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

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

A maurotoxin with constrained standard disulfide bridging: innovative strategy of chemical synthesis,pharmacology, and docking on K+ channels

Maurotoxin (MTX) is a 34-residue toxin that has been isolated initially from the venom of the scorpion Scorpio maurus palmatus. It presents a large number of pharmacological targets, including small conductance Ca2+-activated and voltage-gated K+ channels. Contrary to other toxins of the alpha-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 alpha/beta scaffold of scorpion toxins. Here, we developed an innovative strategy of chemical peptide synthesis to produce an MTX variant (MTXPi1) with a conventional pattern of disulfide bridging without any alteration of the toxin chemical structure. This strategy was used solely to address the impact of half-cystine pairings on MTX structural properties and pharmacology. The data indicate that MTXPi1 displays some marked changes in affinities toward the target K+ channels. Computed docking analyses using molecular models of both MTXPi1 and the various voltage-gated K+ channel subtypes (Shaker B, Kv1.2, and Kv1.3) were found to correlate with MTXPi1 pharmacology. A functional map detailing the interaction between MTXPi1 and Shaker B channel was generated in line with docking experiments.     

Synthesis, 3-D structure, and pharmacology of a reticulated chimeric peptide derived from maurotoxin and Tsk scorpion toxins

Maurotoxin (MTX) is a 34-mer scorpion toxin cross-linked by four disulfide bridges that acts on both Ca(2+)-activated (SK) and voltage-gated (Kv) K(+) channels. A 38-mer chimera of MTX, Tsk-MTX, has been synthesized by the solid-phase method. It encompasses residues from 1 to 6 of Tsk at N-terminal, and residues from 3 to 34 of MTX at C-terminal. As established by enzyme cleavage, Tsk-MTX displays half-cystine pairings of the type C1-C5, C2-C6, C3-C7 and C4-C8 which, contrary to MTX, correspond to a disulfide bridge pattern common to known scorpion toxins. The 3-D structure of Tsk-MTX, solved by (1)H NMR, demonstrates that it adopts the alpha/beta scaffold of scorpion toxins. In vivo, Tsk-MTX is lethal by intracerebroventricular injection in mice (LD(50) value of 0.2 microg/mouse). In vitro, Tsk-MTX is as potent as MTX, or Tsk, to interact with apamin-sensitive SK channels of rat brain synaptosomes (IC(50) value of 2.5 nM). It also blocks voltage-gated K(+) channels expressed in Xenopus oocytes, but is inactive on rat Kv1.3 contrary to MTX.     

Disulfide bridge reorganization induced by proline mutations in maurotoxin

Maurotoxin (MTX) is a 34-residue toxin that has been isolated from the venom of the chactidae scorpion Scorpio maurus palmatus, and characterized. Together with Pi1 and HsTx1, MTX belongs to a family of short-chain four-disulfide-bridged scorpion toxins acting on potassium channels. However, contrary to other members of this family, MTX exhibits an uncommon disulfide bridge organization of the type C1-C5, C2-C6, C3-C4 and C7-C8, versus C1-C5, C2-C6, C3-C7 and C4-C8 for both Pi1 and HsTx1. Here, we report that the substitution of MTX proline residues located at positions 12 and/or 20, adjacent to C3 (Cys(13)) and C4 (Cys(19)), results in conventional Pi1- and HsTx1-like arrangement of the half-cystine pairings. In this case, this novel disulfide bridge arrangement is without obvious incidence on the overall three-dimensional structure of the toxin. Pharmacological assays of this structural analog, [A(12),A(20)]MTX, reveal that the blocking activities on Shaker B and rat Kv1.2 channels remain potent whereas the peptide becomes inactive on rat Kv1.3. These data indicate, for the first time, that discrete point mutations in MTX can result in a marked reorganization of the half-cystine pairings, accompanied with a novel pharmacological profile for the analog.     

Maurotoxin and the Kv1.1 channel: voltage-dependent binding upon enantiomerization of the scorpion toxin disulfide bridge Cys31-Cys34.

Maurotoxin (MTX) is a 34-amino acid polypeptide cross-linked by four disulfide bridges that has been isolated from the venom of the scorpion Scorpio maurus palmatus and characterized. Maurotoxin competed with radiolabeled apamin and kaliotoxin for binding to rat brain synaptosomes and blocked K+ currents from Kv1 channel subtypes expressed in Xenopus oocytes. Structural characterization of the synthetic toxin identified half-cystine pairings at Cys3-Cys24, Cys9-Cys29, Cys13-Cys19 and Cys31-Cys34 This disulfide bridge pattern is unique among known scorpion toxins, particularly the existence of a C-terminal '14-membered disulfide ring' (i.e. cyclic domain 31-34), We therefore studied structure-activity relationships by investigating the structure and pharmacological properties of synthetic MTX peptides either modified at the C-terminus ?i.e. MTX(1-29), [Abu31,34]-MTX and [Cys31,34, Tyr32]D-MTX) or mimicking the cyclic C-terminal domain [i.e. MTX(31-34)]. Unexpectedly, the absence of a disulfide bridge Cys31-Cys34 in [Abu 31,34]-MTX and MTX(1-29) resulted in MTX-unrelated half-cystine pairings of the three remaining disulfide bridges for the two analogs, which is likely to be responsible for their inactivity against Kv1 channel subtypes. Cyclic MTX(31-34) was also biologically inactive. [Cys31,34, Tyr32]D-MTX, which had a 'native', MTX-related, disulfide bridge organization, but a D-residue-induced reorientation of the C-terminal disulfide bridge, was potent at blocking the Kv1.1 channel. This peptide-induced Kv1.1 blockage was voltage-dependent (a property not observed for MTX), maximal in the low depolarization range and associated with on-rate changes in ligand binding. Thus, the cyclic C-terminal domain of MTX seems to be crucial for recognition of Kv1.3, and to a lesser extent, Kv1.2 channels and it may contribute to the stabilization and strength of the interaction between the toxin and the Kv1.1 channel.     

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

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

Increasing the molecular contacts between maurotoxin and Kv1.2 channel augments ligand affinity

Scorpion toxins interact with their target ion channels through multiple molecular contacts. Because a "gain of function" approach has never been described to evaluate the importance of the molecular contacts in defining toxin affinity, we experimentally examined whether increasing the molecular contacts between a toxin and an ion channel directly impacts toxin affinity. For this purpose, we focused on two scorpion peptides, the well-characterized maurotoxin with its variant Pi1-like disulfide bridging (MTX(Pi1)), used as a molecular template, and butantoxin (BuTX), used as an N-terminal domain provider. BuTX is found to be 60-fold less potent than MTX(Pi1) in blocking Kv1.2 (IC(50) values of 165 nM for BuTX versus 2.8 nM for MTX(Pi1)). Removal of its N-terminal domain (nine residues) further decreases BuTX affinity for Kv1.2 by 5.6-fold, which is in agreement with docking simulation data showing the importance of this domain in BuTX-Kv1.2 interaction. Transfer of the BuTX N-terminal domain to MTX(Pi1) results in a chimera with five disulfide bridges (BuTX-MTX(Pi1)) that exhibits 22-fold greater affinity for Kv1.2 than MTX(Pi1) itself, in spite of the lower affinity of BuTX as compared to MTX(Pi1). Docking experiments performed with the 3-D structure of BuTX-MTX(Pi1) in solution, as solved by (1)H-NMR, reveal that the N-terminal domain of BuTX participates in the increased affinity for Kv1.2 through additional molecular contacts. Altogether, the data indicate that acting on molecular contacts between a toxin and a channel is an efficient strategy to modulate toxin affinity.     

Evidence for domain-specific recognition of SK and Kv channels by MTX and HsTx1 scorpion toxins

Maurotoxin (MTX) and HsTx1 are two scorpion toxins belonging to the alpha-KTx6 structural family. These 34-residue toxins, cross-linked by four disulfide bridges, share 59% sequence identity and fold along the classical alpha/beta scaffold. Despite these structural similarities, they fully differ in their pharmacological profiles. MTX is highly active on small (SK) and intermediate (IK) conductance Ca(2+)-activated (K(+)) channels and on voltage-gated Kv1.2 channel, whereas HsTx1 potently blocks voltage-gated Kv1.1 and Kv1.3 channels only. Here, we designed and chemically produced MTX-HsTx1, a chimera of both toxins that contains the N-terminal helical region of MTX (sequence 1-16) and the C-terminal beta-sheet region of HsTx1 (sequence 17-34). The three-dimensional structure of the peptide in solution was solved by (1)H NMR. MTX-HsTx1 displays the activity of MTX on SK channel, whereas it exhibits the pharmacological profile of HsTx1 on Kv1.1, Kv1.2, Kv1.3, and IK channels. These data demonstrate that the helical region of MTX exerts a key role in SK channel recognition, whereas the beta-sheet region of HsTx1 is crucial for activity on all other channel types tested.     

Blockage of human T lymphocyte Kv1.3 channels by Pi1, a novel class of scorpion toxin

Using the patch-clamp technique we determined that Pandinus imperator toxin Pi1, a recently described peptide toxin having four disulfide bridges instead of the usual three in scorpion toxins, blocked Kv1.3 channels of human T lymphocytes from the extracellular side with a 1:1 stoichiometry. Kv1.3 block was instantaneous and removable with toxin-free extracellular solution. The toxin did not influence activation or inactivation of the channels. We found that Pi1 blocked Kv1.3 with less affinity (K(d) = 11.4 nM) than the structurally related three disulfide bridge containing toxins Pi2 (50 pM) and Pi3 (0.5 nM). The fourth disulfide bridge in Pi1 had no influence on the channel binding ability of the toxin; the less effective block was due to differences in amino acid side chain properties at positions 11 and 35.     

Synthesis and characterization of Pi4, a scorpion toxin from Pandinus imperator that acts on K+ channels.

Pi4 is a 38-residue toxin cross-linked by four disulfide bridges that has been isolated from the venom of the Chactidae scorpion Pandinus imperator. Together with maurotoxin, Pi1, Pi7 and HsTx1, Pi4 belongs to the alpha KTX6 subfamily of short four-disulfide-bridged scorpion toxins acting on K+ channels. Due to its very low abundance in venom, Pi4 was chemically synthesized in order to better characterize its pharmacology and structural properties. An enzyme-based cleavage of synthetic Pi4 (sPi4) indicated half-cystine pairings between Cys6-Cys27, Cys12-32, Cys16-34 and Cys22-37, which denotes a conventional pattern of scorpion toxin reticulation (Pi1/HsTx1 type). In vivo, sPi4 was lethal after intracerebroventricular injection to mice (LD50 of 0.2 microg per mouse). In vitro, addition of sPi4 onto Xenopus laevis oocytes heterologously expressing various voltage-gated K+ channel subtypes showed potent inhibition of currents from rat Kv1.2 (IC50 of 8 pm) and Shaker B (IC50 of 3 nm) channels, whereas no effect was observed on rat Kv1.1 and Kv1.3 channels. The sPi4 was also found to compete with 125I-labeled apamin for binding to small-conductance Ca(2+)-activated K+ (SK) channels from rat brain synaptosomes (IC50 value of 0.5 microm). sPi4 is a high affinity blocker of the Kv1.2 channel. The toxin was docked (BIGGER program) on the Kv channel using the solution structure of sPi4 and a molecular model of the Kv1.2 channel pore region. The model suggests a key role for residues Arg10, Arg19, Lys26 (dyad), Ile28, Lys30, Lys33 and Tyr35 (dyad) in the interaction and the associated blockage of the Kv1.2 channel.     

The impact of the fourth disulfide bridge in scorpion toxins of the alpha-KTx6 subfamily

Animal toxins are highly reticulated and structured polypeptides that adopt a limited number of folds. In scorpion species, the most represented fold is the alpha/beta scaffold in which an helical structure is connected to an antiparallel beta-sheet by two disulfide bridges. The intimate relationship existing between peptide reticulation and folding remains poorly understood. Here, we investigated the role of disulfide bridging on the 3D structure of HsTx1, a scorpion toxin potently active on Kv1.1 and Kv1.3 channels. This toxin folds along the classical alpha/beta scaffold but belongs to a unique family of short-chain, four disulfide-bridged toxins. Removal of the fourth disulfide bridge of HsTx1 does not affect its helical structure, whereas its two-stranded beta-sheet is altered from a twisted to a nontwisted configuration. This structural change in HsTx1 is accompanied by a marked decrease in Kv1.1 and Kv1.3 current blockage, and by alterations in the toxin to channel molecular contacts. In contrast, a similar removal of the fourth disulfide bridge of Pi1, another scorpion toxin from the same structural family, has no impact on its 3D structure, pharmacology, or channel interaction. These data highlight the importance of disulfide bridging in reaching the correct bioactive conformation of some toxins.     

Brownian dynamics simulations of the recognition of the scorpion toxin maurotoxin with the voltage-gated potassium ion channels

The recognition of the scorpion toxin maurotoxin (MTX) by the voltage-gated potassium (Kv1) channels, Kv1.1, Kv1.2, and Kv1.3, has been studied by means of Brownian dynamics (BD) simulations. All of the 35 available structures of MTX in the Protein Data Bank (http://www.rcsb.org/pdb) determined by nuclear magnetic resonance were considered during the simulations, which indicated that the conformation of MTX significantly affected both the recognition and the binding between MTX and the Kv1 channels. Comparing the top five highest-frequency structures of MTX binding to the Kv1 channels, we found that the Kv1.2 channel, with the highest docking frequencies and the lowest electrostatic interaction energies, was the most favorable for MTX binding, whereas Kv1.1 was intermediate, and Kv1.3 was the least favorable one. Among the 35 structures of MTX, the 10th structure docked into the binding site of the Kv1.2 channel with the highest probability and the most favorable electrostatic interactions. From the MTX-Kv1.2 binding model, we identified the critical residues for the recognition of these two proteins through triplet contact analyses. MTX locates around the extracellular mouth of the Kv1 channels, making contacts with its beta-sheets. Lys23, a conserved amino acid in the scorpion toxins, protrudes into the pore of the Kv1.2 channel and forms two hydrogen bonds with the conserved residues Gly401(D) and Tyr400(C) and one hydrophobic contact with Gly401(C) of the Kv1.2 channel. The critical triplet contacts for recognition between MTX and the Kv1.2 channel are Lys23(MTX)-Asp402(C)(Kv1), Lys27(MTX)-Asp378(D)(Kv1), and Lys30(MTX)-Asp402(A)(Kv1). In addition, six hydrogen-bonding interactions are formed between residues Lys23, Lys27, Lys30, and Tyr32 of MTX and residues Gly401, Tyr400, Asp402, Asp378, and Thr406 of Kv1.2. Many of them are formed by side chains of residues of MTX and backbone atoms of the Kv1.2 channel. Five hydrophobic contacts exist between residues Pro20, Lys23, Lys30 and Tyr32 of MTX and residues Asp402, Val404, Gly401, and Arg377 of the Kv1.2 channel. The simulation results are in agreement with the previous molecular biology experiments and explain the binding phenomena between MTX and Kv1 channels at the molecular level. The consistency between the results of the BD simulations and the experimental data indicated that our three-dimensional model of the MTX-Kv1.2 channel complex is reasonable and can be used in additional biological studies, such as rational design of novel therapeutic agents blocking the voltage-gated channels and in mutagenesis studies in both the toxins and the Kv1 channels. In particular, both the BD simulations and the molecular mechanics refinements indicate that residue Asp378 of the Kv1.2 channel is critical for its recognition and binding functionality toward MTX. This phenomenon has not been appreciated in the previous mutagenesis experiments, indicating this might be a new clue for additional functional study of Kv1 channels.     

Roles of disulfide bridges in scorpion toxin BmK M1 analyzed by mutagenesis.

The unique fold of scorpion toxins is a natural scaffold for protein engineering, in which multiple disulfide bonds are crucial structural elements. To understand the respective roles of these disulfide bridges, a mutagenesis analysis for the four disulfide bonds, 12-63, 16-36, 22-46 and 26-48, of a representative toxin BmK M1 from the scorpion Buthus martensii Karsch was carried out. All cysteines were replaced by serine with double mutations. The recombinant mutants were expressed in the Saccharomyces cerevisiae S-78 system. Toxic activities of the expressed mutants were tested on ICR mice in vivo and on neuronal Na+ channels (rNav1.2) by electrophysiological analysis. Recombinant variants M1 (C22S,C46S) and M1 (C26S,C48S) were not expressed at all; M1 (C16S,C36S) could be expressed at trace levels but was extremely unstable. Variant M1 (C12S,C63S) could be expressed in an amount comparable with that of unmodified rBmK M1, but had no detectable bioactivities. The results indicated that among the four disulfide bonds for long-chain scorpion toxins, loss of either bridge C22-C46 or C26-C48 is fatal for the general folding of the molecule. Bridge C16-C36 mainly contributes to the global stability of the folded scaffold, and bridge C12-C63 plays an essential role in the functional performance of scorpion toxins.     

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.     

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.     

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.     

Chemical synthesis and characterization of Pi1, a scorpion toxin from Pandinus imperator active on K+ channels.

Pi1 is a 35-residue toxin cross-linked by four disulfide bridges that has been isolated from the venom of the chactidae scorpion Pandinus imperator. Due to its very low abundance in the venom, we have chemically synthesized this toxin in order to study its biological activity. Enzyme-based proteolytic cleavage of the synthetic Pi1 (sPi1) demonstrates half-cystine pairings between Cys4-Cys25, Cys10-Cys30, Cys14-Cys32 and Cys20-Cys35, which is in agreement with the disulfide bridge organization initially reported on the natural toxin. In vivo, intracerebroventricular injection of sPi1 in mice produces lethal effects with an LD50 of 0.2 microgram per mouse. In vitro, the application of sPi1 induces drastic inhibition of Shaker B (IC50 of 23 nM) and rat Kv1.2 channels (IC50 of 0.44 nM) heterologously expressed in Xenopus laevis oocytes. No effect was observed on rat Kv1.1 and Kv1.3 currents upon synthetic peptide application. Also, sPi1 is able to compete with 125I-labeled apamin for binding onto rat brain synaptosomes with an IC50 of 55 pM. Overall, these results demonstrate that sPi1 displays a large spectrum of activities by blocking both SK- and Kv1-types of K+ channels; a selectivity reminiscent of that of maurotoxin, another structurally related four disulfide-bridged scorpion toxin that exhibits a different half-cystine pairing pattern.     

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.     

The investigation of interactions of kappa-Hefutoxin1 with the voltage-gated potassium channels: a computational simulation

Kappa-Hefutoxin1 is a K(+) channel-blocking toxin from the scorpion Heterometrus fluvipes. It is a 22-residue protein that adapts a novel fold of two parallel helices linked by two disulfide bridges without beta-sheets. Recognition of interactions of kappa-Hefutoxin1 with the voltage-gated potassium channels, Kv1.1, Kv1.2, and Kv1.3, was studied by 3D-Dock software package. All structures of kappa-Hefutoxin1 were considered during the simulations, which indicated that even small changes in the structure of kappa-Hefutoxin1 considerably affected both the recognition and the binding between kappa-Hefutoxin1 and the Kv1 channels. kappa-Hefutoxin1 is located around the extracellular part of the Kv1 channels, making contacts with its helices. Lys 19, Tyr 5, Arg 6, Trp 9, or Arg 10 in the toxin and residues Asp 402, His 404, Thr 407,Gly 401, and Asp 386 in each subunit of the Kv potassium channel are the key residues for the toxin-channel recognition. Moreover, the simulation result demonstrates that the hydrophobic interactions are important in interaction of negatively charged toxins with potassium channels. The results of our docking/molecular dynamics simulations indicate that our 3D model structure of the kappa-Hefutoxin1-complex is both reasonable and acceptable and could be helpful for smarter drug design and the blocking agents of Kv1 channels.