Biochemical characterization and insecticidal activity of isolated peptides from the venom of the scorpion Centruroides tecomanus.

The venom of scorpions is a mixture of components that constitute a source of bioactive molecules. The venom of the scorpion Centruroides tecomanus contains peptides toxic to insects, however, to date no toxin responsible for this activity has yet been isolated and fully characterized. This communication describes two new peptides Ct-IT1 and Ct-IT2 purified from this scorpion. Both peptides contain 63 amino acids with molecular weight 6857.85 for Ct-IT1 and 6987.77 Da for Ct-IT2. The soluble venom was separated using chromatographic techniques of molecular size exclusion, cationic exchange, and reverse phase chromatography, allowing the identification of at least 99 components of which in 53 the insecticidal activity was evaluated. The LD50 determined for Ct-IT1 is 3.81 µg/100 mg of cricket weight, but low amounts of peptides (0.8 µg of peptide) already cause paralysis in crickets. The relative abundance of these two peptides in the venom is 2.1% for Ct-IT1 and 1% for Ct-IT2. The molecular masses and N-terminal sequences of both insecticidal toxins were determined by mass spectrometry and Edman degradation. The primary structure of both toxins was compared with other known peptides isolated from other scorpion venoms. The analysis of the sequence alignments revealed the position of a highly conserved amino acid residue, Gly39, exclusively present in anti-insect selective depressant ß-toxins (DBTXs), which in Ct-IT1 and Ct-IT2 is at position Gly40. Similarly, a three-dimensional structure of this toxins was obtained by homology modeling and compared to the structure of known insect toxins of scorpions. An important similarity of the cavity formed by the trapping apparatus region of the depressant toxin LqhIT2, isolated from the scorpion Leiurus quinquestriatus hebraeus, was found in the toxins described here. These results indicate that Ct-IT1 and Ct-IT2 toxins have a high potential to be evaluated on pests that affect economically important crops to eventually consider them as a potential biological control method.

Expression systems of human ß-defensins: vectors, purification and biological activities.

Human beta-defensins are 2-5 kDa, cationic, microbicidal peptides, which represent the first-line host defense against several Gram-negative and Gram-positive bacteria, fungi and viruses. They contain a conserved disulfide-bridge pattern of three pairs of intramolecular cystine bonds. The well-known public health problem related with the growing number of multiresistant bacteria has driven research to look for novel antibiotics, such beta-defensins and a feasible way to produce them. Heterologous expression of beta-defensins could be one way to generate large quantities of beta-defensins for clinical research; however, heterologous expression of beta-defensins has some biochemical problems, such toxicity toward the host cell, peptide degradation by proteolytic cell enzymes, size, folding constrains and low recombinant peptide yields. In this communication, several heterologous systems for producing human beta-defensins are reviewed.

Amino acid substitutions in an alpha-helical antimicrobial arachnid peptide affect its chemical properties and biological activity towards pathogenic bacteria but improves its therapeutic index.

Four variants of the highly hemolytic antimicrobial peptide Pin2 were chemically synthesized with the aim to investigate the role of the proline residue in this peptide, by replacing it with the motif glycine-valine-glycine [GVG], which was found to confer low hemolytic activity in a spider antimicrobial peptide. The proline residue in position 14 of Pin2 was substituted by [V], [GV], [VG] and [GVG]. Only the peptide variant with the proline substituted for [GVG] was less hemolytic compared to that of all other variants. The peptide variant [GVG] kept its antimicrobial activity in Muller-Hilton agar diffusion assays, whereas the other three variants were less effective. However, all Pin2 antimicrobial peptide variants, were active when challenged against a Gram-positive bacteria in Muller-Hilton broth assays suggesting that chemical properties of the antimicrobial peptides such as hydrophobicity is an important indication for antimicrobial activity in semi-solid environments.

Helodermatine, a kallikrein-like, hypotensive enzyme from the venom of Heloderma horridum horridum (Mexican beaded lizard).

We have purified and characterized the major N-benzoyl-L-arginine ethyl ester hydrolase from the venom of Heloderma horridum horridum. The enzyme belongs to the serine proteinase family, and its activity vs. peptide amide substrates and human high-molecular-weight kininogen suggests a similarity to the family of kallikreins. This interpretation is corroborated by its reactivity with the natural inhibitors soybean trypsin inhibitor and Kunitz-type bovine pancreatic trypsin inhibitor (aprotinin). Injection of the enzyme (2-16 micrograms/kg) into anesthetized rabbits leads to a rapid dose-dependent transient decrease of the arterial blood pressure. Like glandular kallikrein it specifically converts single-chain tissue type plasminogen activator into its double chain form. In contrast to other kallikrein-like enzymes from snake venoms it shows no thrombin-like or plasminogen activator activity. The enzyme is a single-chain glycoprotein (Mr 63,000). The N-terminal sequence revealed significant homology to pig pancreatic kallikrein and to kallikrein like enzymes from Crotalus atrox and Crotalus adamanteus venom. This enzyme, which we name Helodermatine, is the first purified from Sauria with kallikrein-like properties.

Biochemical characterization of the venom from the Mexican scorpion Centruroides ornatus, a dangerous species to humans.

Every year in Mexico, around 300,000 people suffer from accidents related to scorpion stings. Among the scorpion species dangerous to human is Centruroides ornatus, whose venom characterization is described here. From this venom, a total of 114 components were found using chromatographic separation and mass spectrometry analysis. The most abundant ones have molecular masses between 3000-4000 Da and 6000-8000 Da respectively, similar to other known K+ and Na+-channel specific scorpion peptides. Using intraperitoneal injections into CD1 mice, we were able to identify and fully sequenced three new lethal toxins. We propose to name them Co1, Co2 and Co3 toxins, which correspond to toxins 1 to 3 of the abbreviated species name (Co). Electrophysiology analysis of these peptides using heterologously expressed human Na+-channels revealed a typical ß-toxin effect. Peptide Co52 (the most abundant peptide in the venom) showed no activity in our in vivo and in vitro model assays. A phylogenetic analysis groups the Co1, Co2 and Co3 among other ß-toxins from Centruroides scorpions. Peptide Co52 segregates among peptides of unknown defined functions.

Cn29, a novel orphan peptide found in the venom of the scorpion Centruroides noxius: Structure and function.

A peptide (Cn29) from the venom of the scorpion Centruroides noxius (about 2% of the soluble venom) was purified and its primary and three-dimensional structures were determined. The peptide contains 27 amino acids with primary sequence: LCLSCRGGDYDCRVKGTCENGKCVCGS. The peptide is tightly packed by three disulfide linkages formed between C2-C23, C5-C18 and C12-C25. Since the native peptide was obtained in limited amounts, the full synthetic peptide was prepared using the standard F-moc-based solid phase synthesis method of Merrifield. The native and synthetic peptides were shown to be identical by sequencing, HPLC separation and mass spectrometry. The solution structure of the peptide solved from NMR data shows that it consists of a well-defined N-terminal region without regular secondary structure extending from Leu 1 to Asp 9, followed by a short helical fragment from Tyr10 to Val14 and two short ß strands (Thr17-Glu19 and Lys22-Val24). The primary and tertiary structures of Cn29 are different from all other scorpion peptides described in the literature. Transcriptome analysis of RNA obtained from C. noxius confirmed the expression of a gene coding for Cn29 in its venom gland. Initial experiments were conducted to identify its possible function: lethality tests in mice and insects as well as ion-channel binding using in vitro electrophysiological assays. None of the physiological or biological tests displayed any activity for this peptide, which at present is considered to be another orphan peptide found in scorpion venoms. The peptide is thus the first example of a novel structural component present in scorpion venoms.

Macrophage activation, phagocytosis and intracellular calcium oscillations induced by scorpion toxins from Tityus serrulatus.

The research described here is focused upon studying the activation of mice peritoneal macrophages when submitted to in vitro effects of Tityus serrulatus scorpion venom and its major toxic peptides. Several functional events were analysed, such as: cytotoxicity, spreading, extent of phagocytosis, vacuole formation and changes of internal calcium concentration. Among the main results observed, when macrophages are subjected to the effects of soluble venom of Tityus serrulatus scorpion venom, a partially purified fraction (FII) or a pure toxin (Ts1), are an increment in the percentage of phagocytosis and vacuole formation, a decrement of the spreading ability, accompanied by oscillations of internal calcium concentration. The net results demonstrate that scorpion venom or its major toxins are effective stimulators of macrophage activity; the effect of whole soluble venom or partially purified fractions is due to the toxic peptides, seen here clearly with Ts1. The possible involvement of Na+-channels in these events is discussed. A basic understanding of the underlying molecular mechanisms responsible for macrophage activation should serve as a foundation for novel drug development aimed at modulating macrophage activity.

Venom characterization of the Amazonian scorpion Tityus metuendus.

The soluble venom from the scorpion Tityus metuendus was characterized by various methods. In vivo experiments with mice showed that it is lethal. Extended electrophysiological recordings using seven sub-types of human voltage gated sodium channels (hNav1.1 to 1.7) showed that it contains both α- and ß-scorpion toxin types. Fingerprint analysis by mass spectrometry identified over 200 distinct molecular mass components. At least 60 sub-fractions were recovered from HPLC separation. Five purified peptides were sequenced by Edman degradation, and their complete primary structures were determined. Additionally, three other peptides have had their N-terminal amino acid sequences determined by Edman degradation and reported. Mass spectrometry analysis of tryptic digestion of the soluble venom permitted the identification of the amino acid sequence of 111 different peptides. Search for similarities of the sequences found indicated that they probably are: sodium and potassium channel toxins, metalloproteinases, hyaluronidases, endothelin and angiotensin-converting enzymes, bradykinin-potentiating peptide, hypothetical proteins, allergens, other enzymes, other proteins and peptides.

Oxidative refolding chromatography: folding of the scorpion toxin Cn5.

We have made an immobilized and reusable molecular chaperone system for oxidative refolding chromatography. Its three components-GroEL minichaperone (191-345), which can prevent protein aggregation; DsbA, which catalyzes the shuffling and oxidative formation of disulfide bonds; and peptidyl-prolyl isomerase-were immobilized on an agarose gel. The gel was applied to the refolding of denatured and reduced scorpion toxin Cn5. The 66-residue toxin, which has four disulfide bridges and a cis peptidyl-proline bond, had not previously been refolded in reasonable yield. We recovered an 87% yield of protein with 100% biological activity.

Design and expression of recombinant toxins from Mexican scorpions of the genus Centruroides for production of antivenoms.

This manuscript describes the design of plasmids containing the genes coding for four main mammalian toxins of scorpions from the genus Centruroides (C.) of Mexico. The genes that code for toxin 2 of C. noxius (Cn2), toxin 2 from C. suffusus (Css2) and toxins 1 and 2 from C. limpidus (Cll1 and Cll2) were included into individual plasmids carrying the genetic construction for expression of fusion proteins containing a leader peptide (pelB) that directs the expressed protein to the bacterial periplasm, a carrier protein (thioredoxin), the cleavage site for enterokinase, the chosen toxin and a poly-histidine tag (6xHis-tag) for purification of the hybrid protein by immobilized metal ion affinity chromatography after expression in Escherichia coli strain BL21 (DE3). The purified hybrid proteins containing the recombinant toxins (abbreviated Thio-EK-Toxin) were used for immunization of three independent groups of ten mice and four rabbits. Challenging the first group of mice, immunized with recombinant Thio-EK-Css2, with three median lethal doses (LD50) of C. suffusus soluble venom resulted in the survival of all the test animals without showing intoxication symptoms. All control mice (none immunized) died. Similar results were obtained with mice previously immunized with Thio-EK-Cn2 and challenged with C. noxius venom. The third group of mice immunized with both Thio-EK-Cll1 and Thio-EK-Cll2 showed an 80% survival ratio when challenged with only one LD50 of C. limpidus venom, all showing symptoms of intoxication. The sera from rabbits immunized with a combination of the four recombinant toxins were collected separately and used to assess their neutralization capacity in vitro (pre-incubating the serum with the respective scorpion venom and injecting the mixture into mice), using six mice for each serum/venom combination tested. The venoms from the six most dangerous scorpion species of Mexico were assayed: C. noxius, C. suffusus, C. limpidus, C. elegans, C. tecomanus and C. sculpturatus. Two hundred and 50 µL of serum from any of the immunized rabbits were enough to neutralize three LD50 of any of the tested venoms, with mice showing no symptoms of intoxication. These results confirm that the recombinant forms of the main toxins from the most dangerous scorpions of Mexico are excellent immunogens for the production of antivenoms to treat scorpion intoxications.

Pi5 and Pi6, two undescribed peptides from the venom of the scorpion Pandinus imperator and their effects on K+-channels.

This work reports the isolation, chemical and functional characterization of two previously unknown peptides purified from the venom of the scorpion Pandinus imperator, denominated Pi5 and Pi6. Pi5 is a classical K+-channel blocking peptide containing 33 amino acid residues with 4 disulfide bonds. It is the first member of a new subfamily, here defined by the systematic number α-KTx 24.1. Pi6 is a peptide of unknown real function, containing only two disulfide bonds and 28 amino acid residues, but showing sequence similarities to the κ-family of K-channel toxins. The systematic number assigned is κ-KTx2.9. The function of both peptides was assayed on Drosophila Shab and Shaker K+-channels, as well as four different subtypes of voltage-dependent K+-channels: hKv1.1, hKv1.2, hKv1.3 and hKv1.4. The electrophysiological assays showed that Pi5 inhibited Shaker B, hKv1.1, hKv1.2 and hKv1.3 channels with Kd = 540 nM, Kd = 92 nM and Kd = 77 nM, respectively, other studied channels were not affected. Of the channels tested only hKv1.2 and hKv1.3 were inhibited at 100 nM concentration of Pi6, the remaining current fractions were 68% and 77%, respectively. Thus, Pi5 and Pi6 are high nanomolar affinity non-selective blockers of hKv1.2 and hKv1.3 channels.

K+ channel blockers: novel tools to inhibit T cell activation leading to specific immunosuppression.

During the last two decades since the identification and characterization of T cell potassium channels great advances have been made in the understanding of the role of these channels in T cell functions, especially in antigen-induced activation. Their limited tissue distribution and the recent discovery that different T cell subtypes carrying out distinct immune functions show specific expression levels of these channels have made T cell potassium channels attractive targets for immunomodulatory drugs. Many toxins of various animal species and a structurally diverse array of small molecules inhibiting these channels with varying affinity and selectivity were found and their successful use in immunosuppression in vivo was also demonstrated. Better understanding of the topological differences between potassium channel pores, detailed knowledge of toxin and small-molecule structures and the identification of the binding sites of blocking compounds make it possible to improve the selectivity and affinity of the lead compounds by introducing modifications based on structural information. In this review the basic properties and physiological roles of the voltage-gated Kv1.3 and the Ca2+-activated IKCa1 potassium channels are discussed along with an overview of compounds inhibiting these channels and approaches aiming at producing more efficient modulators of immune functions for the treatment of diseases like sclerosis multiplex and type I diabetes.

Directed evolution, phage display and combination of evolved mutants: a strategy to recover the neutralization properties of the scFv version of BCF2 a neutralizing monoclonal antibody specific to scorpion toxin Cn2.

BCF2, a monoclonal antibody raised against scorpion toxin Cn2, is capable of neutralizing both, the toxin and the whole venom of the Mexican scorpion Centruroides noxius Hoffmann. The single chain antibody fragment (scFv) of BCF2 was constructed and expressed in Escherichia coli. Although its affinity for the Cn2 toxin was shown to be in the nanomolar range, it was non-neutralizing in vivo due to a low stability. In order to recover the neutralizing capacity, the scFv of BCF2 was evolved by error-prone PCR and the variants were panned by phage display. Seven improved mutants were isolated from three different libraries. One of these mutants, called G5 with one mutation at CDR1 and another at CDR2 of the light chain, showed an increased affinity to Cn2, as compared to the parental scFv. A second mutant, called B7 with a single change at framework 2 of heavy chain, also had a higher affinity. Mutants G5 and B7 were also improved in their stability but they were unable to neutralize the toxin. Finally, we constructed a variant containing the changes present in G5 and B7. The purpose of this construction was to combine the increments in affinity and stability borne by these mutants. The result was a triple mutant capable of neutralizing the Cn2 toxin. This variant showed the best affinity constant (KD=7.5x10(-11) M), as determined by surface plasmon resonance (BIAcore). The k(on) and k(off) were improved threefold and fivefold, respectively, leading to 15-fold affinity improvement. Functional stability determinations by ELISA in the presence of different concentrations of guanidinium hydrochloride (Gdn-HCl) revealed that the triple mutant is significantly more stable than the parental scFv. These results suggest that not only improving the affinity but also the stability of our scFv were important for recovering its neutralization capacity. These findings pave the way for the generation of recombinant neutralizing antisera against scorpion stings based on scFvs.

Serum level of scorpion toxins, electrolytes and electrocardiogram alterations in Mexican children envenomed by scorpion sting.

The scorpion Centruroides limpidus limpidus (C.l.l.) is endemic in México, producing hundreds of accidents in humans; children being one of the most susceptible targets. Few studies reported that severe envenoming by scorpion venom induces cardiac damage and electrolytes abnormalities in children, but the relationship of envenoming severity and toxic blood levels is unknown. The aim of this study was to determine the relationship among clinical status of envenoming, serum electrolyte, electrocardiographic abnormalities, and serum toxin levels in 44 children stung by scorpion over a period of 6 months in the State of Morelos, Mexico. The patients were said to be asymptomatic, when they presented just local symptoms, and were said to be symptomatic when showing local symptoms and at least one systemic symptom. The clinical status was evaluated at the admission at the emergency room of the Hospital, and 30 min after the administration of polyspecific F(ab')2 anti-scorpion therapy to symptomatic children. Forty-one percent of the children were asymptomatic and 59% symptomatic. Potassium and sodium imbalance and an elongation of the QT interval were detected; the rate of hypokalemia was higher in symptomatic than on asymptomatic children (50% and 6%, respectively). Hypokalemia persisted in 19% in symptomatic patients, whereas sodium reached normal levels 30 min after anti-venom therapy. The hypokalemia statistically correlated with elongation of the QT interval. The concentration of the toxic components of C.l.l in serum was significantly higher in symptomatic than asymptomatic children, and the serum levels of the toxic component significantly decreased to undetectable levels after the application of anti-venom therapy. Despite the small size of the sample, this study establishes that severity of envenoming was statistically related to potassium imbalance in serum, QT interval and the concentration of toxic components in serum, which decreased at undetectable levels after specific treatment with the anti-scorpion venom, correlating with clinical disappearance or greatly reduction of symptoms of envenomation.

Comprehensive analysis of venom from the scorpion Centruroides tecomanus reveals compounds with antimicrobial, cytotoxic, and insecticidal activities.

Centruroides tecomanus is a medically important scorpion of the state of Colima (Mexico). This communication reports the identification of venom components of this scorpion with biological activity over insects/crickets (Acheta domestica), crustaceans/fresh water shrimps (Cambarellus montezumae), and mammalians/mice (Mus musculus, strain CD1). It also describes the pharmacological effects on cell lines in culture (L5178Y cells, HeLa cells, HuTu cells and Jurkat E6-1 cells), as well as on several types of bacteria (see below). The soluble venom of this scorpion was fractionated by high-performance liquid chromatography (HPLC) and collected separately in twelve independent fractions collected over 60 min run (5 min time apart each other). The HPLC components of fraction VII were lethal to all three species used for assay. The IVth fraction had a toxic effect on freshwater shrimps. In this species, fractions VI, VII and VIII were all lethal. For crickets, fractions V and VI were toxic and fraction VII was lethal. In mouse, the lethal components were found in fraction VII, whereas fraction VIII was toxic, but not lethal, at the doses assayed. The molecular weight of peptides from the various group of fractions were identified by mass spectrometry determination. Components lethal to mice showed molecular weights from 7013 to 7487 Da. Two peptides were obtained in homogeneous form and shown to be lethal to the three species of animal used for assay. The soluble venom tested on L5178Y cell line survival was shown to be cytotoxic, at 10-100 µg/mL concentration, when compared to control murine splenocytes (p = 0.007). The soluble venom applied to Hela, Hutu and Jurkat cell lines did not show cytotoxic effects at these concentrations. On the contrary, it seems to have a proliferative effect. However the HPLC fractions I, III, VI and XII do have a cytotoxic effect on Jurkat E06-1 cells in culture at 200 µg/mL concentration. The antimicrobial activity of the venom fractions on Staphylococcus aureus (gram-positive), Escherichia coli, Pseudomonas aeruginosa y Salmonella spp (gram-negative) was measured, using the liquid inhibition growth system. The four strains of bacteria used were susceptible to fractions III and IV, affecting all four bacterial strains at concentrations below 5 µg/mL.

Fast K(+) currents from cerebellum granular cells are completely blocked by a peptide purified from Androctonus australis Garzoni scorpion venom.

A novel peptide was purified from the venom of the scorpion Androctonus australis Garzoni (abbreviated Aa1, corresponding to the systematic number alpha KTX4.4). It contains 37 amino acid residues, has a molecular mass of 3850 Da, is closely packed by three disulfide bridges and a blocked N-terminal amino acid. This peptide selectively affects the K(+) currents recorded from cerebellum granular cells. Only the fast activating and inactivating current, with a kinetics similar to I(A)-type current, is completely blocked by the addition of low micromolar concentrations (K(i) value of 150 nM) of peptide Aa1 to the external side of the cell preparation. The blockade is partially reversible in our experimental conditions. Aa1 blocks the channels in both the open and the closed states. The blockage is test potential independent and is not affected by changes in the holding potential. The kinetics of the current are not affected by the addition of Aa1 to the preparation; it means that the block is a simple 'plugging mechanism', in which a single toxin molecule finds a specific receptor site in the external vestibule of the K(+) channel and thereby occludes the outer entry to the K(+) conducting pore.

Anionic currents of chick sensory neurons are affected by a phospholipase A2 purified from the venom of the taipan snake.

A neurotoxic phospholipase A2 was purified from the venom of the taipan snake Oxyuranus scutellatus scutellatus by three consecutive chromatographic steps on ion exchange resins, followed by an affinity column prepared with a phosphatidylcholine derivative attached to Sepharose. The phospholipase was shown to be of type A2 (specific activity of 85 units/mg protein), and an apparent molecular weight of 16,000. Amino acid analysis shows the presence of approx. 150 residues with the N-terminal amino acid sequence: NLAQFGFMIRCANGGSRSALDYADYGC, different from all the phospholipases described until now. This enzyme is lethal to experimental mice (LD50 = 10 micrograms/20 g mouse weight) and affects ionic currents in chick (Gallus domesticus) dorsal root ganglion cells, measured by the whole-cell clamp technique. In symmetrical external/internal ionic solutions, after suppression of Na+, K+ and Ca2+ currents, external application of phospholipase at a low concentration (30 nM) was shown to increase the baseline current in a reversible manner. The augmented response was voltage-dependent and the effect was much greater for negative currents. In the presence of a salt gradient across the membrane (out 40 mM NaCl/in 140 mM CsCl), the current reversal potential revealed a shift in the positive direction typically due to Cl- ion flux through the membrane. External application of a 50 microM concentration of picrotoxin caused a reversible reduction of the phospholipase-induced chloride current. Moreover, no appreciable current block was detected after addition of 50 microM DIDS.

A unified nomenclature for short-chain peptides isolated from scorpion venoms: alpha-KTx molecular subfamilies.

Peptidyl toxins are used extensively to determine the pharmacology of ion channels. Four families of peptides have been purified from scorpion venom. In this article, the classification of K+-channel-blocking peptides belonging to family 2 peptides and comprising 30-40 amino acids linked by three or four disulfide bridges, will be discussed. Evidence is provided for the existence of 12 molecular subfamilies, named alpha-KTx1-12, containing 49 different peptides. Because of the pharmacological divergence of these peptides, the principle of classification was based on a primary sequence alignment, combined with maximum parsimony and Neighbour-Joining analysis.

Modification of Na channel gating by an alpha scorpion toxin from Tityus serrulatus.

The effects of TsIV-5, a toxin isolated from the Brazilian scorpion Tityus serrulatus, on whole-cell and single-channel Na currents were determined in N18 neuroblastoma cells. In whole-cell records at a test potential of -10 mV, external application of 500 nM TsIV-5 slowed inactivation 20-fold and increased peak current by about one-third without changing time-to-peak. Both the steady-state activation and inactivation curves were shifted to more negative potentials. Other alpha scorpion toxins produce similar effects but the single-channel mechanism is not known. TsIV-5 caused a voltage-dependent prolongation of mean single-channel open time such that at a test potential of -60 mV no change was observed, whereas at -20 mV mean open time increased about threefold and prolonged bursting was observed. Macroscopic current reconstructed from summed single-channel records showed a characteristic toxin-induced potentiation of peak current and a 20-fold slowing of the decay phase. TsIV-5 does not discriminate between tissue-specific Na channel subtypes. Prolonged open times and bursting were also observed in toxin-treated Na channels from rat ventricular myocytes, rat cortical neurons, and mouse skeletal muscle. The toxin effects are shown to be consistent with a kinetic model in which TsIV-5 selectively interferes with the ability of the channel to reach the inactivated state.

Solution structure of toxin 2 from centruroides noxius Hoffmann, a beta-scorpion neurotoxin acting on sodium channels.

We have determined the solution structure of Cn2, a beta-toxin extracted from the venom of the New World scorpion Centruroides noxius Hoffmann. Cn2 belongs to the family of scorpion toxins that affect the sodium channel activity, and is very toxic to mammals (LD50=0.4 microg/20 g mouse mass). The three-dimensional structure was determined using 1H-1H two-dimensional NMR spectroscopy, torsion angle dynamics, and restrained energy minimization. The final set of 15 structures was calculated from 876 experimental distance constraints and 58 angle constraints. The structures have a global r. m.s.d. of 1.38 A for backbone atoms and 2.21 A for all heavy atoms. The overall fold is similar to that found in the other scorpion toxins acting on sodium channels. It is made of a triple-stranded antiparallel beta-sheet and an alpha-helix, and is stabilized by four disulfide bridges. A cis-proline residue at position 59 induces a kink of the polypeptide chain in the C-terminal region. The hydrophobic core of the protein is made up of residues L5, V6, L51, A55, and by the eight cysteine residues. A hydrophobic patch is defined by the aromatic residues Y4, Y40, Y42, W47 and by V57 on the side of the beta-sheet facing the solvent. A positively charged patch is formed by K8 and K63 on one edge of the molecule in the C-terminal region. Another positively charged spot is represented by the highly exposed K35. The structure of Cn2 is compared with those of other scorpion toxins acting on sodium channels, in particular Aah II and CsE-v3. This is the first structural report of an anti-mammal beta-scorpion toxin and it provides the necessary information for the design of recombinant mutants that can be used to probe structure-function relationships in scorpion toxins affecting sodium channel activity.