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Background The peptide discrepin from the α-KTx15 subfamily of scorpion toxins preferentially affects transient A-type potassium currents, which regulate many aspects of neuronal function in the central nervous system. However, the specific Kv channel targeted by discrepin and the molecular mechanism of interaction are still unknown. Methods Different variant peptides of discrepin were chemically synthesized and their effects were studied using patch clamp technique on rat cerebellum granular cells (CGC) and HEK cells transiently expressing Kv4.3 channels. Results Functional analysis indicated that nanomolar concentrations of native discrepin blocked Kv4.3 expressed channels, as previously observed in CGC. Similarly, the apparent affinities of all mutated peptides for Kv4.3 expressed channels were analogous to those found in CGC. In particular, in the double variant [V6K, D20K] the apparent affinity increased about 10-fold, whereas in variants carrying a deletion (ΔK13) or substitution (K13A) at position K13, the blockage was removed and the apparent affinity decreased more than 20-fold. Conclusion These results indicate that Kv4.3 is likely the target of discrepin and highlight the importance of the basic residue K13, located in the α-helix of the toxin, for current blockage. [ABSTRACT FROM AUTHOR]

Abstract: This communication is a revision of the state-of-the-art knowledge of the field of scorpion toxins specific for the K+-channels, responsible for the I A currents of granular cells of rat cerebellum, maintained in vitro culture. There are 6 members of the sub-family α-KTx15 known to affect the I A currents. They are: toxins Aa1 from Androctonus australis Garzoni, BmTx3 from Buthus martensi Karch, AmmTx3 from Androctonus mauretanicus mauretanicus, AaTx1 and AaTx2 from A. australis Garzoni and Discrepin from Tityus discrepans. They share high sequence similarity, apart from Discrepin, which causes an irreversible effect on the I A currents and is the most thoroughly studied toxin of the sub-family α-KTx15. The three-dimensional structure of Discrepin was determined and a series of mutants were synthesized and assayed in the system with the aim of identifying possible amino acids or sequence segments responsible for the irreversible effect found. In this revision some unpublished original data are also included to foster future work on the field, as well as a short discussion on some relevant aspects still pending and possible limitations associated with the strategy proposed. [Copyright &y& Elsevier]

Abstract: While α-KTx peptides are generally known for their modulation of the Shaker-type and the Ca2+-activated potassium channels, γ-KTxs are associated with hERG channels modulation. An exception to the rule is BmTx3 which belongs to subfamily α-KTx15 and can block hERG channels. To explain the peculiar behavior of BmTx3, a tentative “hot spot” formed of 2 basic residues (R18 and K19) was suggested but never further studied [Huys I, et al. BmTx3, a scorpion toxin with two putative functional faces separately active on A-type K+ and HERG currents. Biochem J 2004;378:745–52]. In this work, we investigated if the “hot spot” is a commonality in subfamily α-KTx15 by testing the effect of (AmmTx3, Aa1, discrepin). Furthermore, single mutations altering the “hot spot” in discrepin, have introduced for the very first time a hERG blocking activity to a previously non-active α-KTx. Additionally, we could extend our results to other α-KTx subfamily members belonging to α-KTx1, 4 and 6, therefore, the “hot spot” represents a common pharmacophore serving as a predictive tool for yet to be discovered α-KTxs. [Copyright &y& Elsevier]

The affinity of scorpion α-toxins for various voltage-gated sodium channels (Navs) differs considerably despite similar structures and activities. It has been proposed that key bioactive residues of the five-residue-turn (residues 8–12) and the C-tail form the NC domain, whose topology is dictated by a cis or trans peptide-bond conformation between residues 9 and 10, which correlates with the potency on insect or mammalian Navs. We examined this hypothesis using Lqh3, an α-like toxin from Leiurus quinquestriatus hebraeus that is highly active in insects and mammalian brain. Lqh3 exhibits slower association kinetics to Navs compared with other α-toxins and its binding to insect Navs is pH-dependent. Mutagenesis of Lqh3 revealed a bi-partite bioactive surface, composed of the Core and NC domains, as found in other α-toxins. Yet, substitutions at the five-residue turn and stabilization of the 9–10 bond in the cis conformation did not affect the activity. However, substitution of hydrogen-bond donors/acceptors at the NC domain reduced the pH-dependency of toxin binding, while retaining its high potency at Drosophila Navs expressed in Xenopus oocytes. Based on these results and the conformational flexibility and rearrangement of intramolecular hydrogen-bonds at the NC domain, evident from the known solution structure, we suggest that acidic pH or specific mutations at the NC domain favor toxin conformations with high affinity for the receptor by stabilizing the bound toxin-receptor complex. Moreover, the C-tail flexibility may account for the slower association rates and suggests a novel mechanism of dynamic conformer selection during toxin binding, enabling α-like toxins to affect a broad range of Navs. [ABSTRACT FROM AUTHOR]

Magi 5, from the hexathelid spider Macrothele gigas, is a 29-residue polypeptide containing three disulfide bridges. It binds specifically to receptor site 4 on mammalian voltage-gated sodium channels and competes with scorpion β-toxins, such as Css IV from Centruroides suffusus suffusus. As a consequence, Magi 5 shifts the activation voltage of the mammalian rNav1.2a channel to more hyperpolarized voltages, whereas the insect channel, DmNav1, is not affected. To gain insight into toxin-channel interactions, Magi 5 and 23 analogues were synthesized. The three-dimensional structure of Magi 5 in aqueous solution was determined, and its voltage-gated sodium channel-binding surfaces were mapped onto this structure using data from electrophysiological measurements on a series of Ala-substituted analogues. The structure clearly resembles the inhibitor cystine knot structural motif, although the triple-stranded β-sheet typically found in that motif is partially distorted in Magi 5. The interactive surface of Magi 5 toward voltage-gated sodium channels resembles in some respects the Janus-faced atracotoxins, with functionally important charged residues on one face of the toxin and hydrophobic residues on the other. Magi 5 also resembles the scorpion β-toxin Css IV, which has distinct nonpolar and charged surfaces that are critical for channel binding and has a key Glu involved in voltage sensor trapping. These two distinct classes of toxin, with different amino acid sequences and different structures, may utilize similar groups of residues on their surface to achieve the common end of modifying voltage-gated sodium channel function. [ABSTRACT FROM AUTHOR]

Discrepin, isolated from the venom of the Venezuelan scorpion Tityus discrepans, blocks preferentially the IA currents of the voltage-dependent K+ channel of rat cerebellum granular cells in an irreversible way. It contains 38 amino acid residues with a pyroglutamic acid as the N-terminal residue [D'Suze, G., Batista, C. V., Frau, A., Murgia, A. R., Zamudio, F. Z., Sevcik, C., Possani, L. D., and Prestipino, G. (2004) Arch. Biochem. Biophys. 430, 256–63]. It is the most distinctive member of the α-KTx15 subfamily of scorpion toxins. Six members of the α-KTx15 subfamily have been reported so far to be specific for this subtype of the K+ channel; however, none of them have had their three-dimensional structure determined, and no information for the residues possibly involved in channel recognition and binding is available. Natural discrepin (n-discrepin) was prepared from scorpion venom, and its synthetic analogue (s-discrepin) was obtained by solid-phase synthesis. Analysis of two-dimensional ¹H NMR spectra of n- and s-discrepin indicates that both peptides have the same structure. Here we report the solution structure of s-discrepin determined by NMR using 565 meaningful distance constraints derived from the volume integration of the two-dimensional NOESY spectrum, 22 dihedrals, and three hydrogen bonds. Discrepin displays the α/β scaffold, characteristic of scorpion toxins. Some features of the proposed interacting surface between the toxin and channel as well as the opposite ‘α-helix surface’ are discussed in comparison with those of other α-KTx15 members. Both n- and s-discrepin exhibit similar physiological actions as verified by patch-clamp and binding and displacement experiments. [ABSTRACT FROM AUTHOR]

δ-Palutoxins from the spider Paracoelotes luctuosus (Araneae: Amaurobiidae) are 36-37 residue long peptides that show preference for insect sodium channels (NaChs) and modulate their function. Although they slow NaCh inactivation in a fashion similar to that of receptor site 3 modifiers, such as scorpion α-toxins, they actually bind with high affinity to the topologically distinct receptor site 4 of scorpion β-toxins. To resolve this riddle, we scanned by Ala mutagenesis the surface of δ-PaluIT2, a δ-palutoxin variant with the highest affinity for insect NaChs, and compared it to the bioactive surface of a scorpion β-toxin. We found three regions on the surface of δ-PaluIT2 important for activity: the first consists of Tyr-22 and Tyr-30 (aromatic), Ser-24 and Met-28 (polar), and Arg-8, Arg-26, Arg-32, and Arg-34 (basic) residues; the second is made of Trp-12; and the third is made of Asp-l9, whose substitution by Ala uncoupled the binding from toxicity to lepidopteran larvae. Although spider δ-palutoxins and scorpion β-toxins have developed from different ancestors, they show some commonality in their bioactive surfaces, which may explain their ability to compete for an identical receptor (site 4) on voltage-gated NaChs. Yet, their different mode of channel modulation provides a novel perspective about the structural relatedness of receptor sites 3 and 4, which until now have been considered topologically distinct. [ABSTRACT FROM AUTHOR]

Many kinds of venomous principles modulate physiological responses of mammalian signal transduction systems, on which they act selectively as enhancers, inhibitors or some other kind of effectors. These toxins have become useful tools for physiological research. We have characterized paralyzing toxins from the venom of spiders, scorpions, insects, jellyfishes and sea anemones in the subtropical region including the Ryukyu Islands. Venom profiles are screened by MALDI-TOF fingerprinting analysis prior to purification of the venomous components, then marked target toxins of small molecular mass (1000–5000) are characterized directly by means of mass spectrometric techniques such as Frit-FAB MS/MS, PSD/CID-TOF MS, Capil. -HPLC/Q-TOF MS/MS etc. The proteinous toxins of jellyfish or sea anemone are characterized by RT-PCR technique by the information of the cleaved peptides after the protein was hydrolyzed by appropriate peptidase and the sequence of the cleaved peptide was determined by conventional methods. The venom of Araneid spider is mainly composed of a mixture of closely related acylpolyamines. More than 90 polyamine toxins were identified from one venom sac of the Madagascan spider, Nephilengys borbonica, by Frit-Fab MS/MS employing charge remote fragmentation technique. A novel polyamine toxin was also found from the rare wondering spider, Macrothele gigas from Iriomote Island. The structure of the toxin is an analog of polyamine toxin found in trapdoor spiders. Many kinds of cystine-rich peptides showing various types of ion channel antagonism have been isolated from spiders. A series of toxins possessing the same mode of cystine knots was recently isolated from the saliva of assassin bugs, Peirates turpis, Isyndus obscurus, Agriophodrus dohrni. These toxins act as calcium channel blocker. Most of the scorpion toxins reported are from scorpions hazardous to humans, and they belong to the major super family Buthoidea. Among them are the well-known genera, such as Buthus, Androctonus, Centruroides, Leiurus, or Tytius. We have investigated the minor group of scorpions from the super family Chactoidea (Scorpionidae, Ishnuridae). The venoms of these scorpions, involving the genera Heterometrus, Pandinus, Opisthacanthus, and Isometrus, contain different kinds of peptide toxins. Fingerprinting peptide analysis of the toxin profiles for these scorpions showed some difference from the profiles of Buthoidea scorpions. These venoms contain linear pore-forming peptides and 2-cystine-bridged toxins in addition to 4-cystine-bridged toxins. The most hazardous jellyfish in Okinawa, Chiropsalmus quadrigatus, and the related box jellyfishes, Carybdea rastoni, C. alata, contain quite labile proteinaceous toxins, CqTX, CrTX and CaTX, respectively. The toxins were inactivated by adding an organic solvent such as methanol or acetonitrile, by changing the pH of the toxin solution, dialyzing the toxin solution, storing the toxin in a refrigerator, or by lyophilizing the toxin solution. However, the toxic activity was retained in the presence of sodium chloride. We purified the jellyfish toxins by adding sodium chloride through all steps of the purification procedure and finally obtained the whole primary amino acid sequence of the toxin by RT-PCR method. The toxic protein CqTX is homologous to the other box jelly fish toxin, CrTX and CaTX. These toxins belong to a new class of proteins since they show no homology to known proteins. Another notorious and dangerous specimen in the Ryukyu Islands is Phyllodiscus semori. The venom is composed of three kinds of proteins (PsTX-20A, PsTX-60A, PsTX-60B). PsTX-20A shows homology to the proteinaceous toxin actinoporin, a cytolytic protein isolated from the genus Actinia, but PsTX-60s has no homology to any ever cloned proteins. Further elucidation of the mechanism of toxic action of these Coelenterates is in progress. [ABSTRACT FROM AUTHOR]

Six peptide toxins (Magi 1–6) were isolated from the Hexathelidae spider Macrothele gigas. The amino acid sequences of Magi 1, 2, 5 and 6 have low similarities to the amino acid sequences of known spider toxins. The primary structure of Magi 3 is similar to the structure of the palmitoylated peptide named PlTx-II from the North American spider Plectreurys tristis (Plectreuridae). Moreover, the amino acid sequence of Magi 4, which was revealed by cloning of its cDNA, displays similarities to the Na+ channel modifier δ-atracotoxin from the Australian spider Atrax robustus (Hexathelidae). Competitive binding assays using several 125I-labelled peptide toxins clearly demonstrated the specific binding affinity of Magi 1–5 to site 3 of the insect sodium channel and also that of Magi 5 to site 4 of the rat sodium channel. Only Magi 6 did not compete with the scorpion toxin LqhαIT in binding to site 3 despite high toxicity on lepidoptera larvae of 3.1 nmol/g. The Kis of other toxins were between 50 pM for Magi 4 and 1747 nM for Magi 1. In addition, only Magi 5 binds to both site 3 in insects (Ki=267 nM) and site 4 in rat brain synaptosomes (Ki=1.2 nM), whereas it showed no affinities for either mammal binding site 3 or insect binding site 4. Magi 5 is the first spider toxin with binding affinity to site 4 of a mammalian sodium channel. [Copyright &y& Elsevier]

Describes the solution structure of the toxin Ptu1 from the Assassin bug Peirates turpis that blocks the voltage-sensitive N-type calcium channels using the conventional two-dimensional nuclear magnetic resonance techniques followed by distance-geometry and molecular dynamics. Analysis of the electrostatic charge's repartition; Sequence of Ptu1; Comparison of Ptu1 with other related toxins.

Spider venoms include various peptide toxins that modify the ion currents, mainly of excitable insect cells. Consequently, scientific research on spider venoms has revealed a broad range of peptide toxins with different pharmacological properties, even for mammal species. In this work, thirty animal venoms were screened against hKv1.5, a potential target for atrial fibrillation therapy. The whole venom of the spider Oculicosa supermirabilis , which is also insecticidal to house crickets, caused voltage-gated potassium ion channel modulation in hKv1.5. Therefore, a peptide from the spider O. supermirabilis venom, named Osu1, was identified through HPLC reverse-phase fractionation. Osu1 displayed similar biological properties as the whole venom; so, the primary sequence of Osu1 was elucidated by both of N-terminal degradation and endoproteolytic cleavage. Based on its primary structure, a gene that codifies for Osu1 was constructed de novo from protein to DNA by reverse translation. A recombinant Osu1 was expressed using a pQE30 vector inside the E. coli SHuffle expression system. recombinant Osu1 had voltage-gated potassium ion channel modulation of human hKv1.5, and it was also as insecticidal as the native toxin. Due to its novel primary structure, and hypothesized disulfide pairing motif, Osu1 may represent a new family of spider toxins. [ABSTRACT FROM AUTHOR]

Abstract: The three-dimensional structures of the long-chain mammalian scorpion β-toxin CssII from Centruroides suffusus suffusus and of its recombinant form, HisrCssII, were determined by NMR. The neurotoxin CssII (nCssII) is a 66 amino acid long peptide with four disulfide bridges; it is the most abundant and deadly toxin from the venom of this scorpion. Both native and recombinant CssII structures were determined by nuclear magnetic resonance using a total of 828 sequential distance constraints derived from the volume integration of the cross peaks observed in 2D NOESY spectra. Both nCssII and HisrCssII structures display a mixed α/β fold stabilized by four disulfide bridges formed between pairs of cysteines: C1–C8, C2–C5, C3–C6, and C4–C7 (the numbers indicate the relative positions of the cysteine residues in the primary structure), with a distortion induced by two cis-prolines in its C-terminal part. The native CssII electrostatic surface was compared to both the recombinant one and to the Cn2 toxin, from the scorpion Centruroides noxius, which is also toxic to mammals. Structural features such N- and C-terminal differences could influence toxin specificity and affinity towards isoforms of different sub-types of Nav channels. [Copyright &y& Elsevier]

Abstract: CssII is a β-scorpion peptide that modifies preferentially sodium currents of the voltage-dependent Na+ channel (Nav) sub-type 1.6. Previously, we have found that the C-terminal amidation of CssII increases its affinity for Nav, which opens at more negative potentials in the presence of CssII. Although C-terminal amidation in vitro conditions is possible, five CssII peptide toxin variants with C-terminal residues modified were heterogously expressed (rN66S, rN66H, rN66R, r[T64R/N66S] and r[T64R/N66R], in which r stands for recombinant, the capital letters to the amino acid residues and the numbers indicate the position of the given residue into the primary sequence of the toxin) and correctly folded. A secondary structure prediction of CssII agrees with the experimental secondary structure obtained by circular dichroism; so all bacterial expressed neurotoxin variants maintained the typical α/β secondary structure motif of most Na+ channel scorpion toxins. The electrophysiological properties of all recombinant variants were examined, and it was found that substitutions of threonine (T) and asparagine (N) at the C-terminal region for arginine (R) (r[T64R/N66R]) increase their affinity for Nav1.6. Although, the molecular interactions involved in this mechanism are still not clearly determined, there is experimental evidence supporting the suspicion that incorporation of basic charged amino acid residues at the C-terminal tail of a group of α-scorpion toxin was favored by natural selection. [ABSTRACT FROM AUTHOR]

Magi 4, now renamed δ-hexatoxin-Mg1a, is a 43-residue neurotoxic peptide from the venom of the hexathelid Japanese funnel-web spider (Macrothele gigas) with homology to δ-hexatoxins from Australian funnel-web spiders. It binds with high affinity to receptor site 3 on insect voltage-gated sodium (Nay) channels but, unlike δ-hexatoxins, does not compete for the related site 3 in rat brain despite being previously shown to be lethal by intracranial injection. To elucidate differences in Nav channel selectivity, we have undertaken the first characterization of a peptide toxin on a broad range of mammalian and insect Nav channel subtypes showing that 5-hexatoxin-Mgla selectively slows channel inactivation of mammalian Nav1.1, Nav1.3, and Nav1.6 but more importantly shows higher affinity for insect Nav1 (para) channels. Consequently, δ-hexatoxin-Mg1a induces tonic repetitive firing of nerve impulses in insect neurons accompanied by plateau potentials. In addition, we have chemically synthesized and folded δ-hexatoxin-Mg1a, ascertained the bonding pattern of the four disuffides, and determined its three-dimensional solution structure using NMR spectroscopy. Despite modest sequence homology, we show that key residues important for the activity of scorpion α-toxins and δ-hexatoxins are distributed in a topologically similar manner in δ-hexatoxin-Mg1a. However, subtle differences in the toxin surfaces are important for the novel selectivity of δ-hexatoxin-Mg1a for certain mammalian and insect Nav channel subtypes. As such, δ-hexatoxin-Mg1a provides us with a specific tool with which to study channel structure and function and determinants for phylumand tissue-specific activity. [ABSTRACT FROM AUTHOR]

Abstract: Two toxic and one non-toxic recombinant peptide variants of the mammalian neurotoxin CssII was cloned into the expression vector pQE30 containing a 6His-tag and a Factor Xa proteolytic cleavage site. The toxic recombinant peptides rCssII, HisrCssII and the non-toxic rCssIIE15R were expressed under induction with isopropyl thiogalactoside (IPTG), isolated using chromatographic techniques and folded correctly in vitro. The three recombinant variants showed similar secondary structures as the native CssII, but only the rCssIIE15R was not toxic to mice at concentrations up to 30μg/20g mouse body weight when injected intraperitoneally. All three recombinant peptides were capable of displacing the native CssII from their receptor sites in rat brain synaptosomes, suggesting that they had similar structural and functional characteristics of the native peptides. The three recombinant variants of CssII and the native one were used as antigens for immunization of New Zealand rabbits. The antibodies present in the rabbit antisera were able to recognize the native CssII. Additionally and more importantly, the sera of the immunized rabbits were able to neutralize both the native toxin CssII and the whole soluble venom of the scorpion Centruroides suffusus suffusus. These results indicate that the recombinant peptides can be used to produce antidotes against the venom of this species of scorpion. [Copyright &y& Elsevier]

Scorpion β-toxins affect the activation of voltage-sensitive sodium channels (NaChs). Although these toxins have been instrumental in the study of channel gating and architecture, little is known about their active sites. By using an efficient system for the production of recombinant toxins, we analyzed by point mutagenesis the entire surface of the β-toxin, Bj-xtrIT, an anti-insect selective excitatory toxin from the scorpion Buthotus judaicus. Each toxin mutant was purified and analyzed using toxicity and binding assays, as well as by circular dichroism spectroscopy to discern the differences among mutations that caused structural changes and those that specifically affected bioactivity. This analysis highlighted a functional discontinuous surface of 1405 Ų, which was composed of a number of non-polar and three charged amino acids clustered around the main α-helical motif and the C-tail. Among the charged residues, Glu30 is a center of a putative "hot spot" in the toxin-receptor binding-interface and is shielded from bulk solvent by a hydrophobic "gasket" (Tyr26and Val34). Comparison of the Bj-xtrIT structure with that of other fi-toxins that are active on mammals suggests that the hot spot and an adjacent non-polar region are spatially conserved. These results highlight for the first time structural elements that constitute a putative "pharmacophore" involved in the interaction of β-toxins with receptor site-4 on NaChs. Furthermore, the unique structure of the C-terminal region most likely determines the specificity of excitatory toxins for insect NaChs. [ABSTRACT FROM AUTHOR]