Purification, sequence, and pharmacological properties of sea anemone toxins from Radianthus paumotensis. A new class of sea anemone toxins acting on the sodium 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.     

The sodium channel in non-impulsive cells. Interaction with specific neurotoxins.

The cell line C9 used in this paper has a resting potential of --50 mV (+/- 10 mV) but is unable to generate an action potential upon electrical stimulation. The cell membrane has receptors for the selectivity filter toxin tetrodotoxin as well as for the gating system toxins, veratridine, scorpion toxin and sea anemone toxin. The Na+ channel which remains silent to electrical stimulation in the absence of toxins can be chemically activated by the gating system toxins. This has been demonstarted by electrophysiological techniques and by 22Na+ flux studies. The electrophysiological approach has shown that the sea anemone toxin is able to induce a spontaneous slow-wave activity inhibited by tetrodotoxin. 22Na+ influx analyses have shown that veratridine and the sea anemone toxin produce an important increase of the initial rate of 22Na+ influx into the C9 cell. The stimulation of 22Na+ entry by these gating system toxins is similar to that found using spiking neuroblastoma cells. Veratridine and the sea anemone toxin on one hand as well as veratridine and the scorpion toxin on the other hand are synergistic in their action to stabilize an open and highly permeable form of the sodium channel. Stimulation of 22Na+ entry into the cell through the sodium channel maintained open by the gating system neurotoxins is completely suppressed by tetrodotoxin.     

The voltage-dependent Na+ channel of insect nervous system identified by receptor sites for tetrodotoxin, and scorpion and sea anemone toxins

Receptor sites for some of the most important toxins known to be specific for voltage-sensitive Na+ channel in the mammalian nervous system have been identified in a purified membrane preparation of house fly brain. Very high affinities have been found for the association of tetrodotoxin or tetrodotoxin derivatives with the insect Na+ channel (Kd = 0.03 - 0.08 nM). The gamma toxin from the Brazilian scorpion Tityus serrulatus forms a complex with the Na+ channel having a Kd of 6.1 pM. The Kd value for toxin II from the sea anemone Anemonia sulcata is 0.12 microM. These results show a high degree of conservation of the pharmacological properties of the brain Na+ channels between insects and mammals.     

Sea anemone toxin and scorpion toxin share a common receptor site associated with the action potential sodium ionophore.

Toxin II isolated from the sea anemone Anemonia sulcata enhances activation of the action potential sodium ionophore of electrically excitable neuroblastoma cells by veratridine and batrachotoxin. This heterotropic cooperative effect is identical to that observed previously with scorpion toxin but occurs at a 110-fold higher concentration. Depolarization of the neuroblastoma cells inhibits the effect of sea anemone toxin as observed previously for scorpion toxin. Specific scorpion toxin binding is inhibited by sea anemone toxin with KD approximately equal to 90 nM. These results show that the polypeptides scorpion toxin and sea anemone toxin II share a common receptors site associated with action potential sodium ionophores.     

Expression and mutagenesis of the sea anemone toxin Av2 reveals key amino acid residues important for activity on voltage-gated sodium channels

Type I sea anemone toxins are highly potent modulators of voltage-gated Na-channels (Na(v)s) and compete with the structurally dissimilar scorpion alpha-toxins on binding to receptor site-3. Although these features provide two structurally different probes for studying receptor site-3 and channel fast inactivation, the bioactive surface of sea anemone toxins has not been fully resolved. We established an efficient expression system for Av2 (known as ATX II), a highly insecticidal sea anemone toxin from Anemonia viridis (previously named A. sulcata), and mutagenized it throughout. Each toxin mutant was analyzed in toxicity and binding assays as well as by circular dichroism spectroscopy to discern the effects derived from structural perturbation from those related to bioactivity. Six residues were found to constitute the anti-insect bioactive surface of Av2 (Val-2, Leu-5, Asn-16, Leu-18, and Ile-41). Further analysis of nine Av2 mutants on the human heart channel Na(v)1.5 expressed in Xenopus oocytes indicated that the bioactive surfaces toward insects and mammals practically coincide but differ from the bioactive surface of a structurally similar sea anemone toxin, Anthopleurin B, from Anthopleura xanthogrammica. Hence, our results not only demonstrate clear differences in the bioactive surfaces of Av2 and scorpion alpha-toxins but also indicate that despite the general conservation in structure and importance of the Arg-14 loop and its flanking residues Gly-10 and Gly-20 for function, the surface of interaction between different sea anemone toxins and Na(v)s varies.     

Structure-function relationships of sea anemone toxin II from Anemonia sulcata

Chemical modifications of sea anemone toxin II from Anemonia sulcata have been used to study the residues involved in its toxic action on crabs and mice and in its binding properties to the Na+ channel of rat brain synaptosomes. Guanidination of th epsilon-amino groups of lysines 35, 36, and 46 with O-methylisourea hydrogen sulfate did not change the net charge of the toxin molecule and had no effect upon its toxic and binding properties. Either acetylation or fluorescamine treatment of the toxin that destroyed the positive charges of the three epsilon-amino groups and of the alpha-amino function of Gly produced an almost complete loss of toxicity and a considerable decrease in the binding activity. Iodination of the toxin on His induced practically no loss of toxic or binding properties. Carbethoxylation of both histidines 32 and 37 with diethyl pyrocarbonate provoked an important decrease of both the toxicity and the binding activity. Modifications of the guanidine side chain of Arg with 1,2-cyclohexanedione fully destroyed both toxicity and binding of the toxin to the Na+ channel. Modification of the carboxylate functions of Asp, Asp, and of the COOH-terminal Gln with glycine ethyl ester in the presence of a soluble carbodiimide completely abolished the toxicity but left the affinity for the sea anemone toxin receptor unchanged. The antagonist character of this carboxylate-modified derivative was further confirmed by electrophysiological and Na+ flux experiments. The theoretical and practical significance of these results are discussed.     

Purification and chemical and biological characterizations of seven toxins from the Mexican scorpion, Centruroides suffusus suffusus

Seven polypeptides highly toxic to mice were isolated from the venom of the scorpion, Centruroides suffusus suffusus (Css), and their chemical and toxic properties were characterized. It was shown that the most active toxins by intracerebroventricular injection are less active when injected subcutaneously. The complete amino acid sequence (66 residues) of toxin II (Css II) has been determined. The C-terminal end is amidated as found for most other scorpion toxins. Css II is a beta-type toxin, previously used to define the binding site for activation of the sodium channel. Using rat brain synaptosomes, we demonstrated that all Css toxins compete with 125I-Css II to bind to site 4 and should be considered as beta-scorpion toxins. Specific binding parameters for Css VI, one of the most active toxins, were determined: KD = 100 pM; capacity in binding sites, 2.2 pmol of toxin/mg of synaptosomal protein. Css VI was shown to inhibit gamma-aminobutyric acid uptake by synaptosomes: K 0.5 = 100 pM, which agrees with its KD. Competition experiments between the seven Css toxins and 125I-Css II for antiserum raised against Css II demonstrated that all these toxins have common antigenic properties.     

Rapid voltage-dependent dissociation of scorpion alpha-toxins coupled to Na channel inactivation in amphibian myelinated nerves

The voltage-dependent action of several scorpion alpha-toxins on Na channels was studied in toad myelinated nerve under voltage clamp. These toxins slow the declining phase of macroscopic Na current, apparently by inhibiting an irreversible channel inactivation step and thus permitting channels to reopen from a closed state in depolarized membranes. In this article, we describe the rapid reversal of alpha-toxin action by membrane depolarizations more positive than +20 mV, an effect not achieved by extensive washing. Depolarizations that were increasingly positive and of longer duration caused the toxin to dissociate faster and more completely, but only up to a limiting extent. Repetitive pulses had a cumulative effect equal to that of a single pulse lasting as long as their combined duration. When the membrane of a nonperfused fiber was repolarized, the effects of the toxin returned completely, but if the fiber was perfused during the conditioning procedure, recovery was incomplete and occurred more slowly, as it did at lower applied toxin concentrations. Other alpha-type toxins, from the scorpion Centruroides sculpturatus (IVa) and the sea anemone Anemonia sulcata (ATXII), exhibited similar voltage-dependent binding, though each had its own voltage range and dissociation rate. We suggest that the dissociation of the toxin molecule from the Na channel is coupled to the inactivation process. An equivalent valence for inactivation gating, of less than 1 e per channel, is calculated from the voltage-dependent change in toxin affinity.     

Tetrodotoxin-insensitive sodium channels. Binding of polypeptide neurotoxins in primary cultures of rat muscle cells

The binding of 125I-labeled derivatives of scorpion toxin and sea anemone toxin to tetrodotoxin-insensitive sodium channels in cultured rat muscle cells has been studied. Specific binding of 125I-labeled scorpion toxin and 125I-labeled sea anemone toxin was each blocked by either native scorpion toxin or native sea anemone toxin. K0.5 for block of binding by several polypeptide toxins was closely correlated with K0.5 for enhancement of sodium channel activation in rat muscle cells. These results directly demonstrate binding of sea anemone toxin and scorpion toxin to a common receptor site on the sodium channel. Binding of both 125I-labeled toxin derivatives is enhanced by the alkaloids aconitine and batrachotoxin due to a decrease in KD for polypeptide toxin. Enhancement of polypeptide toxin binding by aconitine and batrachotoxin is precisely correlated with persistent activation of sodium channels by the alkaloid toxins consistent with the conclusion that there is allosteric coupling between receptor sites for alkaloid and polypeptide toxins on the sodium channel. The binding of both 125I-labeled scorpion toxin and 125I-labeled sea anemone toxin is reduced by depolarization due to a voltage-dependent increase in KD. Scorpion toxin binding is more voltage-sensitive than sea anemone toxin binding. Our results directly demonstrate voltage-dependent binding of both scorpion toxin and sea anemone toxin to a common receptor site on the sodium channel and introduce the 125I-labeled polypeptide toxin derivatives as specific binding probes of tetrodotoxin-insensitive sodium channels in cultured muscle cells.     

Isolation, characterization, and amino acid sequence of a polypeptide neurotoxin occurring in the sea anemone Stichodactyla helianthus

An aqueous exudate collected from frozen and thawed bodies of a Caribbean sea anemone, Stichodactyla (formerly Stoichactis) helianthus, contained a polypeptide neurotoxin (Sh I) selectively toxic to crustaceans. The polypeptide was purified by G-50 Sephadex, phosphocellulose, and sulfopropyl-Sephadex chromatography and shown to have a molecular size of 5200 daltons and a pI of 8.3. The amino acid sequence determined by automatic Edman degradations of whole RCM Sh I and of its clostripain, staphylococcal protease, and cyanogen bromide digest peptides is A1ACKC5DDEGP10DIRTA15PLTGT20VDLGS25CNAGW30EKCAS35YYTII40ADCCR45KKK . Only 33% of this sequence is identical with the sequence of Anemonia sulcata toxin II, a sea anemone toxin isolated from the taxonomic family Actiniidae. The six half-cystines are located in equivalent positions to those of the actiniid toxins and account for nearly half of the residues common to all of the toxins. However, 69% of the Sh I sequence is identical with that of toxin II from Heteractis paumotensis, another sea anemone belonging to the family Stichodactylidae. Stichodactylid toxins lack the initial N-terminal residue of actiniid toxins and possess three consecutive acidic residues at positions 6-8, a single tryptophan at position 30, and four consecutive basic residues at positions 45-48 (C-terminus). A rabbit IgG prepared by Sh I immunization bound Sh I with a K0.5 of 4.7 nM but failed to bind homologous actiniid (Anemonia sulcata II, Condylactis gigantea III) or bolocerid (Bolocera tuedae II) polypeptide neurotoxins.(ABSTRACT TRUNCATED AT 250 WORDS)     

The sodium channel in non-impulsive cells Interaction with specific neurotoxins

The cell line C₉ used in this paper has a resting potential of ?50 mV (±10 mV) but is unable to generate an action potential upon electrical stimulation. The cell membrane has receptors for the selectivity filter toxin tetrodotoxin as well as for the gating system toxins, veratridine, scorpion toxin and sea anemone toxin. The Na⁺ channel which remains silent to electrical stimulation in the absence of toxins can be chemically activated by the gating system toxins. This has been demonstrated by electrophysiological techniques and by ²²Na⁺ flux studies. The electrophysiological approach has shown that the sea anemone toxin is able to induce a spontaneous slow-wave activity inhibited by tetrodotoxin. ²²Na⁺ influx analyses have shown that veratridine and the sea anemone toxin produce an important increase of the initial rate of ²²Na⁺ influx into the C₉ cell. The stimulation of ²²Na⁺ entry by these gating system toxins is similar to that found using spiking neuroblastoma cells. Veratridine and the sea anemone toxin on one hand as well as veratridine and the scorpion toxin on the other hand are synergistic in their action to stabilize an open and highly permeable form of the sodium channel. Stimulation of ²²Na⁺ entry into the cell through the sodium channel maintained open by the gating system neurotoxins is completely suppressed by tetrodotoxin.     

Binding properties of sea anemone toxins to sodium channels in the crayfish giant axon

1. Effects of four different sea anemone toxins from Anthopleura (AP-A and AP-C), Anemonia (ATX II) and Parasicyonis (PaTX), and a scorpion toxin from Leiurus (LqTX) on crayfish giant axons were studied. 2. These toxins slowed the Na channel inactivation process, inducing a maintained Na current during a depolarizing pulse. 3. The binding rates for these toxins markedly decreased under depolarization. The decrease in AP-A binding was mainly derived from an increased dissociation rate under depolarization whereas that in PaTX binding from a reduced association rate. 4. The potential-dependent toxin binding kinetics seemed to be related to the gating mechanism of the Na channel. 5. Competitive bindings between these toxins were demonstrated.     

Reconstitution of highly purified saxitoxin-sensitive Na+-channels into planar lipid bilayers.

Highly purified Na+-channels isolated from rat brain have been reconstituted into virtually solvent-free planar lipid bilayer membranes. Two different types of electrically excitable channels were detected in the absence of any neurotoxins. The activity of both channels was blocked by saxitoxin. The first channel type is highly selective for Na+ over K+ (approximately 10:1), it shows a bursting behavior, a conductance of 25 pS in Na+-Ringer and undergoes continuous opening and closing events for periods of minutes within a defined range of negative membranes voltages. The second channel type has a conductance of 150 pS and a lower selectivity for Na+ and K+ (2.2:1); only a few opening and closing events are observed with this channel after one voltage jump. The latter type of channel is also found with highly purified Na+-channel from Electrophorus electricus electroplax. A qualitative analysis of the physicochemical and pharmacological properties of the high conductance channel has been carried out. Channel properties are affected not only by saxitoxin but also by a scorpion (Centruroides suffusus suffusus) toxin and a sea anemone (Anemonia sulcata) toxin both known to be selective for the Na+-channel. The spontaneous transformation of the large conductance channel type into the small one has been considered; the two channel types may represent the expression of activity of different conformational states of the same protein.     

Characterization of the Actions of Toxins II-9.2.2 and II-10 from the Venom of the Scorpion Centruroides noxius on Transmitter Release from Mouse Brain Synaptosomes

Toxic peptides II-9.2.2 and II-10, purified from Centruroides noxius venom, bear highly homologous N-terminal amino acid sequences, and both toxins are lethal to mice. However, only toxin II-10 is active on the voltage-clamped squid axon, selectively decreasing the voltage-dependent Na+ current. Here, we have tested toxins II-9 and II-10 on synaptosomes from mouse brain: both toxins increased the release of gamma-[3H]aminobutyric acid ([3H]GABA). Their effect was completely blocked by tetrodotoxin or by the absence of external Na+. Also, both toxins increased Na+ permeability in isolated nerve terminals. Besides the observation that toxin II-9 is active on synaptosomes, the effect of toxin II-10 in this preparation is opposite to that observed in the squid axon. Thus, our results reflect functional differences between the populations of Na+ channels in mouse brain synaptosomes and in the squid axon. The release of GABA evoked by these toxins from synaptosomes required external Ca2+ and was blocked by Ca2+ channel blockers (verapamil and Co2+). This latter observation is in sharp contrast to the releasing action of veratrine, which evoked release even in the absence of external Ca2+. Furthermore, the action of both C. noxius toxins was potentiated by veratrine, a result suggesting they have different mechanisms of action. Among drugs that release neurotransmitters by increasing Na+ permeability, it is noteworthy that scorpion toxins are the only ones yet reported to have a strict requirement for external Ca2+.     

Amino-acid sequence of toxin I from Anemonia sulcata.

Toxin I from Anemonia sulcata, a major component of the sea anemone venom, consists of 46 amino acid residues which are linked by three disulfide bridges. The [14C]carboxymethylated polypeptide was sequenced to position 29 by automated Edman degradation. The remaining sequence was determined from cyanogen bromide peptides and from tryptic peptides of the citraconylated [14C]carboxymethylated toxin. Toxin I is homologous to toxin II from Anemonia sulcata and to anthopleurin A, a toxin from the sea anemone Anthopleura xanthogrammica. These toxins constitute a new class of polypeptide toxins. No significant homologies exist with toxin III from Anemonia sulcata nor with known sequences of neurotoxins or cardiotoxins of various origin.     

Properties of the interaction of the sodium channel with permeant monovalent cations

The use of sea anemone toxin, veratridine and scorpion toxin which specifically interact with the gating system of the sodium channel and maintain the channel in an open conformation has permitted a study of the mechanism of transport of monovalent cations through the selectivity filter of this channel. The initial rate of 22Na+ influx through the tetrodotoxin-sensitive Na+ channels of excitable cells is dependent upon the external concentrations of Na+ and Na+-substitutes with the following properties. (a) It is saturable at high Na+ concentrations and increases with the external Na+ concentration in a cooperative manner (nH = 1.6). (b) At low external Na+ concentrations (1 mM), it is activated and then inhibited by increasing external concentrations of monovalent cations such as Li+, guanidinium, hydrazinium, hydroxylamine and K+. The activating effect of these cations disappears at higher external Na+ concentrations (10 mM). The experimental data are consistent with a model involving at least two allosteric cation-binding sites per Na+ channel. The binding of monovalent cations to Na+ sites is characterized by a high positive homotropic cooperativity. Most of the work describes the properties of the Na+ channel in neuroblastoma cells. The mechanism has also been shown to be valid for excitable cells of other types and origins.     

Binding specificity of sea anemone toxins to Nav 1.1-1.6 sodium channels: unexpected contributions from differences in the IV/S3-S4 outer loop

Sea anemones are an important source of various biologically active peptides, and it is known that ATX-II from Anemonia sulcata slows sodium current inactivation. Using six different sodium channel genes (from Nav1.1 to Nav1.6), we investigated the differential selectivity of the toxins AFT-II (purified from Anthopleura fuscoviridis) and Bc-III (purified from Bunodosoma caissarum) and compared their effects with those recorded in the presence of ATX-II. Interestingly, ATX-II and AFT-II differ by only one amino acid (L36A) and Bc-III has 70% similarity. The three toxins induced a low voltage-activated persistent component primarily in the Nav1.3 and Nav1.6 channels. An analysis showed that the 18 dose-response curves only partially fit the hypothesized binding of Lys-37 (sea anemone toxin Anthopleurin B) to the Asp (or Glu) residue of the extracellular IV/S3-S4 loop in cardiac (or nervous) Na+ channels, thus suggesting the substantial contribution of some nearby amino acids that are different in the various channels. As these channels are atypically expressed in mammalian tissues, the data not only suggest that the toxicity is highly dependent on the channel type but also that these toxins and their various physiological effects should be considered prototype models for the design of new and specific pharmacological tools.     

Photoaffinity labeling of scorpion toxin receptors associated with insect synaptosomal Na+ channels

Photoreactive and radioiodinated derivatives of several scorpion toxins acting on insect Na+ channels were prepared without loss of their pharmacological activities. Photoaffinity experiments were carried out on a synaptosomal fraction from the nerve cord of the cockroach Periplaneta americana: with all toxin derivatives, a single specifically labeled band was obtained with a molecular weight of 188,000 +/- 12,000 (n = 17). These results indicate for the first time the molecular weight of the scorpion toxin receptor from the insect nervous system which is probably associated with voltage sensitive Na+ channels. One of these toxins, toxin VII from Tityus serrulatus venom, has been previously shown to be active both in mammals and in insects, in rat brain synaptosomes this toxin labeled a Mr = 31,000 +/- 4,000 band in contrast, to observations in the insect preparation.     

Effects of several sea anemone and scorpion toxins on excitability and ionic currents in the giant axon of the cockroach

The membrane effects of 4 sea anemone and 6 scorpion toxins have been studied under current clamp and voltage clamp conditions. Micromolar concentrations of the purified toxins were applied externally on single giant axons of the american cockroach. Periplaneta americana in a double oil-gap arrangement and the effects on the resting potential, action potential and underlying currents analysed. The 4 sea anemone toxins (Condylactis toxin, Anemonia toxin 2, Anthopleurin toxin A and Parasicyonis toxin) were found to considerably prolong the action potential. This effect is frequency dependent and long plateau spikes (100-500 ms in duration) are consistently seen for frequencies lower than 0.2 Hz. This effect is due to a considerable delay in the turning-off of the sodium current during square membrane depolarizations associated, for large concentrations, with a decrease in the potassium conductance. Toxin effects on the sodium current are not prevented by pretreatment with STX. From the 4 purified toxins extracted from the venom of the scorpion, Androctonus australis Hector, 3 (Mammal toxins 1 and 2 and crustacean toxin) were found to have sea anemone toxin like effects and to induce long duration plateau action potentials. As for sea anemone toxins, this effect is due to a lengthening of the falling phase of the sodium current associated with a small decrease in the potassium conductance. The 4th toxin (insect toxin or ITAaH) depolarizes the membrane and induces repetitive firing of short action potentials.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of neurotoxins (veratridine, sea anemone toxin, tetrodotoxin) on transmitter accumulation and release by nerve terminals in vitro.

Two of the tree toxic compounds used in this work, veratridine and the sea anemone toxin, provoke neurotransmitter release from synaptosomes; the third one, tetrodotoxin, prevents the action of both veratridine and the sea anemone toxin. The half-maximum effects of veratridine and sea anemone toxin actions on synaptosomes are K0.5 = 10 and 0.02 micronM, respectively. Although veratridine and the sea anemone toxin similarly provoke neurotransmitter release, they act on different receptor structures in the membrane. Tetrodotoxin antagonizes the effects of both veratridine and the sea anemone toxin. The half-maximum inhibitory concentration of tetrodotoxin is K0.5 = 4 nM for veratridine and 7.9 nM for ATXII. It is very similar to the dissociation constant measured from direct binding experiments with the radioactive toxin. The analysis of this antagonistic action offers an easy in vitro assay for tetrodotoxin interaction with its receptor.