Cnidarian Toxins Acting on Voltage-Gated Ion Channels

Voltage-gated ion channels generate electrical activity in excitable cells. As such, they are essential components of neuromuscular and neuronal systems, and are targeted by toxins from a wide variety of phyla, including the cnidarians. Here, we review cnidarian toxins known to target voltage-gated ion channels, the specific channel types targeted, and, where known, the sites of action of cnidarian toxins on different channels.


Cnidarian Ion Channel Toxins
The cnidarians are the earliest extant organisms with a neuromuscular system. They are simple carnivores that are radially symmetrical, with a mouth surrounded by tentacles. These tentacles contain high concentrations of stinging cells called nematocysts (cnidocysts), which are specialized epidermal cells that produce, store, and inject toxins used for protection and predation. Venoms contain a bouquet of substances, including peptides, proteins, phospholipids, phospholipases, glycoproteins, sterols, bioactive amines, and carbohydrates [23]. Thus, the cnidarians represent a rich source of venoms and toxins. The phylum comprises four extant classes: the hydrozoans; the cubozoids, or box jellies; the scyphozoans, or true jellyfish; and the anthozoa, the sea anemones and corals.
Sea anemone toxins dominate in terms of number identified and they are also the best characterized in terms of mechanism of action [20]. To date, many cnidarian toxins that act on Na V and various K V channels have been described in detail; toxins from cnidarians that clearly act on Ca V channels have thus far not been characterized.

Na V Channel Toxins
Some of the most thoroughly studied cnidarian toxins are polypeptide toxins from sea anemones that target Na V channels [20,24,25,26]. These toxins are polypeptides of ~5 kDa, and more than 50 different sea anemone Na V channel toxins have been isolated or cloned from a large variety of species. Particular toxins, including ATX II from Anemonia sulcata [27], and Anthopleurin A (ApA) [28] and Anthopleurin B (ApB) [29] from Anthopleura xanthogrammica, have proven especially useful as probes of Na V channel structure and function. Furthermore, recent studies have provided evidence for in vivo epileptogenic effects of toxins (e.g., cangitoxin from Bunodosoma cangicum) [30].
Norton [25] described the classification of anemone Na V channel toxins into different types based on their amino acid sequences (Fig. 1). Type 1 and Type 2 toxins are polypeptides of 46-49 amino acids; Type 3 peptides, from the genus Entacmaea (family Actiniidae), are shorter, containing 27-32 amino acids. Calitoxins I and II from Calliactis parasitica [31,32] are long peptides that do not fit clearly into Types 1 or 2, and may constitute a fourth class. Members of the Type 1 and Type 2 classes show extensive sequence similarity within each class (≥60%), but sequence similarity between members of the two classes is significantly lower (~30%), and there is no immunological crossreactivity between the two groups. Members of both classes contain six conserved cysteine residues that are cross-linked by three disulfide bridges, and both have basic C-terminal sequences, though more markedly for Type 2 toxins (Fig. 1). The Type 1 and 2 toxins are largely distributed according to taxonomy. Toxins from the Actiniidae family are Type 1, while both types of toxins can be found in the family Stichodactylidae [20,25]. A single species can produce toxins from both classes, as is seen with Gigantoxin II and Gigantoxin III from Stichodactyla gigantea [33].  Identical residues are shaded black. Na V toxins shown are: ApA and ApB from Anthopleura xanthogrammica [28,29]; ATX II from Anemonia sulcata [27]; Bg II from Bunodosoma granulifera [51]; Cangitoxin from Bunodosoma cangicum [30]; Sh I from Stichodactyla helianthus [52]; Rp II and Rp III from Radianthus paumotensis [53,54]; RTX I from Radianthus macrodactylus [55]; PaTX from Parasicyonis actinostoloides [56]; Er I from Entacmaea ramsayi [33]; Da I from Dofleinia armata [33]; and ATX III from Anemonia sulcata [57].
Anemone Na V channel toxins were initially characterized as cardiac stimulants [34] and neurotoxins [35], with their predominant effect based on relative affinities for cardiac or neuronal Na V isoforms. They were subsequently shown to interact with neurotoxin receptor site 3 of Na V channels, with their action being the delay of channel inactivation [36][37][38][39][40]. The open state of the channel is thus prolonged during depolarization. Neurotoxin receptor site 3 is one of at least six neurotoxin receptor sites on Na V channels. It is also a receptor for α-scorpion toxins, which do not share sequence homology with the anemone toxins. Receptor site 3 is a complex receptor site that includes the extracellular loop between transmembrane segments S3 and S4 in domain IV of the channel [41]. The toxins bind to this site via electrostatic interactions, predominantly with the negatively charged E1613 residue in rat brain Na V channels [42] and D1612 in cardiac Na V channels [43]. Contributions from other, nearby residues [44] and unidentified contacts in the IS5-S6 and IVS5-S6 loops [45,46] are also likely involved in interaction with these toxins. Interestingly, unlike toxins that interact with receptor site 4, ApB and other site 3 toxins do not bind phospholipids [47].
Work on the ApB toxin has defined a region critical for interaction with Na V channels. ApB, like other Type 1 toxins, has a conformation that is primarily a β-structure, with a four-stranded antiparallel β sheet linked by β-turns and loops with no α-helix [48]. A disordered region corresponding to residues 8-17 of ApB and found in all active sea anemone toxins is referred to as the Arg-14 loop [49]. This region appears to be critical for interaction with receptor site 3 of the Na V channel. Furthermore, the relative non-selectivity of ApB for cardiac and neuronal channels versus the relative selectivity of ApA for cardiac channels is associated with two cationic residues (R12, K49) found in ApB but not in ApA [50].

K V Channel Toxins
There have been at least 11 K V channel toxins that have been identified in various sea anemones to date. These peptide toxins fall into three classes based on structural and functional differences ( Anthopleura elegantissima 3 [66] Type 1 toxins potently block K V 1 (Shaker) channels. They contain 35-37 amino acids that form two nearly perpendicular stretches of helices and include three disulfide bridges [58,59]. A conserved dyad of lysine (K22 in ShK) and tyrosine (Y23 in ShK) appear to be essential residues required for interaction with rat brain K V channels [60]. A similar result is found for the BgK toxin [56,58,59]. Recently, use of ShK and analogs to selectively block K V 1.3, a lymphocyte K V channel that is important for the activation of terminally differentiated effector memory (TEM) T cells, is being investigated as a candidate treatment for multiple sclerosis and other autoimmune diseases [63].
The Type 2 K V channel toxins, represented by the AsKCs, are made up of 58 or 59 amino acid residues, and also block K V 1 channels, though with less potency than Type 1 toxins [64]. They have sequence homology to dendrotoxins, K V channel toxins from snake venom; they also show homology to Kunitz-type protease inhibitors, and exhibit protease inhibitory activity.
Type 3 K V channel toxins include BDS-I and BDS-II from A. sulcata [65], and APETx1 from Anthopleura elegantissima [66]. BDS-I and II are selective blockers of K V 3 (Shaw) channels, predominantly the fast-inactivating K V 3.4 channel. APETx1 is highly similar (54%) to the BDS toxins.

Concluding remarks
Although sea anemone toxins have provided a wealth of pharmacological probes, toxins from only a relatively few cnidarian species have thus far been characterized. Novel venoms from other anemone species as well as other cnidarians have the potential to provide even more powerful tools, as well as possible pharmaceutical applications. For example, Ca V channels are critical components of the neuromuscular junction and of neurosecretory activity, and are targeted by toxins from a wide variety of organisms. Interestingly, however, purified cnidarian toxins with clear selectivity for Ca V channels have not yet been described. A potential candidate Ca V toxin may be the Bainh toxin isolated from Bunodosoma granulifera [72]. This toxin increases the force of contraction in mammalian ventricular muscle preparations, an effect that appears to be mediated by a voltage-independent, toxin-induced increase in L-type Ca V currents.
Several non-anemone venoms produce perturbations in ion transport across excitable membranes [73], and potential Ca V toxins may be present in the crude venoms of the hydrocoral Millepora complanata [74] and the sea nettle Chrysaora quinquecirrha [75]. SNTX, a toxin also from C. quinquecirrha, creates large cation-selective channels that open and close spontaneously and show voltage-dependence of open probability [76]. It is thus likely that as more venoms from different cnidarian species are examined in greater detail, a wider diversity of ion channel toxins will emerge.