Disulfide bridges and blockage of Shaker B K(+)-channels by another butantoxin peptide purified from the Argentinean scorpion Tityus trivittatus.

A peptide was isolated from the venom of the scorpion Tityus trivittatus. It is an isoform of the toxin TsTX-IV earlier described [Toxicon 37 (1999) 651] and identical to butantoxin [Arch. Biochem. Biophys. 379 (2000) 18], both isolated from the Brazilian scorpion Tityus serrulatus. This newly characterized peptide contains 40 amino acid residues with a molecular mass of [M+H(+)] 4507.0, cross-linked by four disulfide bridges, made between the cysteine pairs: Cys2-Cys5, Cys10-Cys31, Cys16-Cys36 and Cys20-Cys38. It blocks in a completely reversible manner the Shaker B K(+)-channels, with a K(d) around 660nM. It belongs to the sub-family 12 and it is now being classified as alpha-KTx 12.2.     

Amino acid sequence and function of a new alpha-toxin from the Amazonian scorpion Tityus cambridgei.

A toxic peptide earlier denominated Tc48b [Toxicon 40 (2002) 557] was purified to homogeneity and its amino acid sequence determined. It has 64 amino acid residues stabilized by four disulfide bridges with a molecular weight of 7,385.2 atomic mass units (a.m.u.). It affects Na(+)-permeability in pituitary GH3 cells in culture, in a similar fashion as those reported for alpha-scorpion toxins, contrary to most of the New World scorpion toxins that are beta-toxins.     

Differentiation of renal Na(+)-K(+)-ATPase in control and streptozotocin-induced diabetic mice by G-protein acting toxins and phorbol esters.

The specific activity of Na(+)-K(+)-ATPase in the renal medulla and cortex of 50-day-old streptozotocin (STZ)-induced diabetic mice was increased 58% and 50%, respectively, as compared to controls. Km values of Na+ and K+ for this enzyme were unaltered, while that of ATP was decreased in diabetic mice. The Na(+)-K(+)-ATPase in control medulla and cortex was activated by both cholera and pertussis toxins, while this effect was abolished in diabetics. Since dibutyryl cAMP stimulates cortical Na(+)-K(+)-ATPase activity in control mice, the activation effect of cholera toxin on this enzyme might be due to its interaction with a Gs-protein and the persistent stimulation of adenylate cyclase activity, while the effect of pertussis toxin might be due to its masking of the inhibitory action of a Gi-protein on adenylate cyclase activity. However, the protein kinase C (PKC)-associated Na(+)-K(+)-ATPase might also be quiescent in diabetes, because the stimulating effect of phorbol 12,13-dibutyrate (PDBu) and phorbol 12-myristate 13-acetate (PMA) on this enzyme was abolished in diabetic cortex. In addition, nicardipine and ouabain were found to have differential effects on this enzyme derived from control and diabetic mice.     

Scorpion toxins that block transient currents (IA) of rat cerebellum granular cells

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 Buthusmartensi 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.     

Discovery and characterization of cnidarian peptide toxins that affect neuronal potassium ion channels

Peptides have been isolated from several species of sea anemones and shown to block currents through various potassium ion channels, particularly in excitable cells. The toxins can be grouped into four structural classes: type 1 with 35-37 amino acid residues and three disulphide bridges; type 2 with 58-59 residues and three disulphide bridges; type 3 with 41-42 residues and three disulphide bridges; and type 4 with 28 residues and two disulphide bridges. Examples from the first class are BgK from Bunodosoma granulifera, ShK from Stichodactyla helianthus and AsKS (or kaliseptine) from Anemonia sulcata (now A. viridis). These interfere with binding of radiolabelled dendrotoxin to synaptosomal membranes and block currents through channels with various Kv1 subunits and also intermediate conductance K(Ca) channels. Toxins in the second class are homologous to Kunitz-type inhibitors of serine proteases; these toxins include kalicludines (AsKC 1-3) from A. sulcata and SHTXIII from S. haddoni; they block Kv1.2 channels. The third structural group includes BDS-I, BDS-II (from A. sulcata) and APETx 1 (from Anthropleura elegantissima). Their pharmacological specificity differs: BDS-I and -II block currents involving Kv3 subunits, while APETx1 blocks ERG channels. The fourth group comprises the more recently discovered SHTX I and II from S. haddoni. Their channel blocking specificity is not yet known but they displace dendrotoxin binding from synaptosomal membranes. Sea anemones can be predicted to be a continued source of new toxins that will serve as molecular probes of various K⁺ channels.     

Induction of neutralizing antibodies against Tityus serrulatus toxins by immunization with a recombinant nontoxic protein.

An immunogenic nontoxic protein (TsNTxP) was purified from the venom of the scorpion Tityus serrulatus (Ts). This peptide is composed of 63 amino acid residues with a high degree of structural homology with the toxins isolated from Ts. The nucleotide sequence of the gene that encodes TsNTxP was obtained and also showed a high degree of similarity with genes encoding Tityus toxins [Guatimosim, S.C.F., Prado, V.F., Diniz, C.R., Chávez-Olórtegui, C.. Kalapothakis, E., 1999. Molecular cloning and genomic analysis of TsNTxP; an immunogenic protein from Tityus serrulatus scorpion venom. Toxicon 37, 507-517]. In the present study the TsNTxP gene was expressed in E. coli BL21DE3 cells as a fusion protein with maltose-binding protein. The recombinant protein (TsNTxPrec) was purified by affinity chromatography and used as an immunogen in rabbits. The antigenic specificity of anti-TsNTxPrec antibodies was compared by an enzyme-linked immunosorbent assay using TsNTxP, TstFG50 (the fraction of Ts venom that represents most of the toxicity of the crude venom) and the crude venom, to coat microtitration plates. Anti-TsNTxPrec antibodies had a comparable high cross-reactivity for all antigens tested. Concentrations of Ts venom equivalent to 20 LD50 were effectively neutralized by 1 ml of the anti-TsNTxPrec serum. This result provides basic data for the use of such recombinant scorpion protein as an immunogen in the development of antivenoms for clinical use.     

Acute gastric mucosal injury induced by toxins from Tityus serrulatus scorpion venom: a novel experimental model in the rat.

The effect of a partially purified fraction (T1) and toxin gamma purified from Tityus serrulatus scorpion venom, on gastric mucosa were investigated in anesthetized rats. The animals were injected i.v. with the T1 fraction (37.5 micrograms/100 g) or with saline and 60 min later were sacrificed and the stomachs resected. The gastric juice was measured and stereoscopic examination of the stomachs made. In animals injected with the T1 fraction there was an increase in volume, acidity and pepsin output of rat stomach. The T1 fraction also induced acute gastric injuries in the glandular mucosa, consisting of circular or linear ulcers, and punctiform lesions. Intravenous injection of 20 micrograms/100 g of a pure toxin obtained from Tityus serrulatus scorpion venom (toxin gamma) also induced similar lesions in the rat stomach. Our data indicate that the injection of T1 fraction or toxin gamma are good models to induce acute gastric ulcers in a short period of time in anesthetized rats.     

Novel mechanism of blocking axonal Na(+) channels by three macrocyclic polyamine analogues and two spider toxins

1. The mechanism of Na(+) channel block by three macrocyclic polyamine derivatives and two spider toxins was studied with voltage clamp and internal perfusion method in squid axons. 2. All these chemicals specifically block Na(+) channels in the open state only from the internal surface, and do not affect K(+) channels. 3. The blocking effect is enhanced as the depolarizing pulse becomes larger. Blocked channels are unable to shift to the inactivated state. 4. In the case of cyclam and guanidyl-side armed cyclam (G-cyclam), quick release of these chemicals from the binding sites is proven by the increase in the tail current and prolongation of the time course of the off gating current. On the other hand, in the presence of N-4 and the spider toxins, their detachment was delayed significantly. 5. Molecular requirements for the block of Na(+) channels by these molecules are the presence of positive charge and hydrophobicity.     

Interaction of bretylium tosylate with guinea-pig myocardial Na(+)-K(+)-ATPase.

1. The effects of bretylium on myocardial Mg(2+)-dependent, Na(+)-K(+)-ATPase (EC 3.6.1.3) activity were compared with those of ouabain in guinea-pig heart preparations. 2. Both ouabain and bretylium inhibited microsomal Na(+)-K(+)-ATPase activity in a concentration-dependent fashion in the range of 0.01-100 and 1.0-4000 microM, respectively. The IC50 values were 1.93 +/- 0.27 microM for ouabain and 2.45 +/- 0.17 mM for bretylium. 3. In another set of experiments, the effects of bretylium on the enzyme activity were tested in combination with 2.5 or 5.0 microM ouabain. 4. The combined effects of the two drugs resulted in a net reduction in the total inhibitory activities of the individual drugs, which became more marked the higher the bretylium concentration. This trend seems to suggest a competitive mode of interaction of the two drugs in their inhibitory actions on Na(+)-K(+)-ATPase activity. 5. The results demonstrate therefore that bretylium is a potent inhibitor of ouabain-inhibited ATP hydrolysis by myocardial Na(+)-K(+)-ATPase. These actions may be pertinent with regard to some of its cardiac actions.     

Localization of epitopes in the toxins of Tityus serrulatus scorpions and neutralizing potential of therapeutic antivenoms.

Overlapping pentadecapeptides covering the complete amino acid sequence of TsII, TsVII and TsIV toxins from the venom of scorpion Tityus serrulatus (Ts), were prepared by use of the Spot method of multiple peptide synthesis. Horse anti-Ts antisera for therapeutic use were tested for their binding to peptides. All nine antisera tested showed reactivity with several peptides from the three toxins. Three antigenic regions, one in the very N-terminal, the second in the central part and the other in the C-terminal part of the three toxins were frequently, but not constantly recognized, with an intensity that seemed to be related to the neutralizing potency of the tested antivenom. Thus the corresponding peptides (residues 1-15 and 48-62 of TsII; residues 1-15, 16-30 and 48-62 of TsIV and residues 1-15 and 47-61 of TsVII) were synthesized, coupled to KLH and used as antigens to coat the microtitration plates to determine any relationship between their ELISA reactivity with therapeutic horse antivenoms and the neutralizing potential of these antivenoms. The mixture of the N-terminal peptide of TsII, of the N-terminal TsVII peptide and of the C-terminal of TsIV was found to give a linear relationship with the neutralizing titer of horse serum of low neutralizing potency (< or =1 mg/ml). However, high neutralizing antivenoms did not show the expected response in peptide ELISA. This observation is discussed in the context of the occurrence of continuous and discontinuous epitopes on toxins.     

Evidence that diphenylhydantoin does not affect adenosine triphosphatases from brain.

The effects of diphenylhydantoin on brain sodium- and potassium-dependent adenosine triphosphatase (NaK ATPase), p-nitrophenylphosphatase and the calcium-dependent adenosine triphosphatase (Ca ATPase) were examined. The results indicated that: (1) diphenylhydantoin did not stimulate the NaK ATPase of beef brain sodium iodide-treated microsomes or rat brain synaptosomes at nonsaturating or saturating levels of sodium and potassium: (2) the apparent stimulation by diphenylhydantoin under high sodium: potassium ratios reported by other workers can be explained by a potassium contamination in the diphenylhydantoin; (3) diphenylhydantoin did not alter the Michaelis constant for ATP for the NaK ATPase; (4) diphenylhydantoin did not slowly interact with or alter the NaK ATPase; (5) diphenylhydantoin did not alter the calcium inhibition of the enzyme and (6) diphenylhydantoin did not alter the p-nitrophenylphosphatase or Ca ATPase. Based on these considerations, diphenylhydantoin must be exerting its pharmacological effect by altering a membrane constituent other than the NaK ATPase.     

Further characterization of toxins T1IV (TsTX-III) and T2IV from Tityus serrulatus scorpion venom

Toxins T1IV (TsTX-III) and T2IV have been purified to homogeneity from Tityus serrulatus scorpion venom and further characterized. Their amino acid composition and SDS-PAGE reveal an approximate mol. wt of 7000. Their intracisternal LD50 (micrograms/kg) in mice were 12.9 +/- 1.6 and 3.0 +/- 0.5, while their N-terminal amino acid sequences were K-E-G-Y-A-M-D-H-E-G-C-K-F-S- and K-E-G-Y-L-M-D-H-E-G-C-K-L-S-C-F-I-R-P-S-G-Y-C-G-R-E-, respectively. This sequence of T2IV, its amino acid composition and its chromatographic and electrophoretic behaviour identify it as toxin gamma (TsTX-I), which is the major toxin from this venom. TsTX-III (13 to 102 micrograms/kg) produced a long lasting enhancement of the hypertensive effect of noradrenaline and a slight decrease of the hypotensive effect of acetylcholine, while T2IV (115 micrograms/kg) induced a prolonged hypotensive effect on the anesthetized rat. On the isolated guinea-pig vas deferens, TsTX-III (2.1 and 3.0 micrograms/ml) produced a horizontal shift of the dose-response curve for noradrenaline to the left with no change of the maximal response. At a concentration of 1.43 microM, it induced a prolongation of the duration of the B component of the compound action potential. This prolongation was strongly reduced after addition of tetrodotoxin.     

Na+,K(+)-ATPase inhibiting activity of cardiac glycosides from Erysimum cheiranthoides.

We previously reported the isolation of eleven new cardiac glycosides called cheiranthosides I-XI together with two known ones (olitoriside and erysimoside) from the seeds of Erysimum cheiranthoides L. The glycosides were evaluated for their inhibitory activity against Na+,K(+)-ATPase by comparing with typical cardiac glycosides. Two of them, cheiranthoside III and VIII, showed high inhibiting activity which was equivalent to that of digitoxin. Cheiranthoside XI containing a rhamnopyranosyl digitoxopyranosyl moiety and a carboxyl group showed the lowest activity which was similar to that of the inactive aglycone, strophanthidin. Some characteristics in the structure-activity relationship are also discussed.     

Phorbol esters inhibit smooth muscle contractions through activation of Na(+)-K(+)-ATPase.

1. The role of protein kinase C (PKC) in agonist-induced contractions of guinea-pig ileum longitudinal smooth muscle has been investigated. 2. The phorbol esters, phorbol 12,13-dibutyrate (PDBu), phorbol 12,13-diacetate (PDA) and phorbol 12-myristate 13-acetate (PMA), relaxed tissues precontracted by submaximal concentrations of carbachol, histamine or substance P. 3. This inhibitory action of the phorbol esters was reversed following the application of ouabain, a specific inhibitor of Na(+)-K(+)-ATPase. Similarly, pretreatment with ouabain inhibited the ability of phorbol esters to relax tissues precontracted by the above agonists. 4. The slow relaxation of the tonic component of contraction induced by submaximal concentrations of carbachol and histamine, and all concentrations of substance P, was abolished in the presence of ouabain. 5. In Na(+)-loaded tissues, PDBu and carbachol caused a concentration-dependent increase of Na(+)-K(+)-ATPase activity, assessed by ouabain-sensitive 86Rb(+)-uptake. Extrusion of Na+, assessed by the cellular content of the ion, was also stimulated by PDBu (the effect of carbachol was not investigated). 6. We conclude that phorbol esters inhibit the tonic component of contractions induced by submaximal concentrations of these agonists through activation of Na(+)-K(+)-ATPase. We suggest that PKC may exert feedback control over the tonic component of agonist contractions through stimulation of the pump.     

Induction of neutralizing antibodies against Tityus serrulatus scorpion toxins by immunization with a mixture of defined synthetic epitopes.

We have used the Spot method of multiple peptide synthesis to prepare sets of immobilized overlapping peptides of uniform size (15 mer), covering the complete amino acid sequences of TsNTxP a non-toxic and immunogenic protein and TsIV, an alpha-type toxin that is the major lethal component of the venom of scorpion Tityus serrulatus. Anti-TsNTxP antibodies binding to peptides, revealed three antigenic regions, one in the N-terminal, the second in the central part and the other in the C-terminal part of TsNTxP. One peptide epitope in the C-terminal part of TsIV was identified with anti-TsIV neutralizing rabbit antibodies. Anti-peptide antibodies were raised against these four peptides all together covalently coupled to keyhole limpet hemocyanin (KLH) and found to neutralize in vitro the toxic effects of the T. serrulatus venom. Quantities of venom equivalent to 13.5 LD(50) were effectively neutralized by 1ml of the anti-peptide serum. The antigenic specificities of the anti-peptides were compared by an indirect enzyme-linked immunosorbent assay (ELISA) using synthetic peptides and crude venoms from T. serrulatus, T. bahiensis, T. cambridgei, T. stigmurus, Androctonus autralis Hector and Centruroides sculpturatus to coat the microtitration plates. The anti-peptide antibodies had a comparable high reactivity with the crude venom of T. serrulatus, moderate binding to T. bahiensis, T. cambridgei, T. stigmurus and Centruroides sculpturatus venoms but were unable to recognize the venom of Androctonus autralis Hector. These results show that by using peptides derived from the sequence of scorpion toxins, the generation of anti-peptide antibodies able to neutralize the cognate venom appears to be an alternative strategy for the easy preparation of antivenoms.     

Screening of expression libraries using ELISA: identification of immunogenic proteins from Tityus bahiensis and Tityus serrulatus venom.

The present report describes the use of ELISA with cDNA expression libraries in the identification of immunogenic proteins. The methodology described was applied using libraries constructed with mRNA isolated from Tityus serrulatus and Tityus bahiensis venom glands. In addition we describe for the first time the sequence of a neurotoxin from Tityus bahiensis venom gland named TbTx5 whose amino acid sequencing showed 93% similarity with the Tityus bahiensis TbTx IV-5 neurotoxin. The methodology described can be used for the generation of an immunogenic bank in order to contribute to genome and proteome projects.     

Non-conventional toxins from Elapid venoms.

Non-conventional toxins constitute a poorly characterized class of three-finger toxins isolated exclusively from Elapidae venoms. These toxins are monomers of 62-68 amino acid residues and contain five disulfide bridges. However, unlike alpha/kappa-neurotoxins and kappa-neurotoxins which have the fifth disulfide bridge in their middle loop (loop II), the fifth disulfide bridge in non-conventional toxins is located in loop I (N-terminus loop). Overall, non-conventional toxins share approximately 28-42% identity with other three-finger toxins including alpha-neurotoxins, alpha/kappa-neurotoxins and kappa-neurotoxins. Recent structural studies have revealed that non-conventional toxins also display the typical three-finger motif. Non-conventional toxins are typically characterized by a lower order of toxicity (LD(50) approximately 5-80 mg/kg) in contrast to prototype alpha-neurotoxins (LD(50) approximately 0.04-0.3 mg/kg) and hence they are also referred to as 'weak toxins'. Further, it is generally assumed that non-conventional toxins target muscle (alpha(2)beta gamma delta) receptors with low affinities several orders of magnitude lower than alpha-neurotoxins and alpha/kappa-neurotoxins. However, it is now known that some non-conventional toxins also antagonize neuronal alpha 7 nicotinic acetylcholine receptors. Hence, non-conventional toxins are not a functionally homogeneous group and other, yet unknown, molecular targets for this class of snake venom toxins may exist. Non-conventional toxins may therefore be a useful source of ligands with novel biological activity targeting the plethora of neuronal nicotinic receptors as well as other physiological processes.     

Ardiscretin a novel arthropod-selective toxin from Tityus discrepans scorpion venom.

A new arthropod selective toxin was purified from the venom of the Venezuelan scorpion Tityus discrepans, and its amino acid sequence, cDNA clone and biological activity are reported here. The amino acid sequence of this peptide, named ardiscretin (from arthropod toxin of T. discrepans) was completed by Edman degradation and mass spectrometry. It is a single polypeptide composed by 61 amino acids with an amidated cysteine residue at the C-terminal end, closely packed by four disulfide bridges. The atomic mass unit (a.m.u.) experimentally determined was 7103.8 a.m.u. This peptide was shown to be specific for invertebrates (crickets, triatomides, crabs and squids), but non-toxic to mice, at the dose assayed. Ardiscretin inhibits the Na(+)-currents of squid giant axons in an apparent irreversible manner, whose inhibitory effect is reached at 30 microM toxin concentration. Sequence comparison showed that it is phylogenetically closely related to insect-specific scorpion toxins. Ardiscretin produced a small depolarization and induced repetitive firing in squid axons resembling those of DDT [1,1'(p-chlorobenzyl)2-tricloretane] in its ability to slow down action potential, to induce repetitive firing, and in that the concentration required for any effect in squid axon is rather high.