Sea Anemone Toxin Research Articles

Molecular analysis of the sea anemone toxin Av3 reveals selectivity to insects and demonstrates the heterogeneity of receptor site-3 on voltage-gated Na+ channels

Av3 is a short peptide toxin from the sea anemone Anemonia viridis shown to be active on crustaceans and inactive on mammals. It inhibits inactivation of Na(v)s (voltage-gated Na+ channels) like the structurally dissimilar scorpion alpha-toxins and type I sea anemone toxins that bind to receptor site-3. To examine the potency and mode of interaction of Av3 with insect Na(v)s, we established a system for its expression, mutagenized it throughout, and analysed it in toxicity, binding and electrophysiological assays. The recombinant Av3 was found to be highly toxic to blowfly larvae (ED50=2.65+/-0.46 pmol/100 mg), to compete well with the site-3 toxin LqhalphaIT (from the scorpion Leiurus quinquestriatus) on binding to cockroach neuronal membranes (K(i)=21.4+/-7.1 nM), and to inhibit the inactivation of Drosophila melanogaster channel, DmNa(v)1, but not that of mammalian Na(v)s expressed in Xenopus oocytes. Moreover, like other site-3 toxins, the activity of Av3 was synergically enhanced by ligands of receptor site-4 (e.g. scorpion beta-toxins). The bioactive surface of Av3 was found to consist mainly of aromatic residues and did not resemble any of the bioactive surfaces of other site-3 toxins. These analyses have portrayed a toxin that might interact with receptor site-3 in a different fashion compared with other ligands of this site. This assumption was corroborated by a D1701R mutation in DmNa(v)1, which has been shown to abolish the activity of all other site-3 ligands, except Av3. All in all, the present study provides further evidence for the heterogeneity of receptor site-3, and raises Av3 as a unique model for design of selective anti-insect compounds.

The MiRP2-Kv3.4 Potassium Channel: Muscling In on Alzheimer's Disease: Fig. 1.

In this issue of Molecular Pharmacology (p. 665), Pannacione et al. provide evidence of a role for the voltage-gated potassium channel alpha subunit Kv3.4 and its ancillary subunit MiRP2 in beta-amyloid (Abeta) peptide-mediated neuronal death. The MiRP2-Kv3.4 channel complex-previously found to be important in skeletal myocyte physiology-is now argued to be a molecular correlate of the transient outward potassium current up-regulated by Abeta peptide, considered a significant step in the etiology of Alzheimer's disease. The authors conclude that MiRP2 and Kv3.4 are up-regulated by Abeta peptide in a nuclear factor kappaB-dependent fashion at the transcriptional level, and the sea anemone toxin BDS-I is shown to protect against Abeta peptide-mediated cell death by specific blockade of Kv3.4-generated current. The findings lend weight to the premise that specific channels, such as MiRP2-Kv3.4, could hold promise as future therapeutic targets in Alzheimer's disease and potentially other neurodegenerative disorders.

Crotamine inhibits preferentially fast-twitching muscles but is inactive on sodium channels

Crotamine is a peptide toxin from the venom of the rattlesnake Crotalus durissus terrificus that induces a typical hind-limb paralysis of unknown nature. Hind limbs have a predominance of fast-twitching muscles that bear a higher density of sodium channels believed until now to be the primary target of crotamine. Hypothetically, this makes these muscles more sensitive to crotamine and would explain such hind-limb paralysis. To challenge this hypothesis, we performed concentration vs. response curves on fast (extensor digitorum longus (EDL)) and slow (soleus) muscles of adult male rats. Crotamine was tested on various human Na + channel isoforms (Na v1.1–Na v1.6 α-subunits) expressed in HEK293 cells in patch-clamp experiments, as well as in acutely dissociated dorsal root ganglion (DRG) neurons. Also, the behavioral effects of crotamine intoxication were compared with those of a muscle-selective sodium channel antagonist μ-CgTx-GIIIA, and other sodium-acting toxins such as tetrodotoxin α- and β-pompilidotoxins, sea anemone toxin BcIII, spider toxin Tx2-6. Results pointed out that EDL was more susceptible to crotamine than soleus under direct electrical stimulation. Surprisingly, electrophysiological experiments in human Na v1.1 to Na v1.6 Na + channels failed to show any significant change in channel characteristics, in a clear contrast with former studies. DRG neurons did not respond to crotamine. The behavioral effects of the toxins were described in detail and showed remarkable differences. We conclude that, although differences in the physiology of fast and slow muscles may cause the typical crotamine syndrome, sodium channels are not the primary target of crotamine and therefore, the real mechanism of action of this toxin is still unknown.

Vitamin B 1 thiazole derivative reduces transmembrane current through ionic channels formed by toxins from black widow spider venom and sea anemone in planar phospholipid membranes

The vitamin B 1 (thiamine) structural analogue 3-decyloxycarbonylmethyl-4-methyl-5-(β-hydroxyethyl) thiazole chloride (DMHT) (0.1 mM) reversibly reduced transmembrane currents in CaCl 2 and KCl solutions via ionic channels produced by latrotoxins (α-latrotoxin (α-LT) and α-latroinsectotoxin (α-LIT)) from black widow spider venom and sea anemone toxin (RTX) in the bilayer lipid membranes (BLMs). Introduction of DMHT from the cis-side of BLM bathed in 10 mM CaCl 2 inhibited transmembrane current by 31.6 ± 3% and by 61.8 ± 3% from the trans-side of BLM for α-LT channels. Application of DMHT in the solution of 10 mM CaCl 2 to the cis-side of BLM decreased the current through the α-LIT and RTX channels by 52 ± 4% and 50 ± 5%, respectively. Addition of Cd 2+ (1 mM) to the cis- or trans-side of the membrane after the DMHT-induced depression of Ca 2+-current across the α-LT channels caused its further decrease by 85 ± 5% that coincides favorably with the intensity of Cd 2+ blocking in control experiments without DMHT. These data suggest that DMHT inhibiting is not specific for latrotoxin channels only and DMHT may exert its action on α-LT channels without considerable influence on the ionogenic groups of Ca 2+-selective site inside the channel cavity. The binding kinetics of DMHT with the α-LT channel shows no cooperativity and allows to expect that the DMHT binding site of the toxin is formed by one ionogenic group as the slopes of inhibition rate determined in log–log coordinates are 1.25 on the trans-side and 0.68 on the cis-side. Similar pK of binding (5.4 on the trans-side and 5.7 on the cis-side) also suggest that DMHT may interact with the same high affinity site of α-LT channel on either side of the BLM. The comparative analysis of effective radii measured for α-LT, α-LIT and RTX channels on the cis-side (0.9 nm, 0.53 nm and 0.55 nm, correspondingly) and for α-LT channel on the trans-side (0.28 ± 0.18 nm) with the intensity of DMHT inhibitory action obtained on these channels allowed to conclude that the potency of DMHT inhibition increased on toxin pores of smaller lumen.

Site-3 sea anemone toxins: Molecular probes of gating mechanisms in voltage-dependent sodium channels

Sea anemone toxins, whose biological function is the capture of marine prey, are invaluable tools for studying the structure and function of mammalian voltage-gated sodium channels. Their high degree of specificity and selectivity have allowed for detailed analysis of inactivation gating and assignment of molecular entities responsible for this process. Because of their ability to discriminate among channel isoforms, and their high degree of structural conservation, these toxins could serve as important lead compounds for future pharmaceutical design.

Site-3 toxins and cardiac sodium channels

Site-3 toxins are small polypeptide venoms from scorpions, sea anemones, and spiders that bind with a high specificity to the extracellular surface of voltage-gated Na channels. After binding to a site near the S4 segment in domain IV the toxin causes disruption of the normal fast inactivation transition resulting in a marked prolongation of the action potentials of excitable tissues including those of cardiac and skeletal muscle and nerve. In this review we discuss the specific binding interactions between residues of the toxin and those of the Na channel, and the specific modification of Na channel kinetic behavior leading to a change in fast inactivation focusing on interactions deduced primarily from the study of sea anemone toxins and the cardiac Na channel (Na V1.5). We also illustrate the usefulness of site-3 toxins in the study of altered Na channel behavior by drug-modification.

BcIV, a new paralyzing peptide obtained from the venom of the sea anemone Bunodosoma caissarum. A comparison with the Na + channel toxin BcIII

Sea anemones produce a wide variety of biologically active compounds, such as the proteinaceous neurotoxins and cytolysins. Herein we report a new peptide, purified to homogeneity from the neurotoxic fraction of B. caissarum venom, by using gel filtration followed by rp-HPLC, naming it as BcIV. BcIV is a 41 amino acid peptide (molecular mass of 4669 amu) possessing 6 cysteines covalently linked by three disulfide bonds. This toxin has 45 and 48% of identity when compared to APETx1 and APETx2 from Anthopleura elegantissima, respectively, and 42% of identity with Am-II and BDS-I and-II obtained from Antheopsis maculata and Anemonia sulcata, respectively. This neurotoxin presents only a weak-paralyzing action (minimal Lethal Dose close to 2000 μg/kg) in swimming crabs Callinectes danae. This appears to be a different effect to that caused by the type 1 sea anemone toxin BcIII that is lethal to the same animals at lower doses (LD 50 = 219 μg/kg). Circular dichroism spectra of BcIII and BcIV show a high content of β-strand secondary structure in both peptides, very similar to type 1 sodium channel toxins from various sea anemones, and to APETx1 and APETx2 from A. elegantissima, a HERG channel modulator and an ASIC3 inhibitor, respectively. Interestingly, BcIII and BcIV have similar effects on the action potential of the crab leg nerves, suggesting the same target in this tissue. As BcIII was previously reported as a Na + channel effector and BcIV is inactive over Na + currents of mammalian GH3 cells, we propose a species-specific action for this new molecule. A molecular model of BcIV was constructed using the structure of the APETx1 as template and putative key residues are discussed.

Effects of ApC, a sea anemone toxin, on sodium currents of mammalian neurons

We have characterized the actions of ApC, a sea anemone polypeptide toxin isolated from Anthopleura elegantissima, on neuronal sodium currents ( I Na) using current and voltage-clamp techniques. Neurons of the dorsal root ganglia of Wistar rats (P5–9) in primary culture were used for this study. These cells express tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) I Na. In current-clamp experiments, application of ApC increased the average duration of the action potential. Under voltage-clamp conditions, the main effect of ApC was a concentration-dependent increase in the TTX-S I Na inactivation time course. No significant effects were observed on the activation time course or on the current peak-amplitude. ApC also produced a hyperpolarizing shift in the voltage at which 50% of the channels are inactivated and caused a significant decrease in the voltage dependence of Na + channel inactivation. No effects were observed on TTX-R I Na. Our results suggest that ApC slows the conformational changes required for fast inactivation of the mammalian Na + channels in a form similar to other site-3 toxins, although with a greater potency than ATX-II, a highly homologous anemone toxin.

Expression and Mutagenesis of the Sea Anemone Toxin Av2 Reveals Key Amino Acid Residues Important for Activity on Voltage-Gated Sodium Channels

Type I sea anemone toxins are highly potent modulators of voltage-gated Na-channels (Na(v)s) and compete with the structurally dissimilar scorpion alpha-toxins on binding to receptor site-3. Although these features provide two structurally different probes for studying receptor site-3 and channel fast inactivation, the bioactive surface of sea anemone toxins has not been fully resolved. We established an efficient expression system for Av2 (known as ATX II), a highly insecticidal sea anemone toxin from Anemonia viridis (previously named A. sulcata), and mutagenized it throughout. Each toxin mutant was analyzed in toxicity and binding assays as well as by circular dichroism spectroscopy to discern the effects derived from structural perturbation from those related to bioactivity. Six residues were found to constitute the anti-insect bioactive surface of Av2 (Val-2, Leu-5, Asn-16, Leu-18, and Ile-41). Further analysis of nine Av2 mutants on the human heart channel Na(v)1.5 expressed in Xenopus oocytes indicated that the bioactive surfaces toward insects and mammals practically coincide but differ from the bioactive surface of a structurally similar sea anemone toxin, Anthopleurin B, from Anthopleura xanthogrammica. Hence, our results not only demonstrate clear differences in the bioactive surfaces of Av2 and scorpion alpha-toxins but also indicate that despite the general conservation in structure and importance of the Arg-14 loop and its flanking residues Gly-10 and Gly-20 for function, the surface of interaction between different sea anemone toxins and Na(v)s varies.

Structure and Function of the Voltage Sensor of Sodium Channels Probed by a β-Scorpion Toxin

Voltage sensing by voltage-gated sodium channels determines the electrical excitability of cells, but the molecular mechanism is unknown. beta-Scorpion toxins bind specifically to neurotoxin receptor site 4 and induce a negative shift in the voltage dependence of activation through a voltage sensor-trapping mechanism. Kinetic analysis showed that beta-scorpion toxin binds to the resting state, and subsequently the bound toxin traps the voltage sensor in the activated state in a voltage-dependent but concentration-independent manner. The rate of voltage sensor trapping can be fit by a two-step model, in which the first step is voltage-dependent and correlates with the outward gating movement of the IIS4 segment, whereas the second step is voltage-independent and results in shifted voltage dependence of activation of the channel. Mutations of Glu(779) in extracellular loop IIS1-S2 and both Glu(837) and Leu(840) in extracellular loop IIS3-S4 reduce the binding affinity of beta-scorpion toxin. Mutations of positively charged and hydrophobic amino acid residues in the IIS4 segment do not affect beta-scorpion toxin binding but alter voltage dependence of activation and enhance beta-scorpion toxin action. Structural modeling with the Rosetta algorithm yielded a three-dimensional model of the toxin-receptor complex with the IIS4 voltage sensor at the extracellular surface. Our results provide mechanistic and structural insight into the voltage sensor-trapping mode of scorpion toxin action, define the position of the voltage sensor in the resting state of the sodium channel, and favor voltage-sensing models in which the S4 segment spans the membrane in both resting and activated states.

A new toxin from the sea anemone Condylactis gigantea with effect on sodium channel inactivation

A new peptide toxin exhibiting a molecular weight of 5043 Da (av.) and comprising 47 amino acid residues was isolated from the sea anemone Condylactis gigantea. Purification of the peptide was achieved by a multistep chromatographic procedure monitoring its strong paralytic activity on crustacea (LD 50 approx. 1 μg/kg). Complete sequence analysis of the toxic peptide revealed the isolation of a new member of type I sea anemone sodium channel toxins containing the typical pattern of the six cysteine residues. From 11 kg of wet starting material, approximately 1 g of the peptide toxin was isolated. The physiological action of the new toxin from C. gigantea CgNa was investigated on sodium currents of rat dorsal root ganglion neurons in culture using whole-cell patch clamp technique ( n = 60 ). Under current clamp condition (CgNa) increased action potential duration. This effect is due to slowing down of the TTX-S sodium current inactivation, without modifying the activation process. CgNa prolonged the cardiac action potential duration and enhanced contractile force albeit at 100-fold higher concentrations than the Anemonia sulcata toxin ATXII. The action on sodium channel inactivation and on cardiac excitation-contraction coupling resemble previous results with compounds obtained from this and other sea anemones [Shapiro, B.I., 1968. Purification of a toxin from tentacles of the anemone C. gigantea. Toxicon 5, 253–259; Pelhate, M., Zlotkin, E., 1982. Actions of insect toxin and other toxins derived from the venom of scorpion Androtonus australis on isolated giant axons of the cockroach Periplaneta americana. J. Exp. Biol. 97, 67–77; Salgado, V., Kem, W., 1992. Actions of three structurally distinct sea anemone toxins on crustacean and insect sodium channels. Toxicon 30, 1365–1381; Bruhn, T., Schaller, C., Schulze, C., Sanchez-Rodriquez, J., Dannmeier, C., Ravens, U., Heubach, J.F., Eckhardt, K., Schmidtmayer, J., Schmidt, H., Aneiros, A., Wachter, E., Béress, L., 2001. Isolation and characterization of 5 neurotoxic and cardiotoxic polypeptides from the sea anemone Anthopleura elegantissima. Toxicon, 39, 693–702]. Comprehensive analysis of the purified active fractions suggests that CgNa may represent the main peptide toxin of this sea anemone species.

ATX‐II Effects on the Apparent Location of M Cells in a Computational Model of a Human Left Ventricular Wedge

The apparent location of the myocytes (M cells) with the longest action potential duration (APD) in a canine left ventricular (LV) wedge have been reported to shift after application of a sea anemone toxin, ATX-II. This toxin slows inactivation of I(Na) and thus prolongs APD. Thus, M cells may exhibit dynamic functional states, rather than being a static, anatomically discrete, myocyte population. In this study, we attempted to further define and understand this phenomenon using a mathematical model of the human ventricular myocyte action potential incorporated into an in silico "wedge" preparation. Our simulations demonstrate that even under conditions of a fixed population and ratio of epicardial, M, and endocardial myocytes, the apparent anatomical position (transmural location) of the myocytes with the longest APD can shift following ATX-II treatment. This arises because the ATX-II effect, modeled as a small increase in the late or persistent Na(+) current, and consequent prolongation of APD significantly changes the electrotonic interactions between ventricular myocytes in this LV wedge preparation. This LV wedge model is based on bidomain equations. It corresponds to a rectangular tissue immersed in a passive and isotropic medium that represents the superfusion bath. In this theoretical work, the three known different and discrete populations of myocytes in the human left ventricle have been included: the epicardial, M, and endocardial cells. The effects of ATX-II on I(Na) were simulated by altering the voltage-dependent steady-state inactivation of the parameters h (fast gate) and j (slow gate). As a result, in these ATX-II simulations a persistent late Na(+) current was generated in all three types of ventricular myocytes. However, the APDs were prolonged in a heterogeneous pattern. Our simulations demonstrate that after the ATX-II effects develop, alterations in transmural electrotonic interactions can produce changes in the transmural location of myocytes with the longest APD. The combination of intercellular electrotonic interactions, which tend to reduce and smooth out the discrete transmural APD variations, and the heterogeneous effects of ATX-II, which preferentially prolong the APD of M cells, can shift the location of the ventricular myocytes. This shift results in significantly altered transmural patterns of action potential durations, which would be expected to change localized refractory period and excitability. These cellular changes give rise to alterations in the corresponding surface electrograms and may change the overall substrates for conduction and rhythm disturbances.

Anaphylactic Shock Caused by Exposure to Sea Anemones

Since the first report of a dog that developed severe systemic symptoms in response to a second injection of sea anemone toxin by Richet and Portier in 1902, no clear human cases of anaphylaxis related to exposure to sea anemones has been reported in the literature. A 24-year-old man with an episode of local urticaria on his first contact with a sea anemone (Stichodactyla haddoni), developed dyspnea, severe urticaria and hypotension on exposure to water containing the dead bodies of the organism. To study whether this reaction was mediated by antigen-specific IgE, we performed a histamine release test with blood, Western blotting with serum and lymphocyte proliferating test with peripheral blood mononuclear cells of the patient, for the homogenate of sea anemones. The homogenate of sea anemones induced histamine release from the blood of the patient, but it also induced histamine release from the blood of control subjects. Moreover, it also caused hemolysis of blood of all donors. However, Western-blotting demonstrated the presence of an 86 kd protein-specific IgE in the serum of the patient. Protein antigen(s) in sea anemones may cause anaphylactic shock under the influence of the cytolytic effects and/or lymphocyte-stimulating activity elicited by the toxin of sea anemones.

An Increase in Late Sodium Current Potentiates the Proarrhythmic Activities of Low-Risk QT-Prolonging Drugs in Female Rabbit Hearts

Assessment of the proarrhythmic risk associated with drugs that prolong the QT interval is difficult. We hypothesized that the proarrhythmic activities of drugs with very low to moderate risk of causing torsades de pointes would be well differentiated when the late sodium current (I(NaL)) was greater than normal. The effects of selected QT-prolonging drugs on electrical activity of female rabbit isolated hearts were determined in the absence and presence of sea anemone toxin (ATX-II; an enhancer of I(NaL)). I(NaL) recorded from ventricular myocytes isolated from female rabbit hearts was slightly increased by 1 and 3 nM ATX-II (n = 13, P < 0.01). ATX-II (1 nM) prolonged the duration of the monophasic action potential (MAPD(90)) the isolated heart by of 19 +/- 3% (P < 0.001, n = 31) and shifted the concentration-response relationships for cisapride (1-30 nM), ziprasidone (0.01-3 microM), quinidine (0.1-1 microM), and moxifloxacin (0.01-1 microM) to prolong MAPD to the left by 2- to 12-fold. In contrast, the increases in MAPD(90) caused by 1 nM ATX-II and pentobarbital were only additive, and the increases in MAPD(90) caused by ATX-II and ranolazine [(+/-)-N-(2,6-dimethylphenyl)-(4[2-hydroxy-3-(2-methoxyphenoxy)propyl]-1-piperazine] were less than additive. Episodes of arrhythmic activity were commonly observed, and beat-to-beat variability of action potential duration was increased, during exposure of hearts to cisapride, ziprasidone, quinidine, and moxifloxacin but not during exposure of hearts to ranolazine or pentobarbital, in the presence of ATX-II. Thus, in the female rabbit heart, ATX-II potentiated the effects of QT-prolonging drugs to increase MAPD(90) and unmasked the proarrhythmic activities of these drugs at clinically relevant drug concentrations.

Modulation of Kv3 Subfamily Potassium Currents by the Sea Anemone Toxin BDS: Significance for CNS and Biophysical Studies

Kv3 potassium channels, with their ultra-rapid gating and high activation threshold, are essential for high-frequency firing in many CNS neurons. Significantly, the Kv3.4 subunit has been implicated in the major CNS disorders Parkinson's and Alzheimer's diseases, and it is claimed that selectively targeting this subunit will have therapeutic utility. Previous work suggested that BDS toxins ("blood depressing substance," from the sea anemone Anemonia sulcata) were specific blockers for rapidly inactivating Kv3.4 channels, and consequently these toxins are increasingly used as diagnostic agents for Kv3.4 subunits in central neurons. However, precisely how selective are these toxins for this important CNS protein? We show that BDS is not selective for Kv3.4 but markedly inhibits current through Kv3.1 and Kv3.2 channels. Inhibition comes about not by "pore block" but by striking modification of Kv3 gating kinetics and voltage dependence. Activation and inactivation kinetics are slowed by BDS-I and BDS-II, and V(1/2) for activation is shifted to more positive voltages. Alanine substitution mutagenesis around the S3b and S4 segments of Kv3.2 reveals that BDS acts via voltage-sensing domains, and, consistent with this, ON gating currents from nonconducting Kv3.2 are markedly inhibited. The altered kinetics and gating properties, combined with lack of subunit selectivity with Kv3 subunits, seriously affects the usefulness of BDS toxins in CNS studies. Furthermore, our results do not easily fit with the voltage sensor "paddle" structure proposed recently for Kv channels. Our data will be informative for experiments designed to dissect out the roles of Kv3 subunits in CNS function and dysfunction.

A Variable Residue in the Pore of Kv1 Channels Is Critical for the High Affinity of Blockers from Sea Anemones and Scorpions

Animal toxins are associated with well defined selectivity profiles; however the molecular basis for this property is not understood. To address this issue we refined our previous three-dimensional models of the complex between the sea anemone toxin BgK and the S5-S6 region of Kv1.1 (Gilquin, B., Racape, J., Wrisch, A., Visan, V., Lecoq, A., Grissmer, S., Ménez, A., and Gasparini, S. (2002) J. Biol. Chem. 277, 37406-37413) using a docking procedure that scores and ranks the structures by comparing experimental and back-calculated values of coupling free energies DeltaDeltaGint obtained from double-mutant cycles. These models further highlight the interaction between residue 379 of Kv1.1 and the conserved dyad tyrosine residue of BgK. Because the nature of the residue at position 379 varies from one channel subtype to another, we explored how these natural mutations influence the sensitivity of Kv1 channel subtypes to BgK using binding and electrophysiology experiments. We demonstrated that mutations at this single position indeed suffice to abolish or enhance the sensitivity of Kv1 channels for BgK and other sea anemone and scorpion toxins. Altogether, our data suggest that the residue at position 379 of Kv1 channels controls the affinity of a number of blocking toxins.

Molecular components and toxicity of the venom of the solitary wasp, Anoplius samariensis

The solitary spider wasp, Anoplius samariensis, is known to exhibit a unique long-term, non-lethal paralysis in spiders that it uses as a food source for its larvae. However, neither detailed venom components nor paralytic compounds have ever been characterized. In this study, we examined the components in the low molecular weight fraction of the venom and the paralytic activity of the high molecular weight fraction. The major low molecular weight components of the venom were identified as γ-aminobutyric acid and glutamic acid by micro-liquid chromatography/electrospray ionization mass spectrometry and nuclear magnetic resonance spectrometry analysis. The sodium dodecyl sulfate–polyacrylamide gel electrophoresis and mass analysis revealed that the A. samariensis venom contained the various proteins with weights of 4–100 kDa. A biological assay using Joro spiders ( Nephila clavata) clearly showed that the high molecular weight fraction of the venom prepared by ultrafiltration exerted as potent non-lethal long-term paralysis as the whole venom, whereas the low molecular weight fraction was devoid of any paralytic activity. These results indicated that several venomous proteins in the high molecular weight fraction are responsible for the paralytic activity. Furthermore, we determined the primary structure of one component designated As-fr-19, which was a novel multiple-cysteine peptide with high sequence similarity to several sea anemone and snake toxins including dendrotoxins, rather than any insect toxic peptides identified so far. Taken together, our data showed the unprecedented molecular and toxicological profiles of wasp venoms.

Differential Phospholipid Binding by Site 3 and Site 4 Toxins: IMPLICATIONS FOR STRUCTURAL VARIABILITY BETWEEN VOLTAGE-SENSITIVE SODIUM CHANNEL DOMAINS

It has been shown recently that polypeptide toxins that modulate the gating properties of voltage-sensitive cation channels are able to bind to phospholipid membranes, leading to the suggestion that these toxins are able to access a channel-binding site that remains membrane-restricted (Lee, S.-Y., and MacKinnon, R. (2004) Nature 430, 232-235). We therefore examined the ability of anthopleurin B (ApB), a sea anemone toxin that selectively modifies inactivation kinetics of Na(V)1.x channels, and ProTx-II, a spider toxin that modifies activation kinetics of the same channels, to bind to liposomes. Whereas ProTx-II can be quantitatively depleted from solution upon incubation with phosphatidylcholine/phosphatidylserine liposomes, ApB displays no discernible phospholipid binding activity. We therefore examined the activities of structurally unrelated site 3 and site 4 toxins derived from Leiurus and Centruroides venoms, respectively, in the same assay. Like ApB, the site 3 toxin LqqV shows no lipid binding activity, whereas the site 4 toxin Centruroides toxin II, like ProTx-II, is completely bound. We conclude that toxins that modify inactivation kinetics via binding to Na(V)1.x site 3 lack the ability to bind phospholipids, whereas site 4 toxins, which modify activation, have this activity. This inherent difference suggests that the conformation of domain II more closely resembles that of the K(V)AP channel than does the conformation of domain IV.

Precious Natural Peptides from Spider Venoms: New Tools for Studying Potassium Channels

Among the large variety of animal toxins that target potassium channels, spider peptides constitute an unique class of voltage-dependent K+ (Kv) current inhibitors according to their structure and pharmacological properties. Spider toxins that block Kv currents are small basic peptides the include three disuflide bridges and belong to the family of inhibitor cystine knot (ICK) molecules. Unlike snake, bee, scorpion, or sea anemone toxins that block Kv1 or Kv3 channels, ICK spider toxins target Kv2 and Kv4 channels, which are expressed in the central nervous system (CNS) and in the cardiovascular system. Their selective affinities for Kv2 and/or Kv4 subfamilies are very useful for dissecting these currents in neuronal and cardiac cells and for the determination of their contribution in physiological processes. Their mode of action is also original, since they induce a shift of channel opening to more depolarized potentials that alter the voltage-dependent properties of K currents. Then they are called gating modifiers. Structure-function studies of these gating modifiers were recently facilitated by solving their tridimensional structure together with the crystallization of prokaryotic K+ channels. Spider toxins present an active molecular surface, including a hydrophobic patch surrounded by charged residues, which are important for their binding on Kv channels. Gating modifiers interact with important residues in the S3C-S4 external loop via both hydrophobic and electrostatic interactions. Several dynamic interaction models were proposed, but all of them remain putative.

Potassium Channel Blockade by the Sea Anemone Toxin ShK for the Treatment of Multiple Sclerosis and Other Autoimmune Diseases

Expression of the two lymphocyte potassium channels, the voltage-gated channel Kv1.3 and the calcium activated channel IKCa1, changes during differentiation of human T cells. While IKCa1 is the functionally dominant channel in naive and "early" memory T cells, Kv1.3 is crucial for the activation of terminally differentiated effector memory (TEM) T cells. Because of the involvement of TEM cells in autoimmune processes, Kv1.3 is regarded as a promising target for the treatment of T-cell mediated autoimmune diseases such as multiple sclerosis and the prevention of chronic transplant rejection. ShK, a 35-residue polypeptide toxin from the sea anemone, Stichodactyla helianthus, blocks Kv1.3 at low picomolar concentrations. ShK adopts a central helix-kink-helix fold, and alanine-scanning and other mutagenesis studies have defined its channel-binding surface. Models have been developed of how this toxin effects K+-channel blockade and how its docking configuration might differ in ShK-Dap22, which contains a single side chain substitution that confers specificity for Kv1.3 blockade. ShK, ShK-Dap22 and the Kv1.3 blocking scorpion toxin kaliotoxin have been shown to prevent and treat experimental autoimmune encephalomyelitis in rats, a model for multiple sclerosis. A fluoresceinated analog of ShK, ShK-F6CA, has been developed, which allows the detection of activated TEM cells in human and animal blood samples by flow cytometry and the visualization of Kv1.3 channel distribution in living cells. ShK and its analogs are currently undergoing further evaluation as leads in the development of new biopharmaceuticals for the treatment of multiple sclerosis and other T-cell mediated autoimmune disorders.