Elsevier

Toxicon

Volume 54, Issue 8, 15 December 2009, Pages 1089-1101
Toxicon

Sea anemone toxins affecting voltage-gated sodium channels – molecular and evolutionary features

https://doi.org/10.1016/j.toxicon.2009.02.028Get rights and content

Abstract

The venom of sea anemones is rich in low molecular weight proteinaceous neurotoxins that vary greatly in structure, site of action, and phyletic (insect, crustacean or vertebrate) preference. This toxic versatility likely contributes to the ability of these sessile animals to inhabit marine environments co-habited by a variety of mobile predators. Among these toxins, those that show prominent activity at voltage-gated sodium channels and are critical in predation and defense, have been extensively studied for more than three decades. These studies initially focused on the discovery of new toxins, determination of their covalent and folded structures, understanding of their mechanisms of action on different sodium channels, and identification of the primary sites of interaction of the toxins with their channel receptors. The channel binding site for Type I and the structurally unrelated Type III sea anemone toxins was identified as neurotoxin receptor site 3, a site previously shown to be targeted by scorpion α-toxins. The bioactive surfaces of toxin representatives from these two sea anemone types have been characterized by mutagenesis. These analyses pointed to heterogeneity of receptor site 3 at various sodium channels. A turning point in evolutionary studies of sea anemone toxins was the recent release of the genome sequence of Nematostella vectensis, which enabled analysis of the genomic organization of the corresponding genes. This analysis demonstrated that Type I toxins in Nematostella and other species are encoded by gene families and suggested that these genes developed by concerted evolution. The current review provides a brief historical description of the discovery and characterization of sea anemone toxins that affect voltage-gated sodium channels and delineates recent advances in the study of their structure-activity relationship and evolution.

Introduction

Sea anemones (Actinaria, Cnidaria) are ancient sessile predators (Chen et al., 2002) that heavily depend on their venom for survival (Ruppert and Barnes, 1994). They immobilize their prey or deter their foe by using cells called nematocytes for stinging and delivery of venom. Analysis of the venom in many sea anemone species uncovered a rich repertoire of low molecular weight compounds such as serotonin and histamine (Beress, 1982), ∼20 kDa pore-forming polypeptide toxins (Kem, 1988, Anderluh and Macek, 2002), 3.5–6.5 kDa polypeptide toxins active on voltage-gated potassium channels (Castaneda et al., 1995, Schweitz et al., 1995, Gendeh et al., 1997, Yeung et al., 2005) and 3–5 kDa polypeptide toxins active on voltage-gated sodium channels (Beress et al., 1975, Rathmayer and Beress, 1976, Honma and Shiomi, 2006). The combined effects of these compounds has apparently been successful over hundreds of millions of years as is evident by the ability of sea anemones to colonize and thrive in a wide variety of ecological niches. Moreover, the ever changing environment and appearance of new species has probably enforced diversification of toxins in sea anemones. Indeed, not only can a variety of toxin configurations be found in their venom (Honma and Shiomi, 2006), but it has been shown that each toxin is encoded by a gene family (Moran et al., 2008a).

The toxins active on voltage-gated sodium channels are abundant in all sea anemone venoms assayed to date, and are present even in rare and highly unique species (Ishida et al., 1997, Moran and Gurevitz, 2006). The abundance of these toxins and the fact they constitute a major fraction of the proteinaceous content of the venom (Beress et al., 1975) points to their major role in predation and defense.

Section snippets

The voltage-gated sodium channel as a prime target for sea anemone toxins

Voltage-gated sodium channels (Navs) have a pivotal role in excitability of most animals as they enable the initiation and propagation of action potentials. These channels are transmembrane complexes composed of a highly conserved pore-forming α-subunit and auxiliary subunits such as β-subunits in vertebrates (Catterall, 2000) and TipE subunits in insects (Feng et al., 1995). The ∼260 kDa α-subunit protein is composed of four homologous domains (D1–D4), each comprising six transmembrane segments

The discovery of sea anemone toxins active on voltage-gated sodium channels

Crude extracts from the tentacles of the sea anemone Anemonia sulcata (today named Anemonia viridis) were first prepared in 1902 and shown to cause anaphylaxis in dogs (Richet, 1902). The paralytic and lethal activities of a sea anemone venom were investigated in more detail by Narahashi and co-workers (1969) who demonstrated that a toxic fraction from the Caribbean giant anemone Condylactis gigantea delayed the inactivation of sodium currents in the crayfish giant axon. Six years later Beress

Classification of sea anemone toxins active on voltage-gated sodium channels

Comparison of toxins Av1, Av2 and Av3 revealed that they belong to at least two protein families. Over the years, numerous toxins that resemble Av1 and Av2 were found in various sea anemone species (Fig. 2). Schweitz et al. (1985) isolated four toxins (Rp1, Rp2, Rp3 and Rp4) from the species Heteractis paumotensis (previously called Radianthus paumotensis) and categorized them as a new class of sea anemone toxins because antibodies raised against Rp3 recognized other R. paumotensis toxins and

Genomic organization and evolution of sea anemone toxin genes

The data regarding the genes and transcripts encoding sea anemone toxins is scarce. Transcripts for sea anemone toxins were isolated from several species mostly via PCR and degenerate primers (Spagnuolo et al., 1994, Kelso and Blumenthal, 1998, Anderluh et al., 2000, Honma et al., 2005). These toxins seem to be translated as precursors, carrying a leader peptide and a propart region, which have been suggested to have a role in intracellular sorting and delivery to the nematocyst (Anderluh

Expression of neurotoxins across the life cycle of sea anemones

Sea anemones begin their life as free swimming planulae that settle and metamorphose into adult polyps (Ruppert and Barnes, 1994). A thorough survey of the literature reveals that all sea anemone toxins reported thus far were isolated from polyps. Nevertheless, it is well documented that nematocytes are common in planulae and that the precursor cells of nematocytes, the nematoblasts, appear in cnidarian embryos as early as 12 h after fertilization (reviewed by Kass-Simon and Scappaticci, 2002),

The selectivity of sea anemone toxins

Profound differences in selectivity of sea anemone toxins toward various animal groups have been documented. Av1 and Av3 are very active in crustaceans and inactive in mice, while Av2 is highly active in both organisms (Schweitz et al., 1981). In contrast, anthopleurin A (ApA) and anthopleurin B (ApB), Type I sea anemone toxins of Anthopleura xanthogrammica, are more active on mammals than on crustaceans. Other studies using cell lines that express specific mammalian Nav subtypes and

The bioactive surfaces of sea anemone toxins

The initial attempt to uncover residues with a functional role in a sea anemone toxin was by chemical modifications of Av2 (Barhanin et al., 1981). This study raised the putative role in bioactivity of Asp-7, Asp-9, Arg-14, His-32, His-37, Lys-35, Lys-36, Lys-46 and the C-terminus. However, since only charged residues were modified and in many instances the simultaneous modifications could lead to alterations in toxin fold, the significance of these results is questionable. Indeed, mutagenesis

The putative receptor for sea anemone toxins on voltage-gated sodium channels

Determination of receptor site 3 on the Nav requires identification of channel residues or determinants that constitute the face of interaction with toxin ligands defined pharmacologically as site 3 toxins. Since this receptor site is targeted by scorpion, spider, and sea anemone toxins that vary greatly in structure, the face of interaction between each toxin and the Nav evidently differs. Yet, considering that these three toxin types compete in binding (Catterall and Beress, 1978, Gordon and

Acknowledgments

The work conducted at Tel Aviv University was supported by the United States-Israel Binational Agricultural Research and Development grants IS-3928-06 (M.G. and D.G.) and IS-4066-07 (D.G. and M.G.); by the Israeli Science Foundation grants 107/08 (M.G.) and 1008/05 (D.G. and M.G.); by the European Community Integrated Project LSH-2005-1.2.5-2 proposal No. 037592 - CONCO (M.G. and D.G.); and by NIH 1 U01 NS058039-01 (M.G.)

Conflict of interest

None declared.

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