First report on an inotropic peptide activating tetrodotoxin-sensitive, "neuronal" sodium currents in the heart.

BACKGROUND: New therapeutic approaches to improve cardiac contractility without severe risk would improve the management of acute heart failure. Increasing systolic sodium influx can increase cardiac contractility, but most sodium channel activators have proarrhythmic effects that limit their clinical use. Here, we report the cardiac effects of a novel positive inotropic peptide isolated from the toxin of the Black Judean scorpion that activates neuronal tetrodotoxin-sensitive sodium channels. METHODS AND RESULTS: All venoms and peptides were isolated from Black Judean Scorpions (Buthotus Hottentotta) caught in the Judean Desert. The full scorpion venom increased left ventricular function in sedated mice in vivo, prolonged ventricular repolarization, and provoked ventricular arrhythmias. An inotropic peptide (BjIP) isolated from the full venom by chromatography increased cardiac contractility but did neither provoke ventricular arrhythmias nor prolong cardiac repolarization. BjIP increased intracellular calcium in ventricular cardiomyocytes and prolonged inactivation of the cardiac sodium current. Low concentrations of tetrodotoxin (200 nmol/L) abolished the effect of BjIP on calcium transients and sodium current. BjIP did not alter the function of Nav1.5, but selectively activated the brain-type sodium channels Nav1.6 or Nav1.3 in cellular electrophysiological recordings obtained from rodent thalamic slices. Nav1.3 (SCN3A) mRNA was detected in human and mouse heart tissue. CONCLUSIONS: Our pilot experiments suggest that selective activation of tetrodotoxin-sensitive neuronal sodium channels can safely increase cardiac contractility. As such, the peptide described here may become a lead compound for a new class of positive inotropic agents.

The sea anemone toxin AdE-1 modifies both sodium and potassium currents of rat cardiomyocytes.

AdE-1, a cardiotonic peptide recently isolated from the sea anemone Aiptasia diaphana, contains 44 amino acids and has a molecular mass of 4907 Da. It was previously found to resemble other sea anemone type 1 and 2 Na+ channel toxins, enhancing contractions of rat cardiomyocytes and slowing their twitch relaxation; however, it did not induce spontaneous twitches. AdE-1 increased the duration of the cardiomyocyte action potential and decreased its amplitude and its time-to-peak in a concentration-dependent manner, without affecting its threshold and cell resting potential. Nor did it generate the early and delayed after-depolarizations characteristic of sea anemone Na+ channel toxins. To further understand its mechanism of action we investigated the effect of AdE-1 on the major ion currents of rat cardiomyocytes. In the present study we show that AdE-1 markedly slowed inactivation of the Na+ current, enhancing and prolonging the current influx with no effect on current activation, possibly through direct interaction with the site 3 receptor of the Na+ channel. No significant effect of AdE-1 on the Ca2+ current was observed, but, unexpectedly, AdE-1 significantly increased the amplitude of the transient component of the K+ current, shifting the current threshold to more negative membrane potentials. This effect on the K+ current has not been found in any other sea anemone toxin and may explain the exclusive reduction in action potential amplitude and the absence of the action potential disorders found with other toxins, such as early and delayed after-depolarizations.

AdE-1, a new inotropic Na(+) channel toxin from Aiptasia diaphana, is similar to, yet distinct from, known anemone Na(+) channel toxins.

Heart failure is one of the most prevalent causes of death in the western world. Sea anemone contains a myriad of short peptide neurotoxins affecting many pharmacological targets, several of which possess cardiotonic activity. In the present study we describe the isolation and characterization of AdE-1 (ion channel modifier), a novel cardiotonic peptide from the sea anemone Aiptasia diaphana, which differs from other cnidarian toxins. Although AdE-1 has the same cysteine residue arrangement as sea anemone type 1 and 2 Na(+) channel toxins, its sequence contains many substitutions in conserved and essential sites and its overall homology to other toxins identified to date is low (<36%). Physiologically, AdE-1 increases the amplitude of cardiomyocyte contraction and slows the late phase of the twitch relaxation velocity with no induction of spontaneous twitching. It increases action potential duration of cardiomyocytes with no effect on its threshold and on the cell's resting potential. Similar to other sea anemone Na(+) channel toxins such as Av2 (Anemonia viridis toxin II), AdE-1 markedly inhibits Na(+) current inactivation with no significant effect on current activation, suggesting a similar mechanism of action. However, its effects on twitch relaxation velocity, action potential amplitude and on the time to peak suggest that this novel toxin affects cardiomyocyte function via a more complex mechanism. Additionally, Av2's characteristic delayed and early after-depolarizations were not observed. Despite its structural differences, AdE-1 physiologic effectiveness is comparable with Av2 with a similar ED(50) value to blowfly larvae. This finding raises questions regarding the extent of the universality of structure-function in sea anemone Na(+) channel toxins.

The tale of a resting gland: transcriptome of a replete venom gland from the scorpion Hottentotta judaicus.

cDNA libraries are increasingly being used for high-throughput interrogation of animal venomes. Most previous studies have focused on discovery of new venom toxins, whereas the dynamics of toxin transcription and associated cellular processes have received much less attention. Here we provide, for the first time, an analysis of a transcriptome from the venom gland of a scorpion (Hottentotta judaicus) that is not actively engaged in regenerating its venom. We demonstrate a low abundance of toxin-encoding transcripts coupled with a previously unobserved proliferation of protease sequences. Additionally, we identified several low abundance, toxin-like sequences that may represent decommissioned toxins that are unlikely to be translated. These sequences are not evenly distributed across all toxin families, but rather appear more frequently in transcripts related to α-toxins and ß-toxins that are known to target voltage-gated sodium channels. The transcriptomic profile of the replete venom gland is very different to that obtained previously from scorpion venom glands actively engaged in venom regeneration, and it highlights our lack of knowledge as to how the dynamics of transcription changes as the gland progresses from venom regeneration to a "resting" state. This study therefore provides an important foundation for future studies into the dynamics of transcription in the venom glands of scorpions and other venomous animals.

A hydra with many heads: protein and polypeptide toxins from hydra and their biological roles.

Hydra have been classical model organisms for over 250 years, yet little is known about the toxins they produce, and how they utilize these toxins to catch prey, protect themselves from predators and fulfill other biological roles necessary for survival. Unlike typical venomous organisms the hydra allomonal system is complex and "holistic", produced by various stinging cells (in the hunting tentacles and body ectoderm) as well as by non-nematocystic tissue. Toxic proteins also fulfill novel, non-allomonal roles in hydra. This review described the toxins produced by hydra within the context of their biology and natural history. Hydra nematocyst venom contains a high-molecular weight (>100 kDa) hemolytic and paralytic protein and a protein of approximately 30 kDa which induces a long-lasting flaccid paralysis. No low-molecular weight toxicity is observed, suggesting the lack of "classical" 4-7 kDa neurotoxins. The occurrence of a potent phospholipase activity in the venom is supported by the detection of several venom-like phospholipase A2 genes expressed by hydra. Hydra also produce toxins which are not part of the nematocyst venom. In the green hydra, Hydralysins, a novel family of Pore-Forming Proteins, are secreted into the gastrovascular cavity during feeding, probably helping in disintegration of the prey. Other putative non-nematocystic "toxins" may be involved in immunity, development or regulation of behavior. As the first venomous organism for which modern molecular tools are available, hydra provide a useful model to answer many outstanding questions on the way venomous organisms utilize their toxins to survive.

Cnidarian internal stinging mechanism.

Stinging mechanisms generally deliver venomous compounds to external targets. However, nematocysts, the microscopic stinging organelles that are common to all members of the phylum Cnidaria, occur and act in both external and internal tissue structures. This is the first report of such an internal piercing mechanism. This mechanism identifies prey items within the body cavity of the sea anemone and actively injects them with cytolytic venom compounds. Internal tissues isolated from sea anemones caused the degradation of live Artemia salina nauplii in vitro. When examined, the nauplii were found to be pierced by discharged nematocysts. This phenomenon is suggested to aid digestive phagocytic processes in a predator otherwise lacking the means to masticate its prey.

Expulsion of symbiotic algae during feeding by the green hydra--a mechanism for regulating symbiont density?

BACKGROUND: Algal-cnidarian symbiosis is one of the main factors contributing to the success of cnidarians, and is crucial for the maintenance of coral reefs. While loss of the symbionts (such as in coral bleaching) may cause the death of the cnidarian host, over-proliferation of the algae may also harm the host. Thus, there is a need for the host to regulate the population density of its symbionts. In the green hydra, Chlorohydra viridissima, the density of symbiotic algae may be controlled through host modulation of the algal cell cycle. Alternatively, Chlorohydra may actively expel their endosymbionts, although this phenomenon has only been observed under experimentally contrived stress conditions. PRINCIPAL FINDINGS: We show, using light and electron microscopy, that Chlorohydra actively expel endosymbiotic algal cells during predatory feeding on Artemia. This expulsion occurs as part of the apocrine mode of secretion from the endodermal digestive cells, but may also occur via an independent exocytotic mechanism. SIGNIFICANCE: Our results demonstrate, for the first time, active expulsion of endosymbiotic algae from cnidarians under natural conditions. We suggest this phenomenon may represent a mechanism whereby cnidarians can expel excess symbiotic algae when an alternative form of nutrition is available in the form of prey.

A pharmacological solution for a conspecific conflict: ROS-mediated territorial aggression in sea anemones.

Venomous organisms are usually resistant to their own venoms, and utilize mechanical behavioral means to resolve intra-specific conflicts, such as those occurring over territory, mates or social status. The present study deals with a venom apparatus, which has been specifically designed for conspecific aggression, by the aid of a unique pharmacology. Actinarian sea anemones such as Actinia equina utilize vesicular organs termed acrorhagi in order to deter conspecific territorial competitors. The territorial aggression was shown to be performed by the aid of acrorhagial cnidocysts, which inflict localized tissue necroses on the body of the approaching-threatening anemone. In view of the fact that sea anemones were shown to resist mechanical injuries and their own cytolytic, necrosis-inducing pore-forming substances-the above acrorhagial injuries are ambiguous. Using an electrical device to collect acrorhagial cnidocyst-derived venom, we have shown that the venom is devoid of paralytic-neurotoxic activity, contains heat denaturable hemolytic polypeptides of a low molecular weight and is capable of inducing intracellular formation of reactive oxygen species (ROS) upon medium application to various cultured cells. The ROS formation phenomenon provides a reasonable pharmacological solution to the, above-mentioned, paradoxical conspecific self-intoxication by triggering a preexisting global endogenous mechanism of oxygen toxicity common to aerobic organisms.

Osmotically driven prey disintegration in the gastrovascular cavity of the green hydra by a pore-forming protein.

Pore-forming proteins (PFPs) are water-soluble proteins able to integrate into target membranes to form transmembrane pores. They are common determinants of bacterial pathogenicity and are often found in animal venoms. We recently isolated and characterized Hydralysins (Hlns), paralytic PFPs from the venomous green hydra Chlorohydra viridissima that are not found within the nematocytes, suggesting they are not involved in prey capture. The present study aimed to decipher the biological role of Hlns. Using in situ hybridization and immunohistochemistry, we show that Hlns are expressed by digestive cells surrounding the gastrovascular cavity (GVC) of Chlorohydra and secreted onto the prey during feeding. At biologically relevant concentrations, Hlns bind prey membranes and form pores, lysing the cells and disintegrating the prey tissue. Hlns are unable to bind Chlorohydra membranes, thus protecting the producing animal from the destructive effect of its own cytolytic protein. We suggest that osmotic disintegration of the prey within the GVC by Hlns, followed by phagocytosis and intracellular digestion, allows the soft-bodied green hydra to feed on hard, cuticle-covered prey while lacking the physical means to mechanically disintegrate it. Our results extend the biological significance of PFPs beyond the commonly expected offensive or defensive roles.

Toxic polypeptides of the hydra--a bioinformatic approach to cnidarian allomones.

Cnidarians such as hydrae and sea anemones are sessile, predatory, soft bodied animals which depend on offensive and defensive allomones for prey capture and survival. These allomones are distributed throughout the entire organism both in specialized stinging cells (nematocytes) and in the body tissues. The cnidarian allomonal system is composed of neurotoxins, cytolysins and toxic phospholipapses. The present bioinformatic survey was motivated by the fact that while hydrae are the most studied model cnidarian, little is known about their allomones. A large-scale EST database from Hydra magnipapillata was searched for orthologs of known cnidarian allomones, as well as for allomones found in other venomous organisms. We show that the hydrae express orthologs of cnidarian phospholipase A2 toxins and cytolysins belonging to the actinoporin family, but could not find orthologs of the 'classic' short chain neurotoxins affecting sodium and potassium conductance. Hydrae also express proteins similar to elapid-like phospholipases, CRISP proteins, Prokineticin-like polypeptides and toxic deoxyribonucleases. Our results illustrate a high level of complexity in the hydra allomonal system, suggest that several toxins represent a basal component of all cnidarian allomones, and raise the intriguing possibility that similar proteins may fulfill both endogenous and allomonal roles in cnidaria.

Hydralysins, a new category of beta-pore-forming toxins in cnidaria.

Cnidaria are venomous animals that produce diverse protein and polypeptide toxins, stored and delivered into the prey through the stinging cells, the nematocytes. These include pore-forming cytolytic toxins such as well studied actinoporins. In this work, we have shown that the non-nematocystic paralytic toxins, hydralysins, from the green hydra Chlorohydra viridissima comprise a highly diverse group of beta-pore-forming proteins, distinct from other cnidarian toxins but similar in activity and structure to bacterial and fungal toxins. Functional characterization of hydralysins reveals that as soluble monomers they are rich in beta-structure, as revealed by far UV circular dichroism and computational analysis. Hydralysins bind erythrocyte membranes and form discrete pores with an internal diameter of approximately 1.2 nm. The cytolytic effect of hydralysin is cell type-selective, suggesting a specific receptor that is not a phospholipid or carbohydrate. Multiple sequence alignment reveals that hydralysins share a set of conserved sequence motifs with known pore-forming toxins such as aerolysin, epsilon-toxin, alpha-toxin, and LSL and that these sequence motifs are found in and around the poreforming domains of the toxins. The importance of these sequence motifs is revealed by the cloning, expression, and mutagenesis of three hydralysin isoforms that strongly differ in their hemolytic and paralytic activities. The correlation between the paralytic and cytolytic activities of hydralysin suggests that both are a consequence of receptor-mediated pore formation. Hydralysins and their homologues exemplify the wide distribution of beta-pore formers in biology and provide a useful model for the study of their molecular mode of action.

BjalphaIT: a novel scorpion alpha-toxin selective for insects--unique pharmacological tool.

Long-chain neurotoxins derived from the venom of the Buthidae scorpions, which affect voltage-gated sodium channels (VGSCs) can be subdivided according to their toxicity to insects into insect-selective excitatory and depressant toxins (beta-toxins) and the alpha-like toxins which affect both mammals and insects. In the present study by the aid of reverse-phase HPLC column chromatography, RT-PCR, cloning and various toxicity assays, a new insect selective toxin designated as BjalphaIT was isolated from the venom of the Judean Black Scorpion (Buthotus judaicus), and its full primary sequence was determined: MNYLVVICFALLLMTVVESGRDAYIADNLNCAYTCGSNSYCNTECTKNGAVSGYCQWLGKYGNACWCINLPDKVPIRIPGACR (leader sequence is underlined). Despite its lack of toxicity to mammals and potent toxicity to insects, BjalphaIT reveals an amino acid sequence and an inferred spatial arrangement that is characteristic of the well-known scorpion alpha-toxins highly toxic to mammals. BjalphaITs sharp distinction between insects and mammals was also revealed by its effect on sodium conductance of two cloned neuronal VGSCs heterloguously expressed in Xenopus laevis oocytes and assayed with the two-electrode voltage-clamp technique. BjalphaIT completely inhibits the inactivation process of the insect para/tipE VGSC at a concentration of 100 nM, in contrast to the rat brain Na(v)1.2/beta1 which is resistant to the toxin. The above categorical distinction between mammal and insect VGSCs exhibited by BjalphaIT enables its employment in the clarification of the molecular basis of the animal group specificity of scorpion venom derived neurotoxic polypeptides and voltage-gated sodium channels.

Endogenous regulation of the functional duality of pahutoxin, a marine trunkfish surfactant.

Pahutoxin (PHN) is a long chain detergent-like quaternary ammonium cationic substance derived from defensive skin secretions of trunkfish. A recent study has revealed that PHN's ichthyotoxicity and its phospholipid membrane disruption effect are produced by two separate mechanisms, which presumably represent two separate physicochemical domains in the PHN molecule [Kalmanzon, E., Rahamim, Y., Barenholz, Y., Carmeli, S., Zlotkin, E., 2003. Receptor-mediated toxicity of pahutoxin, a marine trunkfish surfactant. Toxicon 42, 63-71]. Here we report on the occurrence of a natural endogenous mechanism, which regulates the above PHN's functional duality. The regulation is performed by the aid of two separates constituents of the trunkfish secretion, which either selectively amplify PHN's ichthyotoxicity (factor I) or suppress its phospholipids permeabilization effect (factor II). The ecological significance of such endogenous regulation is discussed.

Receptor-mediated toxicity of pahutoxin, a marine trunkfish surfactant.

Pahutoxin (PHN, choline chloride ester of 3-acetoxypalmitic acid) is a natural fish-killing (ichthyotoxic) agent derived from the defensive secretions of trunkfish. In spite of its obvious structural resemblance to synthetic cationic long-chain quaternary ammonium detergents, we show that PHN's action does not rely on its surfactant properties and is in fact, receptor-mediated. The above conclusion is supported by the following data: 1. Ichthyotoxicity is not related to its detergency or surfactivity, as indicated by the fact that the lethal concentration is about 1.5 orders of magnitude below its critical micelle concentration value (69 microM) and its liposomal/seawater partition coefficient is low (62-85); 2. The trunkfish is tolerant to its own pahutoxin; 3. Ichthyotoxicity occurs only upon application to the surrounding water, suggesting the existence of externally located receptors; 4. The receptor hypothesis was supported by the aid of equilibrium saturation binding assays revealing the presence of specific binding sites to PHN on the fish gill membranes; 5. The PHN tolerant trunkfish was shown to be devoid of PHN-binding sites. Some chemo-ecological, and environmental implications are discussed.

Hydralysin, a novel animal group-selective paralytic and cytolytic protein from a noncnidocystic origin in hydra.

In Cnidaria, the production of neurotoxic polypeptides is attributed to the ectodermal stinging cells (cnidocytes), which are discharged for offensive (prey capture) and/or defensive purposes. In this study, a new paralysis-inducing (neurotoxic) protein from the green hydra Chlorohydra viridissima was purified, cloned, and expressed. This paralytic protein is unique in that it (1) is derived from a noncnidocystic origin, (2) reveals a clear animal group-selective toxicity, (3) possesses an uncommon primary structure, remindful of pore-forming toxins, and (4) has a fast cytotoxic effect on insect cells but not on the tested mammalian cells. The possible biological role of such a noncnidocystic toxin is discussed.

Domain 2 of Drosophila para voltage-gated sodium channel confers insect properties to a rat brain channel.

The ability of the excitatory anti-insect-selective scorpion toxin AahIT (Androctonus australis hector) to exclusively bind to and modify the insect voltage-gated sodium channel (NaCh) makes it a unique tool to unravel the structural differences between mammalian and insect channels, a prerequisite in the design of selective pesticides. To localize the insect NaCh domain that binds AahIT, we constructed a chimeric channel composed of rat brain NaCh alpha-subunit (rBIIA) in which domain-2 (D2) was replaced by that of Drosophila Para (paralytic temperature-sensitive). The choice of D2 was dictated by the similarity between AahIT and scorpion beta-toxins pertaining to both their binding and action and the essential role of D2 in the beta-toxins binding site on mammalian channels. Expression of the chimera rBIIA-ParaD2 in Xenopus oocytes gave rise to voltage-gated and TTX-sensitive NaChs that, like rBIIA, were sensitive to scorpion alpha-toxins and regulated by the auxiliary subunit beta(1) but not by the insect TipE. Notably, like Drosophila Para/TipE, but unlike rBIIA/beta(1), the chimera gained sensitivity to AahIT, indicating that the phyletic selectivity of AahIT is conferred by the insect NaCh D2. Furthermore, the chimera acquired additional insect channel properties; its activation was shifted to more positive potentials, and the effect of alpha-toxins was potentiated. Our results highlight the key role of D2 in the selective recognition of anti-insect excitatory toxins and in the modulation of NaCh gating. We also provide a methodological approach to the study of ion channels that are difficult to express in model expression systems.