKir1.1 channel gathered to form a narrow pore entryway for TPN_Q toxin recognition. The Phe146 and Phe148 residues in the channel pore region formed strong hydrophobic protrusions, and produced dominant nonpolar interactions with toxin residues. These specific structure features of rKir1.1 channel vestibule well matched the binding of potent TPN_Q toxin, and likely restricted the binding of the classical animal toxins.

Conclusions/Significance

The TPN_Q toxin-rKir1.1 channel complex structure not only revealed their unique interaction mechanism, but also would highlight the diverse animal toxin-potassium channel interactions, and elucidate the relative insensitivity of rKir1.1 channel towards animal toxins.     

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.     

Expanding the pharmacological profile of κ-hefutoxin 1 and analogues: A focus on the inhibitory effect on the oncogenic channel Kv10.1

Peptide toxins, such as scorpion peptides, are interesting lead compounds in the search for novel drugs. In this paper, the focus is on the scorpion peptide κ-hefutoxin 1. This peptide displays a cysteine-stabilized helix-loop-helix fold (CSα/α) and is known to be a weak K_v1.x inhibitor. Due to the low affinity of κ-hefutoxin 1 for these channels, it is assumed that the main target(s) of κ-hefutoxin 1 remain(s) unknown. In order to identify novel targets, electrophysiological measurements and antifungal assays were performed. The effect of κ-hefutoxin 1 was previously evaluated on a panel of 11 different voltage-gated potassium channels. Here, we extended this target screening with the oncogenic potassium channel K_v10.1. κ-Hefutoxin 1 was able to inhibit this channel in a dose-dependent manner (IC₅₀ ∼ 26 μM). Although the affinity is rather low, this is the first peptide toxin ever described to be a K_v10.1 inhibitor. The structure-activity relationship of κ-hefutoxin 1 on K_v10.1 was investigated by testing eight κ-hefutoxin 1 variants using the two-electrode voltage clamp technique. Several important amino acid residues were identified; the functional dyad residues (Tyr⁵ and Lys¹⁹), N-terminal residues (Gly¹ and His²) and the amidated C-terminal residue (Cys²²). Since the CSα/α fold is also found in a class of antifungal plant peptides, the α-hairpinines, we investigated the antifungal activity of κ-hefutoxin 1. κ-Hefutoxin 1 showed low activity against the plant pathogen Fusarium culmorum and no activity against three other yeast and fungal species, even at high concentrations (∼100 μM).     

Refined solution structure of the anti-mammal and anti-insect LqqIII scorpion toxin: Comparison with other scorpion toxins

The solution structure of the anti-mammal and anti-insect LqqIII toxin from the scorpion Leiurus quinquestriatus quinquestriatuswas refined and compared with other long-chain scorpion toxins. This structure, determined by ¹H-NMR and molecular modeling, involves an α-helix (18–29) linked to a three-stranded β-sheet (2–6, 33–39, and 43–51) by two disulfide bridges. The average RMSD between the 15 best structures and the mean structure is 0.71 Å for Cα atoms. Comparison between LqqIII, the potent anti-mammal AaHII, and the weakly active variant-3 toxins revealed that the LqqIII three-dimensional structure is closer to that of AaHII than to the variant-3 structure. Moreover, striking analogies were observed between the electrostatic and hydrophobic potentials of LqqIII and AaHII. Several residues are well conserved in long-chain scorpion toxin sequences and seem to be important in protein structure stability and function. Some of them are involved in the CSαβ (Cysteine Stabilized α-helix β-sheet) motif. A comparison between the sequences of the RII rat brain and the Drosophila extracellular loops forming scorpion toxin binding-sites of Na⁺ channels displays differences in the subsites interacting with anti-mammal or anti-insect toxins. This suggests that hydrophobic as well as electrostatic interactions are essential for the binding and specificity of long-chain scorpion toxins. Proteins 28:360–374, 1997 © 1997 Wiley-Liss, Inc.     

Analysis of the interacting surface of maurotoxin with the voltage‐gated Shaker B K+ channel

Maurotoxin (MTX) is a 34‐residue toxin that was isolated initially from the venom of the scorpion Scorpio maurus palmatus. Unlike the other toxins of the α‐KTx6 family (Pi1, Pi4, Pi7, and HsTx1), MTX exhibits a unique disulfide bridge organization of the type C₁? C₅, C₂? C₆, C₃? C₄, and C₇? C₈ (instead of the conventional C₁? C₅, C₂? C₆, C₃? C₇, and C₄? C₈, herein referred to as Pi1‐like) that does not prevent its folding along the classic α/β scaffold of scorpion toxins. MTX_Pi1 is an MTX variant with a conventional pattern of disulfide bridging without any primary structure alteration of the toxin. Here, using MTX and/or MTX_Pi1 as models, we investigated how the type of folding influences toxin recognition of the Shaker B potassium channel. Amino acid residues of MTX that were studied for Shaker B recognition were selected on the basis of their homologous position in charybdotoxin, a three disulfide‐bridged scorpion toxin also active on this channel type. These residues favored either an MTX‐ or MTX_Pi1‐like folding. Our data indicate clearly that Lys²³ and Tyr³² (two out of ten amino acid residues studied) are the most important residues for Shaker B channel blockage by MTX. For activity on SKCa channels, the same amino acid residues also affect, directly or indirectly, the recognition of SK channels. The molecular modeling technique and computed docking indicate the existence of a correlation between the half cystine pairings of the mutated analogs and their activity on the Shaker B K⁺ channel. Overall, mutations in MTX could, or could not, change the reorganization of disulfide bridges of this molecule without affecting its α/β scaffold. However, changing of the peptide backbone (cross linking disulfide bridges from MTX‐like type vs MTX_Pi1‐like type) appears to have less impact on the molecule activity than mutation of certain key amino acids such as Lys²³ and Tyr³² in this toxin. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Hg1, novel peptide inhibitor specific for Kv1.3 channels from first scorpion Kunitz-type potassium channel toxin family

The potassium channel Kv1.3 is an attractive pharmacological target for autoimmune diseases. Specific peptide inhibitors are key prospects for diagnosing and treating these diseases. Here, we identified the first scorpion Kunitz-type potassium channel toxin family with three groups and seven members. In addition to their function as trypsin inhibitors with dissociation constants of 140 nM for recombinant LmKTT-1a, 160 nM for LmKTT-1b, 124 nM for LmKTT-1c, 136 nM for BmKTT-1, 420 nM for BmKTT-2, 760 nM for BmKTT-3, and 107 nM for Hg1, all seven recombinant scorpion Kunitz-type toxins could block the Kv1.3 channel. Electrophysiological experiments showed that six of seven scorpion toxins inhibited ~50-80% of Kv1.3 channel currents at a concentration of 1 μM. The exception was rBmKTT-3, which had weak activity. The IC(50) values of rBmKTT-1, rBmKTT-2, and rHg1 for Kv1.3 channels were ~129.7, 371.3, and 6.2 nM, respectively. Further pharmacological experiments indicated that rHg1 was a highly selective Kv1.3 channel inhibitor with weak affinity for other potassium channels. Different from classical Kunitz-type potassium channel toxins with N-terminal regions as the channel-interacting interfaces, the channel-interacting interface of Hg1 was in the C-terminal region. In conclusion, these findings describe the first scorpion Kunitz-type potassium channel toxin family, of which a novel inhibitor, Hg1, is specific for Kv1.3 channels. Their structural and functional diversity strongly suggest that Kunitz-type toxins are a new source to screen and design potential peptides for diagnosing and treating Kv1.3-mediated autoimmune diseases.     

Structural and functional diversity of acidic scorpion potassium channel toxins

Background

Although the basic scorpion K⁺ channel toxins (KTxs) are well-known pharmacological tools and potential drug candidates, characterization the acidic KTxs still has the great significance for their potential selectivity towards different K⁺ channel subtypes. Unfortunately, research on the acidic KTxs has been ignored for several years and progressed slowly.

Principal Findings

Here, we describe the identification of nine new acidic KTxs by cDNA cloning and bioinformatic analyses. Seven of these toxins belong to three new α-KTx subfamilies (α-KTx28, α-KTx29, and α-KTx30), and two are new members of the known κ-KTx2 subfamily. ImKTx104 containing three disulfide bridges, the first member of the α-KTx28 subfamily, has a low sequence homology with other known KTxs, and its NMR structure suggests ImKTx104 adopts a modified cystine-stabilized α-helix-loop-β-sheet (CS-α/β) fold motif that has no apparent α-helixs and β-sheets, but still stabilized by three disulfide bridges. These newly described acidic KTxs exhibit differential pharmacological effects on potassium channels. Acidic scorpion toxin ImKTx104 was the first peptide inhibitor found to affect KCNQ1 channel, which is insensitive to the basic KTxs and is strongly associated with human cardiac abnormalities. ImKTx104 selectively inhibited KCNQ1 channel with a K_d of 11.69 µM, but was less effective against the basic KTxs-sensitive potassium channels. In addition to the ImKTx104 toxin, HeTx204 peptide, containing a cystine-stabilized α-helix-loop-helix (CS-α/α) fold scaffold motif, blocked both Kv1.3 and KCNQ1 channels. StKTx23 toxin, with a cystine-stabilized α-helix-loop-β-sheet (CS-α/β) fold motif, could inhibit Kv1.3 channel, but not the KCNQ1 channel.

Conclusions/Significance

These findings characterize the structural and functional diversity of acidic KTxs, and could accelerate the development and clinical use of acidic KTxs as pharmacological tools and potential drugs.     

Binding of scorpion and sea anemone neurotoxins to a common site related to the action potential Na+ ionophore in neuroblastoma cells.

The protein neurotoxin II from the venom of the scorpion $\text{Androctonus}\text{australis}$ Hector was labeled with ¹²⁵I by the lactoperoxidase method to a specific radioactivity of about 100 μCi/μg without loss of biological activity. The labeled neurotoxin binds specifically to a single class of non intereacting binding sites of high affinity (K_D = 0.3 – 0.6 nM) and low capacity (4000 – 8000 sites/cell) to electrically excitable neuroblastoma cells. Relation of these sites to the action potential Na⁺ channel is derived from identical concentration dependence of scorpion toxin binding and increase in duration and amplitude of action potential. The protein neurotoxin II from the sea anemone $\text{Anemona sulcata}$ also affects the closing of the action potential Na⁺ ionophore in nerve axons. The unlabelled sea anemone toxin modifies ¹²⁵I-labeled scorpion toxin II binding to neuroblastoma cells by increasing the apparent K_D for labeled scorpion toxin without modification of the number of binding sites. It is concluded that both $\text{Androctonus}$ scorpion toxin II and $\text{Anemona}$ sea anemone toxin II interact competitively with a regulatory component of the action potential Na⁺ channel.     

hERG Potassium Channel Blockage by Scorpion Toxin BmKKx2 Enhances Erythroid Differentiation of Human Leukemia Cells K562

Background

The hERG potassium channel can modulate the proliferation of the chronic myelogenous leukemic K562 cells, and its role in the erythroid differentiation of K562 cells still remains unclear.

Principal Findings

The hERG potassium channel blockage by a new 36-residue scorpion toxin BmKKx2, a potent hERG channel blocker with IC₅₀ of 6.7±1.7 nM, enhanced the erythroid differentiation of K562 cells. The mean values of GPA (CD235a) fluorescence intensity in the group of K562 cells pretreated by the toxin for 24 h and followed by cytosine arabinoside (Ara-C) treatment for 72 h were about 2-fold stronger than those of K562 cells induced by Ara-C alone. Such unique role of hERG potassium channel was also supported by the evidence that the effect of the toxin BmKKx2 on cell differentiation was nullified in hERG-deficient cell lines. During the K562 cell differentiation, BmKKx2 could also suppress the expression of hERG channels at both mRNA and protein levels. Besides the function of differentiation enhancement, BmKKx2 was also found to promote the differentiation-dependent apoptosis during the differentiation process of K562 cells. In addition, the blockage of hERG potassium channel by toxin BmKKx2 was able to decrease the intracellular Ca²⁺ concentration during the K562 cell differentiation, providing an insight into the mechanism of hERG potassium channel regulating this cellular process.

Conclusions/Significance

Our results revealed scorpion toxin BmKKx2 could enhance the erythroid differentiation of leukemic K562 cells via inhibiting hERG potassium channel currents. These findings would not only accelerate the functional research of hERG channel in different leukemic cells, but also present the prospects of natural scorpion toxins as anti-leukemic drugs.     

Biochemical and physiological characterization of a new Na+-channel specific peptide from the venom of the Argentinean scorpion Tityus trivittatus

A new peptide with 61 amino acids cross-linked by 4 disulfide bridges, with molecular weight of 6938.12 Da, and an amidated C-terminal amino acid residue was purified and characterized. The primary structure was obtained by direct Edman degradation and sequencing its gene. The peptide is lethal to mammals and was shown to be similar (95% identity) to toxin Ts1 (gamma toxin) from the Brazilian scorpion Tityus serrulatus; it was named Tt1g (from T. trivittatus toxin 1 gamma-like). Tt1g was assayed on several sub-types of Na⁺-channels showing displacement of the currents to more negative voltages, being the hNav1.3 the most affected channel. This toxin displays characteristics typical to the β-type sodium scorpion toxins. Lethality tests and physiological assays indicate that this peptide is probably the most important toxic component of this species of scorpion, known for causing human fatalities in the South American continent.     

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.     

The binding of the insect selective neurotoxin (AaIT) from scorpion venom to locust synaptosomal membranes

The binding of the radioiodinated insect selective neurotoxin from the venom of the scorpion Androctonus australis (AaIT), to synaptic plasma membrane vesicles derived from osmotically shocked insect synaptosomes was studied under kinetic and equilibrium conditions. The integrity of these vesicles and the existence of membrane potential and its modifiability were demonstrated by assays of the uptake of the lipophilic cation tetraphenylphosphonium. It has been shown that ¹²⁵I-labeled AaIT binds specifically and reversibly to a single class of noninteracting binding sites of high affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}{}^1\text{= 1.2–3}\text{nM}$) and low capacity (1.2–2.0 pmol/mg protein). The values of the rate association and dissociation constants k₁ and k_?1 are, respectively, 1.36 · 10⁶ M^?1 · s^?1 and 1.9 · 10^?3 s^?1, and are in a good accordance with the equilibrium constant. The use of various ionophores and changes in external potassium concentration shown to modify the membrane potential of the present neuronal preparation, did not affect the binding of ¹²⁵I-AaIT, thus indicating its voltage-independence. Veratridine, tetrodotoxin, sea anemone toxin and the α and β scorpion toxins specific for vertebrates did not affect the binding of ¹²⁵I-AaIT. Furthermore, the above scorpion toxins were devoid of specific binding to the present insect neuronal preparation. Two additional insect toxins derived from the venom of the scorpion Buthotus judaicus, BjIT₁ (spastic-excitatory toxin, homologus to the AaIT) and BjIT₂ (flaccidity inducing-depressory toxin), were both shown to displace the ¹²⁵I-AaIT with a high affinity (K_d = 2.2 and 1.3 nM, respectively). These data are compared and discussed in light of the information concerning the interaction of scorpion venom toxins affecting vertebrates with mammalian neuronal tissues.     

Structural basis of a potent peptide inhibitor designed for Kv1.3 channel, a therapeutic target of autoimmune disease

The potassium channel Kv1.3 is an attractive pharmacological target for immunomodulation of T cell-mediated autoimmune diseases. Potent and selective blockers of Kv1.3 are potential therapeutics for treating these diseases. Here we describe the design of a new peptide inhibitor that is potent and selective for Kv1.3. Three residues (Gly(11), Ile(28), and Asp(33)) of a scorpion toxin BmKTX were substituted by Arg(11), Thr(28), and His(33), resulting in a new peptide, named ADWX-1. The ADWX-1 peptide blocked Kv1.3 with picomolar affinity (IC(50), 1.89 pM), showing a 100-fold increase in activity compared with the native BmKTX toxin. The ADWX-1 also displayed good selectivity on Kv1.3 over related Kv1.1 and Kv1.2 channels. Furthermore, alanine-scanning mutagenesis was carried out to map the functional residues of ADWX-1 in blocking Kv1.3. Moreover, computational simulation was used to build a structural model of the ADWX-1-Kv1.3 complex. This model suggests that all mutated residues are favorable for both the high potency and selectivity of ADWX-1 toward Kv1.3. While Arg(11) of ADWX-1 interacts with Asp(386) in Kv1.3, Thr(28) and His(33) of ADWX-1 locate right above the selectivity filter-S6 linker of Kv1.3. Together, our data indicate that the specific ADWX-1 peptide would be a viable lead in the therapy of T cell-mediated autoimmune diseases, and the successful design of ADWX-1 suggests that rational design based on the structural model of the peptide-channel complex should accelerate the development of diagnostic and therapeutic agents for human channelopathies.     

Reconstitution of High-Affinity Binding of a β-Scorpion Toxin to Neurotoxin Receptor Site 4 on Purified Sodium Channels

Abstract: Reconstitution of purified sodium channels into phospholipid vesicles restores many aspects of sodium channel function including high-affinity neurotoxin binding and action at neurotoxin receptor sites 1–3 and 5, but neurotoxin binding and action at receptor site 4 has not previously been demonstrated in purified and reconstituted preparations. Toxin IV from the venom of the American scorpion Centruroides suffusus suffusus (Css IV), a β-scorpion toxin, shifts the voltage dependence of sodium channel activation by binding with high affinity to neurotoxin receptor site 4. Sodium channels were purified from rat brain and reconstituted into phospholipid vesicles composed of phosphatidylcholine and phosphatidylethanolamine (65:35). ¹²⁵I-Css IV, purified by reversed-phase HPLC, bound rapidly and specifically to reconstituted sodium channels. Dissociation of the bound toxin was biphasic with half-times of 0.22 min^?1 and 0.015 min^?1. At equilibrium, the toxin bound to two classes of specific high-affinity sites, a variable minor class with K_D of ～0.1 nM and a major class with a K_D of ～5 nM. Approximately 0.8 mol ¹²⁵I-Css IV was bound per mole of reconstituted, right-side-out sodium channels, as assessed from comparison of binding of saxitoxin and Css IV. Binding of Css IV was unaffected by membrane potential or by neurotoxins that bind at sites 1–3 or 5, consistent with the characteristics of binding of β-scorpion toxins to sodium channels in cells and membrane preparations. Our results show that specific, high-affinity binding at neurotoxin receptor site 4 on purified sodium channels can be restored by reconstitution into phospholipid vesicles and provide an experimental approach to analysis of the peptide components of the toxin receptor site.     

Structural and functional consequences of the presence of a fourth disulfide bridge in the scorpion short toxins: solution structure of the potassium channel inhibitor HsTX1

We have determined the three-dimensional structure of the potassium channel inhibitor HsTX1, using nuclear magnetic resonance and molecular modeling. This protein belongs to the scorpion short toxin family, which essentially contains potassium channel blockers of 29 to 39 amino acids and three disulfide bridges. It is highly active on voltage-gated Kv1.3 potassium channels. Furthermore, it has the particularity to possess a fourth disulfide bridge. We show that HsTX1 has a fold similar to that of the three-disulfide-bridged toxins and conserves the hydrophobic core found in the scorpion short toxins. Thus, the fourth bridge has no influence on the global conformation of HsTX1. Most residues spatially analogous to those interacting with voltage-gated potassium channels in the three-disulfide-bridged toxins are conserved in HsTX1. Thus, we propose that Tyr21, Lys23, Met25, and Asn26 are involved in the biological activity of HsTX1. As an additional positively charged residue is always spatially close to the aromatic residue in toxins blocking the voltage-gated potassium channels, and as previous mutagenesis experiments have shown the critical role played by the C-terminus in HsTX1, we suggest that Arg33 is also important for the activity of the four disulfide-bridged toxin. Docking calculations confirm that, if Lys23 and Met25 interact with the GYGDMH motif of Kv1.3, Arg33 can contact Asp386 and, thus, play the role of the additional positively charged residue of the toxin functional site. This original configuration of the binding site of HsTX1 for Kv1.3, if confirmed experimentally, offers new structural possibilities for the construction of a molecule blocking the voltage-gated potassium channels.     

Characterization of three "Birtoxin-like" toxins from the Androctonus amoreuxi scorpion venom

The venom of the North African scorpion Androctonus amoreuxi (Aam) was analyzed using a combination of gel filtration, C18 reverse phase HPLC together with mass spectrometry analysis and bioassays. Three novel Birtoxin-like (BTX-L) peptides of 58 amino acid residues comprising three disulfide bridges were isolated and chemically characterized. One peptide, AamBTX-L3, induced serious toxic symptoms in mice and was lethal at nanogram quantities using intracerebroventricular injection. The three BTX-L peptides were tested in competition experiments on rat brain synaptosomes against the ¹²⁵I-labeled “classical” α- and β-toxins of reference, as well as with the ¹²⁵I-KTX, a voltage-gated potassium channel blocker. Only AamBTX-L3 was able to prevent the equilibrium binding of the β-toxin ¹²⁵I-Css IV to its receptor site 4 with a IC₅₀ value of 189 nM. Even if previous electrophysiological data allowed the classification of other BTX-L peptides among the β-type toxins, this report clearly shows that AamBTX-L3 is pharmacologically a β-toxin, which recognizes the voltage-gated Na⁺ (Na_v) channels from central mammalian neurons. In order to uncover the residues functionally essential for interaction between the AamBTX-L3 with the putative receptor site of ¹²⁵I-Css IV on Na_v1.2, molecular models of the three novel Aam BTX-L molecules were made and their surfaces were compared to the already described Css IV biologically interactive surfaces. A hypothesis is given that in BTX-L3, three residues found in the α-helix play a key role during target binding.     

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.     

Computational simulations of interactions of scorpion toxins with the voltage-gated potassium ion channel

Based on a homology model of the Kv1.3 potassium channel, the recognitions of the six scorpion toxins, viz. agitoxin2, charybdotoxin, kaliotoxin, margatoxin, noxiustoxin, and Pandinus toxin, to the human Kv1.3 potassium channel have been investigated by using an approach of the Brownian dynamics (BD) simulation integrating molecular dynamics (MD) simulation. Reasonable three-dimensional structures of the toxin-channel complexes have been obtained employing BD simulations and triplet contact analyses. All of the available structures of the six scorpion toxins in the Research Collaboratory for Structural Bioinformatics Protein Data Bank determined by NMR were considered during the simulation, which indicated that the conformations of the toxin significantly affect both the molecular recognition and binding energy between the two proteins. BD simulations predicted that all the six scorpion toxins in this study use their beta-sheets to bind to the extracellular entryway of the Kv1.3 channel, which is in line with the primary clues from the electrostatic interaction calculations and mutagenesis results. Additionally, the electrostatic interaction energies between the toxins and Kv1.3 channel correlate well with the binding affinities (-logK(d)s), R(2) = 0.603, suggesting that the electrostatic interaction is a dominant component for toxin-channel binding specificity. Most importantly, recognition residues and interaction contacts for the binding were identified. Lys-27 or Lys-28, residues Arg-24 or Arg-25 in the separate six toxins, and residues Tyr-400, Asp-402, His-404, Asp-386, and Gly-380 in each subunit of the Kv1.3 potassium channel, are the key residues for the toxin-channel recognitions. This is in agreement with the mutation results. MD simulations lasting 5 ns for the individual proteins and the toxin-channel complexes in a solvated lipid bilayer environment confirmed that the toxins are flexible and the channel is not flexible in the binding. The consistency between the results of the simulations and the experimental data indicated that our three-dimensional models of the toxin-channel complex are reasonable and can be used as a guide for future biological studies, such as the rational design of the blocking agents of the Kv1.3 channel and mutagenesis in both toxins and the Kv1.3 channel. Moreover, the simulation result demonstrates that the electrostatic interaction energies combined with the distribution frequencies from BD simulations might be used as criteria in ranking the binding configuration of a scorpion toxin to the Kv1.3 channel.