Sea anemone toxin:a tool to study molecular mechanisms of nerve conduction and excitation-secretion coupling.

Abstract

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...

Developmental Biology of Neoplastic Growth

“…They possess specialized stinging organelles (cnidocysts) for capturing prey and self-protection. Cnidocysts contain potent paralysing toxins which have been isolated during the last 35 years from numerous sea anemone species (Beress et al 1975;Kelso and Blumenthal 1998;Norton 1991;Romey et al 1976;Schweitz et al 1981). They are polypeptides of molecular weights between 3,000 Da and 6,500 Da that are crosslinked by several disulfide bridges.…”

“…At low concentrations (< 40 μg/kg) these toxins, after in vivo intravenous, intracisternal (mammals) or intramuscular (crustaceans) injections, induce severe toxic syndromes including paralysis, general hyperexcitability, cardiac disorders, convulsions and death (Alsen et al 1978;Schweitz 1984). These toxins interact with a large variety of excitable cells including neurons, cardiac and skeletal muscle cells (Abita et al 1977;Bergman et al 1976;Kelso et al 1996;Renaud et al 1986;Romey et al 1976;Shibata et al 1976). In cardiac tissues they induce positive inotropic effects, arrhythmias or cell fibrillation (Hanck and Sheets 1995;Khera et al 1995;Reimer et al 1985;Renaud et al 1986;Shibata et al 1976).…”

“…Most of the excitatory and paralyzing effects of the sea anemone toxins acting on Nav channels in excitable membranes are due to a prolongation of action potential duration. By binding to a specific receptor (site 3) they alter the gating of Nav channels and delay the Na + current inactivation without altering activation kinetics Cestele and Catterall 2000;Norton 1991;Romey et al 1976;Vincent et al 1980). Structure-activity relationship studies using mutants of toxins, and the comparison of their relative toxicity and Nav channel activities have been largely investigated and described in more recent publications (Benzinger et al 1998;Gallagher and Blumenthal 1994;Loret et al 1994;Norton 1991;Rogers et al 1996).…”

Supersensitivity to tetrodotoxin and lignocaine of sea anemone toxin II‐treated sodium channel in guinea‐pig ventricular muscle

“…;man Romey et Lignocaine and other local anaesthetics are known to block ar cells (Honerjdger, 1982; sodium channels differently according to the state of the channels; i.e. resting, open and inactivated states (Hille, 1977;Hon-was shortened by the addideghem & Katzung, 1977;Matsubara et al, 1987 …”

“…This toxin, like other polypeptide toxins (a-scorpion toxins, anthopleurin A), binds to receptor site 3 among the six distinct sites mentioned above, resulting in a slowing or block of the inactivation process of sodium channels (Romey et al, 1976;Isenberg & Ravens, 1984; & Narahashi, 1988). ATX II has also been reported to enhance the binding and action of lipid-soluble toxins such as batrachotoxin acting at receptor site 2 (Catterall, 1984;Sheldon et al, 1986).…”