Sea anemone venom is known to contain toxins that are active on voltage-sensitive Na؉ channels, as well as on delayed rectifier K ؉ channels belonging to the Kv1 family. This report describes the properties of a new set of peptides from Anemonia sulcata that act as blockers of a specific member of the Kv3 potassium channel family. These toxins, blood depressing substance (BDS)-I and BDS-II, are 43 amino acids long and differ at only two positions. They share no sequence homologies with other K ؉ channel toxins from sea anemones, such as AsKS, AsKC, ShK, or BgK. In COS-transfected cells, the Kv3.4 current was inhibited in a reversible manner by BDS-I, with an IC 50 value of 47 nM. This inhibition is specific because BDS-I failed to block other K ؉ channels in the Kv1, Kv2, Kv3, and Kv4 subfamilies. Inward rectifier K ؉ channels are also insensitive to BDS-I. BDS-I and BDS-II share the same binding site on brain synaptic membranes, with K 0.5 values of 12 and 19 nM, respectively. We observed that BDS-I and BDS-II have some sequence homologies with other sea anemone Na ؉ channels toxins, such as AsI, AsII, and AxI. However, they had a weak effect on tetrodotoxin-sensitive Na ؉ channels in neuroblastoma cells and no effect on Na ؉ channels in cardiac and skeletal muscle cells. BDS-I and BDS-II are the first specific blockers identified so far for the rapidly inactivating Kv3.4 channel.

Potassium channels have an essential role in repolarization phases of action potentials and in the fine regulation of the resting potential. Molecular cloning has recently led to the identification of a large number (over 15) of genes for voltagesensitive, non inward-rectifier, K ϩ (Kv) channels (1, 2) which, when expressed in Xenopus oocytes, generate a variety of K ϩ channel activities with different kinetics, voltage dependences, conductances, and regulation properties. Surprisingly, only a relatively small number of toxins active on these channels has yet been discovered (3, 4). They are MCD peptide from bee venom (5, 6), charybdotoxin and analogs from different scorpion species (7-14), ␤-bungarotoxin (15, 16), and dendrotoxins from mamba venoms (3,5,(17)(18)(19)(20)(21)(22).These different toxins only block the expression of four of the cloned Kv channels (Kv1.1, Kv1.2, and Kv1.6 for MCD peptide and dendrotoxin, Kv1.1, Kv1.2, Kv1.3, and Kv1.6 for charybdotoxin) (reviewed in Ref. 23). Binding studies using radioiodinated derivatives of these toxins have been essential for the identification, purification, and determination of the subunit structure (6, 24 -26) of these Kv channels. These toxins have also been important for the first brain localizations of Kv channels (16,27) and are particularly interesting inducers of long term potentiation (28).Sea anemones produce toxins with which they paralyze their prey. They are particularly important as sources of toxins active on voltage-dependent Na ϩ channel which have been essential tools for studying the structure, the mechanism, and the diversity of this channel type (29 -38).This paper reports the isolation, structure, and properties of a series of new toxins from Anemonia sulcata which behave as blockers of Kv channels. EXPERIMENTAL PROCEDURES Materials-Trypsin, the Kunitz trypsin inhibitor (BPTI),1 and N ␣ -benzoyl-DL-arginine p-nitroanilide (BAPNA) were obtained from Sigma. Sephadex G-25, Sephadex G-50, SP Sephadex C-25 were obtained from Pharmacia, Fractogel TSK HW-50 (F), Fractogel EMD SO 3 -650 (M), and RP18 Lichrocart were from Merck. For HPLC columns, TSK SP 5PW was from Toyosoda. Ultrasphere ODS was from Beckman, Hypersil BDS was from SFCC Shandon, and Alltima was from Alltech. HPLC purifications were performed with a Waters system.Purification of Anemonia Sulcata Peptides-The first steps of this purification were performed with slight modifications of a method previously described for the isolation of Na ϩ channel toxins of A. sulcata (39). In this procedure 12 g of the crude sea anemone toxin (Ref. 39; Fig. 2B1) was dissolved in 120 ml of NaCl 1 M and regelfiltered in two parts on a Sephadex G-50 medium column (7 ϫ 140 cm) equilibrated in 1 M NaCl. The crab paralyzing fractions of these gel filtrations were combined, dialyzed in a Visking Dialysis tube (molecular weight cutoff 12,000 -14,000) for 5 h, concentrated at reduced pressure, and desalted on a Sephadex G-25 column (7 ϫ 70 cm) equilibrated with 0.3 M acetic acid. After a concentration at red...

The venom of the black mamba contains a

(3,12). This paper reports the isolation of a toxin, calcicludine (CaC), from the venom of the green mamba Dendroaspis angusticeps. This toxin is a potent blocker of all types of HVA Ca2+ channels (L-, N-, and P-type). However, L-type Ca2+ channels from cerebellar granule cells appear to be the preferential target of CaC. MATERIALS AND METHODSPurification of CaC. The crude venom (500 mg) of D. angusticeps (Latoxan, Rosans, France) was dissolved in 1% acetic acid and chromatographed onto a Sephadex G50 column. The peptidic fraction was directly loaded onto a TSK (Toyosoda, Japan) SP 5PW (21.5 x 150 mm) column equilibrated with 1% acetic acid. Peptide fractions were then eluted (Fig. 1 Top), with a linear gradient from 1% acetic acid to 1 M ammonium acetate at a flow rate of 8 ml/min. The fractions obtained (horizontal bars) were designated A-R. Fraction Q was lyophilized, redissolved in 1 ml of 0.5% trifluoroacetic acid plus 0.9%6 triethylamine in water, and loaded on a Lichrosorb RP18 7-ikm (250 x 10 mm) column (Merck, Darmstadt, Germany) and eluted ( Fig. 1 Middle) at aflow rate of3 ml/min with a lineargradient from 10%6 to 40% of0.5% trifluoroacetic acid plus 0.9% triethylamine in acetonitrile. Fraction Q1 is CaC.The primary structure of CaC (Fig.

The effects of a polypeptide neurotoxin from Anemonia sulcata on nerve conduction in crayfish giant axons and on frog myelinated fibers have been analyzed. The main features of toxin action are the following: (i) the toxin acts at very low doses and its action is apparently irreversible. (il) The toxin selectively affects the closing (inactivation) of the Na+ channel by slowing it down considerably; it does not alter the opening mechanism of the Na+ channel or the steady-state potassium conductance. (iii) The tetrodotoxin-receptor association is unaffected by previous treatment of the axonal membrane with the sea anemone toxin. (iv) Conversely, the sea anemone toxin can only associate with the membrane when the Na+ channel is o en for Na+; it does not bind when the channel is previously blocked by tetrodotoxin. (v) Besides its effect on the action potential, the sea anemone toxin displays a veratridinetype depolarizing action at low Ca2+ concentration which can be suppressed by tetrodotoxin. The sea anemone toxin greatly stimulates the release of {Y43H]aminobutyric acid from neurotransmitter-loaded rat brain synaptosomes. The apparent dissociation constant of the neurotoxin-receptor complex in this system is 20 nM. The sea anemone toxin effect is antagonized by tetrodotoxin.Neurotoxins are essential tools for the analysis of molecular aspects of nerve conduction and transmission. Toxic molecules already available for study of molecular aspects of conduction include: (i) tetrodotoxin and saxitoxin, which are highly specific for blocking the Na+ channel in most axons (1, 2); (ii) veratridine and batrachotoxin, which depolarize nerve membrane by a selective increase in the resting sodium permeability (2-5); and (iii) scorpion neurotoxin, a miniprotein which affects reversibly the closing of the Na+ channel and the opening of the K+ channel (6-8).A series of neurotoxins was recently isolated in the pure form froln the sea anemone Anemonia sulcata (9-11). The toxins all are small polypeptides. MATERIALS AND METHODS Purification of sea anemone toxins (Anemonia sulcata) was carried out according to Beress et al. (9,10). ATX11 is the most abundant of the three neurotoxic polypeptides (9, 10, 12).Giant axons used in this work were those of the crayfish Astacus leptodactylus and of a cephalopod, the cuttlefish Sepia offlicnalis (axon diameter 200-400 Mum). Giant axons from crustacea were isolated from circumesophageal nerve connectives, those of Sepia from stellar nerves (13). Resting and action potential recordings and voltage clamp experiments have been previously described (8). When the nerve is bathed in a solution containing 0.1 nM ATX11 some of the thin axons begin to fire spontaneously (Fig. IA). More axons are affected at a concentration of 1 nM of ATX11.The giant axon having the maximum diameter (about 100 Mm), which has been used for microelectrode and voltage clamp analysis, is sensitive to ATX11 at concentrations higher than 0.1 AM (Fig. iC). Toxin action on this axon provokes a marked plateau phase of...

Mamba intestinal toxin (MIT 1 ) isolated from Dendroaspis polylepis venom is a 81 amino acid polypeptide crosslinked by five disulphide bridges. MIT 1 has a very potent action on guinea-pig intestinal contractility. MIT 1 (1 nM) potently contracts longitudinal ileal muscle and distal colon, and this contraction is equivalent to that of 40 mM K + . Conversely MIT 1 relaxes proximal colon again as potently as 40 mM K + . The MIT 1 -induced effects are antagonised by tetrodotoxin (1 W WM) in proximal and distal colon but not in longitudinal ileum. The MIT 1 -induced relaxation of the proximal colon is reversibly inhibited by the NO synthase inhibitor L-NAME (200 W WM). 125 Ilabelled MIT 1 binds with a very high affinity to both ileum and brain membranes (K d = 1.3 pM and 0.9 pM, and B max = 30 fmol/ mg and 26 fmol/mg, respectively). MIT 1 is a very highly selective toxin for a receptor present both in the CNS and in the smooth muscle and which might be an as yet unidentified K + channel.z 1999 Federation of European Biochemical Societies.

Eight different polypeptide toxins from sea anemones of four different origins (Anemonia sulcata, Anthopleura xanthogrammica, Stoichactis giganteus, and Actinodendron plumosum) have been studied. Three of these toxins are new; the purification procedure for the five other ones has been improved. Sea anemone toxins were assayed (i) for their toxicity to crabs and mice, (ii) for their affinity for the specific sea anemone toxin receptor situated on the Na+ channels of rat brain synaptosomes, and (iii) for their capacity to increase, in synergy with veratridine, the rate of 22Na+ entry into neuroblastoma cells via the Na+ channel. Some of the toxins are more active on crustaceans, whereas others are more toxic to mammals. A very good correlation exists between the toxic activity to mice, the affinity of the toxin for the Na+ channel in rat brain synaptosomes, and the stimulating effect on 22 Na+ uptake by neuroblastoma cells. The observation has also been made that the most cationic toxins are also the most active on mammals and the least active on crustaceans. Toxicities (LD50) to mice of the most active sea anemone toxins and of the most active scorpion toxins are similar, and sea anemone toxins at high enough concentrations prevent binding of scorpion toxins to their receptor. However, scorpion toxins have affinities for the Na+ channel which are approximately 60 times higher than those found for the most active sea anemone toxins. Three sea anemone toxins appear to be more interesting than toxin II from A. sulcata (the "classical" sea anemone toxin) for studies of the Na+ channel structure and mechanism when the source of the channel is of a mammalian origin. Two of these three toxins can be radiolabeled with iodine while retaining their toxic activity; they appear to be useful tools for future biochemical studies of the Na+ channel.

Four new toxins have been isolated from the sea anemone Radianthus paumotensis: RpI, RpII, RpIII, and RpIV. They are polypeptides comprised of 48 or 49 amino acids; the sequence of RpII has been determined. Toxicities of these toxins in mice and crabs are similar to those of the other known sea anemone toxins, but they fall into a different immunochemically defined class. The sequence of RpII shows close similarities with the N-terminal end (up to residue 20) of the previously sequenced long sea anemone toxins, but most of the remaining part of the molecule is completely different. Like the other sea anemone toxins, Radianthus toxins are active on sodium channels; they slow down the inactivation process. Through their Na+ channel action, Radianthus toxins stimulate Na+ influx into tetrodotoxin-sensitive neuroblastoma cells and tetrodotoxin-resistant rat skeletal myoblasts. The efficiency of the toxins is similar in the two cellular systems. In that respect, Radianthus toxins behave much more like scorpion neurotoxins than sea anemone toxins from Anemonia sulcata or Anthopleura xanthogrammica. In binding experiments to synaptosomal Na+ channels, Radianthus toxins compete with toxin II from the scorpion Androctonus australis but not with toxins II and V from Anemonia sulcata.