Purification and characterization of Ts15, the first member of a new α-KTX subfamily from the venom of the Brazilian scorpion Tityus serrulatus

Voltage-gated potassium channel toxins (KTxs) are basic short chain peptides comprising 23-43 amino acid residues that can be cross-linked by 3 or 4 disulfide bridges. KTxs are classified into four large families: α-, β-, γ- and κ-KTx. These peptides display varying selectivity and affinity for K_v channel subtypes. In this work, a novel toxin from the Tityus serrulatus venom was isolated, characterized and submitted to a wide electrophysiological screening on 5 different subtypes of Na_V channels (Na_V1.4; Na_V1.5; Na_V1.6; Na_V1.8 and DmNa_V1) and 12 different subtypes of K_V channels (K_V1.1 - K_V1.6; K_V2.1; K_V3.1; K_V4.2; K_V4.3; Shaker IR and ERG). This novel peptide, named Ts15, has 36 amino acids, is cross-linked by 3 disulfide bridges, has a molecular mass of 3956 Da and pI around 9. Electrophysiological experiments using patch clamp and the two-electrode voltage clamp techniques show that Ts15 preferentially blocks K_V1.2 and K_V1.3 channels with an IC₅₀ value of 196 ± 25 and 508 ± 67 nM, respectively. No effect on Na_V channels was observed, at all tested concentrations. Since Ts15 shows low amino acid identity with other known KTxs, it was considered a bona fide novel type of scorpion toxin. Ts15 is the unique member of the new α-Ktx21 subfamily and therefore was classified as α-Ktx21.1.     

Turkish scorpion Buthacus macrocentrus: General characterization of the venom and description of Bu1, a potent mammalian Na+-channel α-toxin

The venom of the scorpion Buthacus macrocentrus of Turkey was fractionated by high performance liquid chromatography (HPLC) and its mass finger print analysis was obtained by spectrometry. More than 70 different fractions were obtained, allowing the determination of the molecular masses of at least 60 peptides ranging between 648 and 44,336 Da. The venom is enriched with peptides containing molecular masses between 3200–4500 Da, and 6000–7500 Da. They very likely correspond to K⁺-channel and Na⁺-channel specific peptides, respectively, as expected from venoms of scorpions of the family Buthidae, already determined for other species. The major component obtained from HPLC was shown to be lethal to mice and was further purified and characterized. It contains 65 amino acid residues maintained closely packed by 4 disulfide bridges, and shows a molecular weight of 7263 Da. Additionally, a cDNA from the venomous glands of this scorpion was used in conjunction with sequence data from Edman degradation and mass spectrometry for cloning the gene that codes for Bu1 as we named this toxin. This gene codes for a 67 amino acid residues peptide, where the two last are eliminated post-translationally for production of an amidated C-terminal arginine. Its sequence is closely related to toxins from the species Leiurus quinquestriatus, as revealed by a phylogenetic tree analysis. Electrophysiological results conducted with Bu1 using patch-clamp techniques indicate that it modifies the Na⁺ currents, in a similar way as other well known α-scorpion toxins. These results support the conclusion that this species of scorpions is dangerous to humans, having an epidemiological interest for the country.     

Tityus serrulatus venom peptidomics: Assessing venom peptide diversity

MALDI-TOF-TOF and de novo sequencing were employed to assess the Tityus serrulatus venom peptide diversity. Previous works has shown the cornucopia of molecular masses, ranging from 800 to 3000 Da, present in the venom from this and other scorpions species. This work reports the identification/sequencing of several of these peptides. The majority of the peptides found were fragments of larger venom toxins. For instance, 28 peptides could be identified as fragments from Pape proteins, 10 peptides corresponded to N-terminal fragments of the TsKβ (scorpine-like) toxin and fragments of potassium channel toxins (other than the k-beta) were sequenced as well. N-terminal fragments from the T. serrulatus hypotensins-I and II and a novel hypotensin-like peptide could also be found. This work also reports the sequencing of novel peptides without sequence similarities to other known molecules.     

Chemical synthesis and structure–activity relationships of Ts κ, a novel scorpion toxin acting on apamin-sensitive SK channel

Abstract: Tityus kappa (Ts κ), a novel toxin from the venom of the scorpion Tityus serrulatus, is a 35-residue polypeptide cross-linked by three disulphide bridges and acts on small-conductance calcium-activated potassium channels (SK channels). Ts κ was chemically synthesized using the solid-phase method and characterized. The synthetic product, sTs κ, was indistinguishable from the natural toxin when tested in vitro in competition assay with radiolabelled apamin for binding to rat brain synaptosomes (IC₅₀ = 3 nm ). The sTs κ was further tested in vivo for lethal activity to mice following intracerebroventricular inoculation (LD₅₀ = 70 ng per mouse). The half-cystine pairings were formerly established by enzyme-based cleavage of sTs κ; they were between Cys₇–Cys₂₈, Cys₁₃–Cys₃₃ and Cys₁₇–Cys₃₅, which is a disulphide bridge pattern similar to that of other short scorpion toxins. According to previous studies on SK channel-acting toxins, the putative influence of certain basic residues of Ts κ (i.e. Arg₆, Arg₉, Lys₁₈, Lys₁₉) in its pharmacological activity was investigated using synthetic point-mutated analogues of the toxin with an Ala substitution at these positions. Data from binding assay, together with conformational analysis of the synthetic analogues by ¹H-NMR, suggest that Arg₆, and to a lesser extent Arg₉, are important residues for an high-affinity interaction of this toxin with SK channels; interestingly these residues are located outside the α-helical structure, whereas the pharmacologically important basic residues from other SK channel-specific toxins had been located inside the α-helix.     

Negative-shift activation, current reduction and resurgent currents induced by β-toxins from Centruroides scorpions in sodium channels

The β-toxins purified from the New World scorpion venoms of the Centruroides species affect several voltage-gated sodium channels (VGSCs) and thus are essential tools not only for the discrimination of different channel sub-types but also for studying the structure-function relationship between channels and toxins. This communication reports the results obtained with four different peptides purified from three species of Centruroides scorpions and assayed on seven distinct isoforms of VGSC (Na_v1.1-Na_v1.7) by specific functional analysis conducted through single cell electrophysiology. The toxins studied were CssII from Centruroides suffusus suffusus, Cll1 and Cll2 from Centruroides limpidus limpidus and a novel toxin from Centruroides noxius, which was characterized for the first time here. It has 67 amino acid residues and four disulfide bridges with a molecular mass of 7626 Da. Three different functional features were identified: current reduction of macroscopic conductance, left shift of the voltage-dependent activation and induction of resurgent currents at negative voltages following brief, strong depolarizations. The isoforms which revealed to be more affected resulted to be Na_v1.6 > 1.1 > 1.2 and, for the first time, a β-toxin is here shown to induce resurgent current also in isoforms different from Na_v1.6. Additionally, these results were analyzed with molecular modelling. In conclusion, although the four toxins have a high degree of identity, they display tri-modal function, each of which shows selectivity among the different sub-types of Na⁺-channels. Thus, they are invaluable as tools for structure-function studies of β-toxins and offer a basis for the design of novel ion channel-specific drugs.     

Inhibitory effects of scorpion venom on the uptake of amino acids by synaptosomes and synaptosomal membrane vesicles

Scorpion (Tityus serrulatus) venom strongly inhibited the Na⁺-dependent uptake of [¹⁴C]proline by rat brain synaptosomal preparations. In addition, the efflux of proline was enhanced markedly by scorpion venom. The inhibitory effects of the venom were also demonstrated in synaptosomal vesicle preparations where proline uptake was energized by an artificially imposed Na⁺ gradient. In both preparations, the effect of scorpion venom was additive with the inhibitory effect of veratridine on Na⁺-dependent amino acid uptake. The inhibitory effects of both compounds were abolished by tetrodotoxin. The Na⁺-dependent uptakes of amino acids (e.g. proline, glutamic acid, and γ-aminobutyric acid) were much more sensitive to inhibition by the toxin than the Na⁺⁶-independent uptakes (e.g. leucine and phenylalanine). The results of the present study indicate that the scorpion venom mav exert its inhibitory effect on Na⁺-dependent transport by decreasing the transmembrane Na⁺ gradient. Efflux of accumulated proline, which is presumably controlled by maintenance of this Na⁺ gradient, was stimulated 3- to 4-fold by the scorpion venom.     

Effect of maurotoxin,a four disulfide‐bridged toxin from the chactoid scorpion Scorpio maurus,on Shaker K+ channels

Abstract: Maurotoxin is a 34‐residue toxin isolated from the venom of the Tunisian chactoid scorpion Scorpio maurus palmatus and contains four disulfide bridges that are normally found in long‐chain toxins of 60–70 amino acid residues, which affect voltage‐gated sodium channels. However, despite the unconventional disulfide‐bridge pattern of maurotoxin, the conformation of this toxin remains similar to that of other toxins acting on potassium channels. Here, we analyzed the effects of synthetic maurotoxin on voltage‐gated Shaker potassium channels (ShB) expressed in Xenopus oocytes. Maurotoxin produces a strong, but reversible, inhibition of the ShB K⁺ current with an IC₅₀ of 2 nm . Increasing concentrations of the toxin induce a progressively higher block at saturating concentrations. At nonsaturating concentrations of the toxin (5–20 nm ), the channel block appears slightly more pronounced at threshold potentials suggesting that the toxin may have a higher affinity for the closed state of the channel. At the single channel level, the toxin does not modify the unitary current amplitude, but decreases ensemble currents by increasing the number of depolarizing epochs that failed to elicit any opening. A point mutation of Lys²³ to alanine in maurotoxin produces a 1000‐fold reduction in the IC₅₀ of block by the toxin suggesting the importance of this charged residue for the interaction with the channel. Maurotoxin does not affect K⁺ currents carried by Kir2.3 channels in oocytes or Na⁺ currents carried by the α_IIa channel expressed in CHO cells.     

Nine novel precursors of Buthus martensii scorpion α-toxin homologues

The cDNAs encoding nine novel α-toxin homologues were isolated from the venom gland cDNA library of the Chinese scorpion Buthus martensii Karsch (BmK). They are rich in AAAA and TTTT elements at the 5′ UTRs. The flanking region of the translation initiation codon ATG is AAAATGAA, which is highly conserved in scorpion Na⁺, K⁺ and Cl⁻ channel toxin genes. These putative scorpion α-toxins shared 45.5–98.4% homology with the characterized BmK α-toxins, and were completely conserved in the positions of all eight cysteines. This showed, together with higher homology at nucleotide level than that at amino acid level, that these toxins may originate from a common ancestor. The discovery of a series of homologues of scorpion α-toxin with a different degree of natural mutation in the primary structure will provide us with a valuable system for studying the structure–function relationship of scorpion toxins.     

Identification of BmKAPi, a novel type of scorpion venom peptide with peculiar disulfide bridge pattern from Buthus martensii Karsch

A novel cDNA sequence encoding a new type of scorpion venom peptide (BmKAPi) was first isolated from the venom gland of Buthus martensiiKarsch by cDNA library screening combined with 5′-race. The encoded precursor of BmKAPi consisted of 89 amino acid residues including a signal peptide of 24 residues, a putative mature peptide of 64 residues (BmKAPi) and an extra basic residue at the C-terminus which might be removed in the post-translational processing. BmKAPi is stabilized by five disulfide bridges, whereas all other disulfide-bridged scorpion toxins described are cross-linked by three or four disulfide bridges. It suggested the three-dimensinal scaffold of BmKAPi might be different from other scorpion toxins. The amino acid sequence of BmKAPi showed no homology with other scorpion venom peptides, but shared a little similarity with some anticoagulant peptides and proteinase inhibitors isolated from hookworm, honeybee or European frog, respectively. RT-PCR analysis showed that BmKAPi mRNA could be induced by venom extraction suggesting BmKAPi might be a component of scorpion venom. These results suggest that BmKAPi is a new type of scorpion venom peptide different from other described scorpion toxins in structural and functional aspects.     

Actions of sea anemone type 1 neurotoxins on voltage-gated sodium channel isoforms

As voltage-gated Na⁺ channels are responsible for the conduction of electrical impulses in most excitable tissues in the majority of animals (except nematodes), they have become important targets for the toxins of venomous animals, from sea anemones to molluscs, scorpions, spiders and even fishes. During their evolution, different animals have developed a set of cysteine-rich peptides capable of binding different extracellular sites of this channel protein. A fundamental question concerning the mechanism of action of these toxins is whether they act at a common receptor site in Na⁺ channels when exerting their different pharmacological effects, or at distinct receptor sites in different Na_v channels subtypes whose particular properties lead to these pharmacological differences. The α-subunits of voltage-gated Na⁺ channels (Na_v1.x) have been divided into at least nine subtypes on the basis of amino acid sequences. Sea anemones have been extensively studied from the toxinological point of view for more than 40 years. There are about 40 sea anemone type 1 peptides known to be active on Na_v1.x channels and all are 46-49 amino acid residues long, with three disulfide bonds and their molecular weights range between 3000 and 5000 Da. About 12 years ago a general model of Na_v1.2-toxin interaction, developed for the α-scorpion toxins, was shown to fit also to action of sea anemone toxin such as ATX-II. According to this model these peptides are specifically acting on the type 3 site known to be between segments 3 and 4 in domain IV of the Na⁺ channel protein. This region is indeed responsible for the normal Na⁺ currents fast inactivation that is potently slowed by these toxins. This fundamental “gain-of-function” mechanism is responsible for the strong increase in the action potential duration. They constitute a class of tools by means of which physiologists and pharmacologists can study the structure/function relationships of channel proteins. As most of the structural and electrophysiological studies were performed on type 1 sea anemone sodium channel toxins, we will present a comprehensive and updated review on the current understanding of the physiological actions of these Na channel modifiers.     

Biochemical and molecular characterization of the venom from the Cuban scorpion Rhopalurus junceus

This communication describes the first general biochemical, molecular and functional characterization of the venom from the Cuban blue scorpion Rhopalurus junceus, which is often used as a natural product for anti-cancer therapy in Cuba. The soluble venom of this arachnid is not toxic to mice, injected intraperitoneally at doses up to 200 μg/20 g body weight, but it is deadly to insects at doses of 10 μg per animal. The venom causes typical alpha and beta-effects on Na⁺ channels, when assayed using patch-clamp techniques in neuroblastoma cells in vitro. It also affects K⁺ currents conducted by ERG (ether-a-go-go related gene) channels. The soluble venom was shown to display phospholipase, hyaluronidase and anti-microbial activities. High performance liquid chromatography of the soluble venom can separate at least 50 components, among which are peptides lethal to crickets. Four such peptides were isolated to homogeneity and their molecular masses and N-terminal amino acid sequence were determined. The major component (RjAa12f) was fully sequenced by Edman degradation. It contains 64 amino acid residues and four disulfide bridges, similar to other known scorpion toxins. A cDNA library prepared from the venomous glands of one scorpion allowed cloning 18 genes that code for peptides of the venom, including RjA12f and eleven other closely related genes. Sequence analyses and phylogenetic reconstruction of the amino acid sequences deduced from the cloned genes showed that this scorpion contains sodium channel like toxin sequences clearly segregated into two monophyletic clusters. Considering the complex set of effects on Na⁺ currents verified here, this venom certainly warrant further investigation.     

Revealing the Function and the Structural Model of Ts4: Insights into the “Non-Toxic” Toxin from Tityus serrulatus Venom

The toxin, previously described as a “non-toxic” toxin, was isolated from the scorpion venom of Tityus serrulatus (Ts), responsible for the most severe and the highest number of accidents in Brazil. In this study, the subtype specificity and selectivity of Ts4 was investigated using six mammalian Nav channels (Nav1.2→Nav1.6 and Nav1.8) and two insect Nav channels (DmNav1 and BgNav). The electrophysiological assays showed that Ts4 specifically inhibited the fast inactivation of Nav1.6 channels, the most abundant sodium channel expressed in the adult central nervous system, and can no longer be classified as a “non-toxic peptide”. Based on the results, we could classify the Ts4 as a classical α-toxin. The Ts4 3D-structural model was built based on the solved X-ray Ts1 3D-structure, the major toxin from Ts venom with which it shares high sequence identity (65.57%). The Ts4 model revealed a flattened triangular shape constituted by three-stranded antiparallel β-sheet and one α-helix stabilized by four disulfide bonds. The absence of a Lys in the first amino acid residue of the N-terminal of Ts4 is probably the main responsible for its low toxicity. Other key amino acid residues important to the toxicity of α- and β-toxins are discussed here.     

Covalent structure of toxins I and II from the scorpion Buthus occitanus tunetanus

The amino acid sequences of neurotoxins I and II, which are active on mammals, purified from the venom of Buthus occitanus tunetanus have been determined using standard methods, including mainly automatic phenylisothiocyanate degradation of S-carboxymethylated derivatives of the two proteins and peptides derived by enzymatic hydrolyses. Both toxins are made of sixty-five amino acid residues cross-linked with four disulfide bridges. For toxin II, the complete covalent structure, including the positions of the four disulfide bridges was determined: the positions are similar to those previously found in toxin II of another scorpion from Africa, Androctonus australis Hector. This finding is in favor of a similar structure for all of the scorpion neurotoxins active on mammals.     

Identification of BmKAPi, a novel type of scorpion venom peptide with peculiar disulfide bridge pattern from Buthus martensii Karsch.

A novel cDNA sequence encoding a new type of scorpion venom peptide (BmKAPi) was first isolated from the venom gland of Buthus martensiiKarsch by cDNA library screening combined with 5′-race. The encoded precursor of BmKAPi consisted of 89 amino acid residues including a signal peptide of 24 residues, a putative mature peptide of 64 residues (BmKAPi) and an extra basic residue at the C-terminus which might be removed in the post-translational processing. BmKAPi is stabilized by five disulfide bridges, whereas all other disulfide-bridged scorpion toxins described are cross-linked by three or four disulfide bridges. It suggested the three-dimensinal scaffold of BmKAPi might be different from other scorpion toxins. The amino acid sequence of BmKAPi showed no homology with other scorpion venom peptides, but shared a little similarity with some anticoagulant peptides and proteinase inhibitors isolated from hookworm, honeybee or European frog, respectively. RT-PCR analysis showed that BmKAPi mRNA could be induced by venom extraction suggesting BmKAPi might be a component of scorpion venom. These results suggest that BmKAPi is a new type of scorpion venom peptide different from other described scorpion toxins in structural and functional aspects.     

Tst26, a novel peptide blocker of Kv1.2 and Kv1.3 channels from the venom of Tityus stigmurus

Using high-performance liquid chromatography Tst26, a novel potassium channel blocker peptide, was purified from the venom of the Brazilian scorpion Tityus stigmurus. Its primary structure was determined by means of automatic Edman degradation and mass spectrometry analysis. The peptide is composed of 37 amino acid residues and tightly folded through three disulfide bridges, similar to other K⁺ channel blocking peptides purified from scorpion venoms. It contains the “essential dyad” for K⁺ channel recognition comprised of a lysine at position 27 and a tyrosine at position 36. Electrophysiological assays revealed that Tst26 blocked hKv1.2 and hKv1.3 channels with high affinity (K_d = 1.9 nM and 10.7 nM, respectively) while it did not affect several other ion channels (mKv1.1, hKv1.4, hKv1.5, hERG, hIKCa1, hBK, hNav1.5) tested at 10 nM concentration. The voltage-dependent steady-state parameters of K⁺ channel gating were unaffected by the toxin in both channels, but due to the fast association and dissociation kinetics Tst26 slowed the rate of inactivation of Kv1.3 channels. Based on the primary structure, the systematic nomenclature proposed for this peptide is α-KTx 4.6.     

Selective alteration of sodium channel gating by Australian funnel-web spider toxins

The actions of potent mammalian neurotoxins isolated from the venom of two Australian funnel-web spiders were investigated using both electrophysiological and neurochemical techniques. Whole-cell patch clamp recording of sodium currents in rat dorsal root ganglion neurons revealed that versutoxin (VTX), isolated from the venom of Hadronyche versuta, produced a concentration-dependent slowing or removal of tetrodotoxin-sensitive (TTX-S) sodium current inactivation and a reduction in peak TTX-S sodium current. In contrast, VTX had no effect on tetrodotoxin-resistant (TTX-R) sodium currents or potassium currents. VTX also shifted the voltage dependence of sodium channel activation in the hyperpolarizing direction and increased the rate of recovery from inactivation. Ion flux studies performed in rat brain synaptosomes also revealed that robustoxin (RTX), from the venom of Atrax robustus, and VTX both produced a partial activation of ²²Na ⁺ flux and an inhibition of batrachotoxin-activated ²²Na⁺ flux. This inhibition of flux through batrachotoxin-activated channels was not due to an interaction with neurotoxin receptor site 1 since [³H]saxitoxin binding was unaffected. In addition, the partial activation of ²²Na⁺ flux was not enhanced in the presence of α-scorpion toxin and further experiments suggest that VTX also enhances [³H]batrachotoxin binding. These selective actions of funnel-web spider toxins on sodium channel function are comparable to those of α-scorpion and sea anemone toxins which bind to neurotoxin receptor site 3 on the channel to slow channel inactivation profoundly. Also, these modifications of sodium channel gating and kinetics are consistent with actions of the spider toxins to produce repetitive firing of action potentials.     

Antibacterial activity of six novel peptides from Tityus discrepans scorpion venom. A fluorescent probe study of microbial membrane Na permeability changes

Six novel peptides (named bactridines) were isolated from Tityus discrepans scorpion venom. From mass spectrometry molecular masses were 6916, 7362, 7226, 7011, 7101 and 7173 Da (bactridines 1–6). Bactridines 1 and 2 were sequenced by Edman degradation. The sequences and in silico analysis, indicated that they are positively charged polypeptides comprised of 61 and 64 amino acids (AA), respectively, bactridine 1 and bactridine 2 containing 4 disulfide bridges. Bactridine 1 was only toxic to cockroaches and crabs, and bactridine 2–6 were only toxic to mice. Bactridine 1 has a 78% sequence identity with ardiscretin. Ardisctretin is an insect specific sodium toxin which also produces a small depolarization and induces repetitive firing in squid axons resembling those of DDT [1,10(pchlorobenzyl) 2-trichloretane] in its ability to slow down action potential, to induce repetitive firing. Measured as the minimal inhibitory concentration, bactridines had high antibacterial activity against a wide range of Gram positive and Gram negative bacteria. Complete bacterial growth inhibition occurred at concentrations from 20 to 80 μM depending on the bacteria and peptide tested. Effects on membrane Na⁺ permeability induced by bactridines were observed on Yersinia enterocolitica loaded with 1 μM CoroNa™ Red. CoroNa™ Red fluorescence leakage from bacteria was observed after exposure to 0.3 μM of any bactridine tested, indicating that they modified Na⁺ membrane permeability. This effect was blocked by 10 μM amiloride and by 25 μM mibefradil drugs that affect Na⁺ and Ca²⁺ channels respectively. We found no evidence of changes of K⁺ or Ca²⁺ concentrations neither inside nor outside the bacteria in experiments using the fluorescent dyes Fluo 4AM (10 μM) and PBFI (20 μM).     

The Venom of the Spider Selenocosmia Jiafu Contains Various Neurotoxins Acting on Voltage-Gated Ion Channels in Rat Dorsal Root Ganglion Neurons

Selenocosmia jiafu is a medium-sized theraphosid spider and an attractive source of venom, because it can be bred in captivity and it produces large amounts of venom. We performed reversed-phase high-performance liquid chromatography (RP-HPLC) and matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analyses and showed that S. jiafu venom contains hundreds of peptides with a predominant mass of 3000–4500 Da. Patch clamp analyses indicated that the venom could inhibit voltage-gated Na⁺, K⁺ and Ca²⁺ channels in rat dorsal root ganglion (DRG) neurons. The venom exhibited inhibitory effects on tetrodotoxin-resistant (TTX-R) Na⁺ currents and T-type Ca²⁺ currents, suggesting the presence of antagonists to both channel types and providing a valuable tool for the investigation of these channels and for drug development. Intra-abdominal injection of the venom had severe toxic effects on cockroaches and caused death at higher concentrations. The LD₅₀ was 84.24 μg/g of body weight in the cockroach. However, no visible symptoms or behavioral changes were detected after intraperitoneal injection of the venom into mice even at doses up to 10 mg/kg body weight. Our results provide a basis for further case-by-case investigations of peptide toxins from this venom.     

Effect of Tityus serrulatus scorpion venom on the rabbit isolated corpus cavernosum and the involvement of NANC nitrergic nerve fibres

1. The effect of Tityus serrulatus scorpion venom and its toxin components on the rabbit isolated corpus cavernosum was investigated by use of a bioassay cascade.
2. Tityus serrulatus venom (3–100 μg), acetylcholine (ACh; 0.3–30 nmol) and glyceryl trinitrate (GTN; 0.5–10 nmol) dose-dependently relaxed rabbit isolated corpus cavernosum preparations precontracted with noradrenaline (3 μM). The selective soluble guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo [4,3,-alquinoxalin-1-one] (ODQ; 30 μM) increased the basal tone of the rabbit isolated corpus cavernosum and abolished the relaxations induced by the agents mentioned above. Methylene blue (30 μM) also inhibited the relaxations induced by Tityus serrulatus venom but, in contrast to ODQ, the inhibition was irreversible.
3. The non-selective NO synthase (NOS) inhibitors N^Ω-nitro-L-arginine methyl ester (L-NAME; 10 μM) and N^G-iminoethyl-L-ornithine (L-NIO; 30 μM) also increased the tone of the rabbit isolated corpus cavernosum and markedly reduced both ACh- and Tityus serrulatus venom-induced relaxations without affecting those evoked by GTN. The inhibitory effect was reversed by infusion of L-arginine (300 μM), but not D-arginine (300 μM). The neuronal NOS inhibitor 1-(2-trifluoromethylphenyl) imidazole (TRIM, 100 μM) did not affect either the tone of the rabbit isolated corpus cavernosum or the relaxations induced by ACh, bradykinin (Bk), Tityus serrulatus venom and GTN. TRIM was approximately 1,000 times less potent than L-NAME in inhibiting rabbit cerebellar NOS in vitro, as measured by the conversion of [³H]-L-arginine to [³H]-L-citrulline.
4. The protease inhibitor aprotinin (Trasylol; 10 μg ml⁻¹) and the bradykinin B₂ receptor antagonist Hoe 140 (D-Arg-[Hyp³,Thi⁵,D-Tic⁷, Oic⁸]-BK; 50 nM) did not affect the rabbit isolated corpus cavernosum relaxations induced by Tityus serrulatus venom. The ATP-dependent K⁺ channel antagonist glibenclamide (10 μM) and the Ca²⁺-activated K⁺  channel antagonists apamin (0.1 μM) and charybdotoxin (0.1 μM) also failed to affect the venom-induced relaxations. Similarly, the K⁺ channel blocker tetraethylammonium (TEA; 10 μM) had no effect on the venom-induced relaxations.
5. Capsaicin (3 and 10 nmol) relaxed the rabbit isolated corpus cavernosum in a dose-dependent and non-tachyphylactic manner. Ruthenium red (30 μM), an inhibitor of capsaicin-induced responses, markedly reduced the relaxations caused by capsaicin, but failed to affect those induced by Tityus serrulatus venom. L-NAME (10 μM) had no effect on the capsaicin-induced relaxations of the rabbit isolated corpus cavernosum.
6. The sodium channel blocker tetrodotoxin (TTX; 1 μM) abolished the relaxations of the rabbit isolated corpus cavernosum induced by Tityus serrulatus venom without affecting those evoked by capsaicin, ACh and GTN. Tetrodotoxin (1 μM) also promptly reversed the response to the venom when infused during the relaxation phase.
7. The bioassay cascade of the toxin components purified from Tityus serrulatus venom revealed that only fractions X, XI and XII caused dose-dependent relaxations of the rabbit isolated corpus cavernosum and these were markedly reduced by either TTX (1 μM) or L-NAME (10 μM).
8. Our results indicate that Tityus serrulatus scorpion venom (and the active fractions X, XI and XII) relaxes rabbit corpus cavernosum via the release of NO. This release is specifically triggered by the activation of capsaicin-insensitive cavernosal non-adrenergic non-cholinergic (NANC) fibres, that may possibly be nitrergic neurones. Tityus serrulatus venom may therefore provide an important tool for understanding further the mechanism of NANC nitrergic nerve activation.

     

Spider Neurotoxins Targeting Voltage-Gated Sodium Channels

The voltage-gated sodium (Na_v) channel is a target for a number of drugs, insecticides, and neurotoxins. These bind to at least seven identified neurotoxin binding sites and either block conductance or modulate sodium channel gating and/or kinetics. A number of polypeptide toxins from the venoms of araneomorph and mygalomorph spiders have been isolated and characterized that interact with several of these sites. Certain huwentoxins and hainantoxins appear to target site 1 to block Na_v channel conductance. The δ -atracotoxins and Magi 4 slow Na_v-channel inactivation via an interaction with neurotoxin site 3. The δ -palutoxins, and most likely μ -agatoxins and curtatoxins, target site 4. However, their action is complex with the μ -agatoxins causing a hyperpolarizing shift in the voltage-dependence of activation, an action analogous to scorpion β -toxins, but with both δ -palutoxins and μ -agatoxins slowing Na_v channel inactivation, a site 3-like action. Many spider toxins target undefined sites, while others are likely to cross-react with other ion channels due to conserved structures within domains of voltage-gated ion channels. It is already clear, however, that many spider toxins represent highly potent and specific molecular tools to define novel links between sites modulating channel activation and inactivation. Other spider toxins show phyla specificity and are being considered as lead compounds for the development of biopesticides. Others display tissue specificity via interactions with specific Na_v channel subtypes and should provide useful tools to delineate the molecular determinants to target ligands to these channel subtypes. These studies are being greatly assisted by the determination of the pharmacophore of these toxins, but without precise identification of their binding site and mode of action their potential in the mentioned areas remains underdeveloped.