Charybdotoxin is a new member of the K+ channel toxin family that includes dendrotoxin I and mast cell degranulating peptide

A polypeptide was identified in the venom of the scorpion Leiurus quinquestriatus hebraeus by its potency to inhibit the high-affinity binding of the radiolabeled snake venom toxin dendrotoxin I (125I-DTX1) to its receptor site. It has been purified, and its properties investigated by different techniques were found to be similar to those of MCD and DTXI, two polypeptide toxins active on a voltage-dependent K+ channel. However, its amino acid sequence was determined, and it was shown that this toxin is in fact charybdotoxin (ChTX), a toxin classically used as a specific tool to block one class of Ca2+-activated K+ channels. ChTX, DTXI, and MCD are potent convulsants and are highly toxic when injected intracerebroventricularly in mice. Their toxicities correlate well with their affinities for their receptors in rat brain. These three structurally different toxins release [3H]GABA from preloaded synaptosomes, the efficiency order being DTXI greater than ChTX greater than MCD. Both binding and cross-linking experiments of ChTX to rat brain membranes and to the purified MCD/DTXI binding protein have shown that the alpha-subunit (Mr = 76K-78K) of the MCD/DTXI-sensitive K+ channel protein also contains the ChTX binding sites. Binding sites for DTXI, MCD, and ChTX are in negative allosteric interaction. Our results show that charybdotoxin belongs to the family of toxins which already includes the dendrotoxins and MCD, which are blockers of voltage-sensitive K+ channels. ChTX is clearly not selective for Ca2+-activated K+ channel.     

BTK-2, a new inhibitor of the Kv1.1 potassium channel purified from Indian scorpion Buthus tamulus

A novel inhibitor of voltage-gated potassium channel was isolated and purified to homogeneity from the venom of the red scorpion Buthus tamulus. The primary sequence of this toxin, named BTK-2, as determined by peptide sequencing shows that it has 32 amino acid residues with six conserved cysteines. The molecular weight of the toxin was found to be 3452 Da. It was found to block the human potassium channel hKv1.1 (IC(50)=4.6 microM). BTK-2 shows 40-70% sequence similarity to the family of the short-chain toxins that specifically block potassium channels. Multiple sequence alignment helps to categorize the toxin in the ninth subfamily of the K+ channel blockers. The modeled structure of BTK-2 shows an alpha/beta scaffold similar to those of the other short scorpion toxins. Comparative analysis of the structure with those of the other toxins helps to identify the possible structure-function relationship that leads to the difference in the specificity of BTK-2 from that of the other scorpion toxins. The toxin can also be used to study the assembly of the hKv1.1 channel.     

Motions and structural variability within toxins: implication for their use as scaffolds for protein engineering

Animal toxins are small proteins built on the basis of a few disulfide bonded frameworks. Because of their high variability in sequence and biologic function, these proteins are now used as templates for protein engineering. Here we report the extensive characterization of the structure and dynamics of two toxin folds, the "three-finger" fold and the short alpha/beta scorpion fold found in snake and scorpion venoms, respectively. These two folds have a very different architecture; the short alpha/beta scorpion fold is highly compact, whereas the "three-finger" fold is a beta structure presenting large flexible loops. First, the crystal structure of the snake toxin alpha was solved at 1.8-A resolution. Then, long molecular dynamics simulations (10 ns) in water boxes of the snake toxin alpha and the scorpion charybdotoxin were performed, starting either from the crystal or the solution structure. For both proteins, the crystal structure is stabilized by more hydrogen bonds than the solution structure, and the trajectory starting from the X-ray structure is more stable than the trajectory started from the NMR structure. The trajectories started from the X-ray structure are in agreement with the experimental NMR and X-ray data about the protein dynamics. Both proteins exhibit fast motions with an amplitude correlated to their secondary structure. In contrast, slower motions are essentially only observed in toxin alpha. The regions submitted to rare motions during the simulations are those that exhibit millisecond time-scale motions. Lastly, the structural variations within each fold family are described. The localization and the amplitude of these variations suggest that the regions presenting large-scale motions should be those tolerant to large insertions or deletions.     

Localization of the voltage-sensor toxin receptor on KvAP

A variety of venomous animals produce small protein toxins that impair the function of voltage-dependent cation channels by affecting the motions of the voltage-sensor domains and altering the energetics of the opening of the channel. In this study, we investigate the location of the receptor for tarantula venom voltage-sensor toxins on the voltage-dependent K+ channel from Aeropyrum pernix (KvAP), an archeabacterial channel that is functionally inhibited by members of this toxin family. We show that it is possible to purify the same set of toxins from venom of the tarantula Grammostola spatulata using either the purified KvAP voltage-sensor domain or the full-length KvAP channel. The equivalence of toxin retention profiles for the two channel proteins implies that the tarantula voltage-sensor toxin receptor resides exclusively on the voltage-sensor domain and that the pore is not required for the toxin-channel interaction. We have identified and characterized the functional properties of a subset of the tarantula toxins that bind to the KvAP voltage-sensor domain. Some of these toxins, VSTX1 and GSMTX4, have been previously isolated, while others, VSTX2 and VSTX3, are new members of the tarantula voltage-sensor toxin family. Some but not all toxins that bind to the voltage-sensor domain affect voltage-dependent gating of KvAP channels in lipid membranes.     

A hot spot for the interaction of gating modifier toxins with voltage-dependent ion channels

The gating modifier toxins are a large family of protein toxins that modify either activation or inactivation of voltage-gated ion channels. omega-Aga-IVA is a gating modifier toxin from spider venom that inhibits voltage-gated Ca(2+) channels by shifting activation to more depolarized voltages. We identified two Glu residues near the COOH-terminal edge of S3 in the alpha(1A) Ca(2+) channel (one in repeat I and the other in repeat IV) that align with Glu residues previously implicated in forming the binding sites for gating modifier toxins on K(+) and Na(+) channels. We found that mutation of the Glu residue in repeat I of the Ca(2+) channel had no significant effect on inhibition by omega-Aga-IVA, whereas the equivalent mutation of the Glu in repeat IV disrupted inhibition by the toxin. These results suggest that the COOH-terminal end of S3 within repeat IV contributes to forming a receptor for omega-Aga-IVA. The strong predictive value of previous mapping studies for K(+) and Na(+) channel toxins argues for a conserved binding motif for gating modifier toxins within the voltage-sensing domains of voltage-gated ion channels.     

东亚钳蝎蝎毒素BmKBT基因组序列的克隆及其分析

东亚钳蝎 (ButhusmartensiiKarsch ,BmK)蝎毒素BmKBT(又名BmKabT)是一个在初级结构上相似于β类哺乳动物毒素和功能接近于α类哺乳动物毒素的Na+ 通道毒素 .基于从毒腺cDNA文库中筛选得到的全长BmKBT前体核苷酸序列设计引物 ,以蝎基因组总DNA为模板进行聚合酶链式反应 (PCR) ,将PCR产物克隆至T载体、测序 .序列分析表明 :在BmKBT信号肽编码区的 3′端的- 4位Gly密码子的第 1位与第 2位碱基中有 1个长 2 2 5nt的内含子 ,插入位点距离该基因的起始密码子 4 6nt ,AT含量为 78 7% ,其内含子可能的剪接分枝位点距离 3′剪接受体位点 4 7nt.内含子的大小及其基因组织结构分析表明 :BmKBT具有与α类哺乳动物毒素类似的基因组织结构 ,进一步说明BmKBT是一个介于α类和β类Na+ 通道毒素之间的中间型蝎毒素 ,可以作为研究蝎毒素分子进化的合适材料     

BF9, the First Functionally Characterized Snake Toxin Peptide with Kunitz‐Type Protease and Potassium Channel Inhibiting Properties

Although numerous Kunitz‐type toxins were isolated from snake venom, no bifunctional Kunitz‐type snake toxins with protease and potassium channel inhibiting properties have been reported till now. With the help of bioinformatics analyses and biological experiments, we characterized Kunitz‐type snake toxin BF9 as a bifunctional peptide. Enzyme and inhibitor reaction kinetics experiments showed that BF9 inhibited α‐chymotrypsin with Ki value of 1.8 × 10^?8 M. Electrophysiological experiments showed that BF9 inhibited the Kv1.3 potassium channel with an IC₅₀ of 120.0 nM, which demonstrated that serine protease inhibitor BF9 could also inhibit potassium channels. In addition, the key amino acids of BF9 responsible for the unique bifunctional mechanism are further investigated. To the best of our knowledge, BF9 is the first Kunitz‐type snake peptide with the unique bifunctionality of potassium channel and serine protease inhibiting properties, providing novel insights into divergent evolution and functional applications of snake Kunitz‐type peptides.     

The impact of the fourth disulfide bridge in scorpion toxins of the alpha-KTx6 subfamily

Animal toxins are highly reticulated and structured polypeptides that adopt a limited number of folds. In scorpion species, the most represented fold is the alpha/beta scaffold in which an helical structure is connected to an antiparallel beta-sheet by two disulfide bridges. The intimate relationship existing between peptide reticulation and folding remains poorly understood. Here, we investigated the role of disulfide bridging on the 3D structure of HsTx1, a scorpion toxin potently active on Kv1.1 and Kv1.3 channels. This toxin folds along the classical alpha/beta scaffold but belongs to a unique family of short-chain, four disulfide-bridged toxins. Removal of the fourth disulfide bridge of HsTx1 does not affect its helical structure, whereas its two-stranded beta-sheet is altered from a twisted to a nontwisted configuration. This structural change in HsTx1 is accompanied by a marked decrease in Kv1.1 and Kv1.3 current blockage, and by alterations in the toxin to channel molecular contacts. In contrast, a similar removal of the fourth disulfide bridge of Pi1, another scorpion toxin from the same structural family, has no impact on its 3D structure, pharmacology, or channel interaction. These data highlight the importance of disulfide bridging in reaching the correct bioactive conformation of some toxins.     

Isolation and molecular characterization of LVP1 lipolysis activating peptide from scorpion Buthus occitanus tunetanus

LVP1, a novel protein inducing lipolytic response in adipose cells, was purified from scorpion Buthus occitanus tunetanus venom. It represented 1% of crude venom proteins, with pHi approximately 6 and molecular mass of 16170 Da. In contrast to well-characterized scorpion toxins, reduction and alkylation of LVP1 revealed an heterodimeric structure. Isolated alpha and beta chains of LVP1 have a respective molecular mass of 8877 and 8807 Da as determined by mass spectrometry. The N-terminal and some internal peptide sequences of LVP1alpha and beta were determined by Edman degradation. The full amino acid sequences of both chains were deduced from nucleotide sequences of the corresponding cDNAs prepared based on peptide sequences and the 3' and 5' RACE methodologies. LVP1alpha and beta cDNAs encode a signal peptide of 22 residues and a mature peptide of 69 and 73 residues, respectively. Each mature peptide contains seven cysteines, which are compatible with an interchain disulfide bridge. The cDNA deduced protein structures share a high similarity with those of some Na+ channel scorpion toxins. LVP1 was not toxic to mice after intracerebro-ventricular injection. LVP1 stimulated lipolysis on freshly dissociated rat adipocytes in a dose-dependent manner with EC50 of approximately 1+0.5 microg/ml. LVP1 subunits did not display any lipolytic activity. As previously described for venom, beta adrenergic receptor (beta AR) antagonists interfere with LVP1 activity. Furthermore, it is shown that LVP1 competes with [3H]-CGP 12177 (beta1/beta2 antagonist) for binding to adipocyte plasma membrane with an IC50 of about 10(-7) M. These results demonstrate the existence of a new type of scorpion venom nontoxic peptides that are structurally related to Na+ channel toxins but can exert a distinct biological activity on adipocyte lipolysis through a beta-type adrenoreceptor pathway.     

Purification and pharmacological properties of eight sea anemone toxins from Anemonia sulcata, Anthopleura xanthogrammica, Stoichactis giganteus, and Actinodendron plumosum

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.     

Tityus serrulatus venom contains two classes of toxins. Tityus gamma toxin is a new tool with a very high affinity for studying the Na+ channel

The interaction of TiTx gamma, the major toxin in the venom of the scorpion Tityus serrulatus, with its receptor in excitable membranes was studied with the use of 125I-TiTx gamma. This derivative retains biological activity, and its specific binding to both brain synaptosomes and electroplaque membranes from Electrophorus electricus is characterized by a dissociation constant equal to that of the native toxin-receptor complex, about 2 to 5 pM. This very high affinity results mainly from a very slow rate of dissociation, equivalent to a half-life longer than 10 h at 4 degrees C. There is a 1:1 stoichiometry between TiTx gamma binding and tetrodotoxin binding to the membranes, but neither tetrodotoxin nor any of 7 other neurotoxins that are representative of 4 different classes of effectors of the Na+ channel interfere with TiTx gamma binding. Similarly, local anesthetics and other molecules that affect other types of ionic channels or neurotransmitter receptors have no effect on TiTx gamma binding. However, toxin II from Centruroides suffusus suffusus does compete with TiTx gamma, though its affinity for the receptor is much lower. Since the Centruroides toxin II is known to affect Na+ channel function, these two scorpion toxins must be put into a fifth class of Na+ channel effectors.     

Investigation of the relationship between the structure and function of Ts2, a neurotoxin from Tityus serrulatus venom

Scorpion toxins targeting voltage-gated sodium (Na(V)) channels are peptides that comprise 60-76 amino acid residues cross-linked by four disulfide bridges. These toxins can be divided in two groups (α and β toxins), according to their binding properties and mode of action. The scorpion α-toxin Ts2, previously described as a β-toxin, was purified from the venom of Tityus serrulatus, the most dangerous Brazilian scorpion. In this study, seven mammalian Na(V) channel isoforms (rNa(V)1.2, rNa(V)1.3, rNa(V)1.4, hNa(V)1.5, mNa(V)1.6, rNa(V)1.7 and rNa(V)1.8) and one insect Na(V) channel isoform (DmNa(V)1) were used to investigate the subtype specificity and selectivity of Ts2. The electrophysiology assays showed that Ts2 inhibits rapid inactivation of Na(V)1.2, Na(V)1.3, Na(V)1.5, Na(V)1.6 and Na(V)1.7, but does not affect Na(V)1.4, Na(V)1.8 or DmNa(V)1. Interestingly, Ts2 significantly shifts the voltage dependence of activation of Na(V)1.3 channels. The 3D structure of this toxin was modeled based on the high sequence identity (72%) shared with Ts1, another T. serrulatus toxin. The overall fold of the Ts2 model consists of three β-strands and one α-helix, and is arranged in a triangular shape forming a cysteine-stabilized α-helix/β-sheet (CSαβ) motif.     

Positive inotropic effects of Tityus cambridgei and T. serrulatus scorpion venoms on skeletal muscle

Toxins that block voltage-dependent K+ channels and those that modify Na+ channel gating exhibit positive inotropic effect on skeletal muscle. We compared the effect of the venom of Tityus cambridgei (Tc) and Tityus serrulatus (Ts) scorpions on mouse diaphragm force, in vitro. In indirect and direct (using D-tubocurarine 7.3 microM) stimulation, Tc, 10microg/mL, increased the contractile force, an effect prevented by tetrodotoxin (TTX) while Ts, 0.5 microg/mL, potentiated only indirectly stimulated diaphragm, thus indicating its activity is mainly mediated through acetylcholine release from nerve terminal. This effect is prevented by TTX and attenuated by the K+ channel opener cromakalim. In conclusion, our data show that while the positive inotropic effect of both venoms appears associated to the activity of Na+ and K+ channels, only Tc venom acts also directly on skeletal muscle. This finding call for further studies on Tc venom to identify the toxin responsible for its direct inotropic activity as it may have clinical applications.     

Effect of maurotoxin, a four disulfide-bridged toxin from the chactoid scorpion Scorpio maurus, on Shaker K+ channels.

Maurotoxin is a 34-residue toxin isolated from the venom of the Tunisian chactoid scorpion Scorpio maurus palmatus and contains four disulfide bridges that are normally found in long-chain toxins of 60-70 amino acid residues, which affect voltage-gated sodium channels. However, despite the unconventional disulfide-bridge pattern of maurotoxin, the conformation of this toxin remains similar to that of other toxins acting on potassium channels. Here, we analyzed the effects of synthetic maurotoxin on voltage-gated Shaker potassium channels (ShB) expressed in Xenopus oocytes. Maurotoxin produces a strong, but reversible, inhibition of the ShB K+ current with an IC50 of 2 nM. Increasing concentrations of the toxin induce a progressively higher block at saturating concentrations. At nonsaturating concentrations of the toxin (5-20 nM), the channel block appears slightly more pronounced at threshold potentials suggesting that the toxin may have a higher affinity for the closed state of the channel. At the single channel level, the toxin does not modify the unitary current amplitude, but decreases ensemble currents by increasing the number of depolarizing epochs that failed to elicit any opening. A point mutation of Lys23 to alanine in maurotoxin produces a 1000-fold reduction in the IC50 of block by the toxin suggesting the importance of this charged residue for the interaction with the channel. Maurotoxin does not affect K+ currents carried by Kir2.3 channels in oocytes or Na+ currents carried by the alphaIIa channel expressed in CHO cells.     

Stimulation of sodium and calcium uptake by scorpion toxin in chick embryo heart cells.

Scorpion toxins, the basic miniproteins of scorpion venom, stimulated the passive uptake of Na+ and Ca2+ in chick embryo heart cells. Half-maximum stimulation was obtained for 20-30 nM Na+ and 40-50 nM Ca2+. Scorpion toxin-activated Na+ and Ca2+ uptakes were fully inhibited by tetrodotoxin, a specific inhibitor of the action potential Na+ ionophore in excitable membranes. Half-maximum inhibition was obtained with the same concentration of tetrodotoxin (10 nM) for both Na+ and Ca2+. Scorpion toxin-stimulated Ca2+ uptake was dependent on extracellular Na+ concentration and was not inhibited by Ca2+ channel blocking drugs which are inactive on heart cell action potential. Thus, in heart cells scorpion toxin affects the passive Ca2+ transport, which is coupled to passive Na+ ionphore. Other results suggest that (1) tetrodotoxin and scorpion toxin bind to different sites of the sarcolemma and (2) binding of scorpion toxin to its specific sites may unmask latent tetrodotoxin - sensitive fast channels.     

Molecular cloning and nucleotide sequence analysis of encoded anti-insect toxin BotIT2 from the scorpion Buthus occitanus tunetanus venom

Numerous toxins from scorpion venoms are much more toxic to insects than to other animal classes, and possess high affinity to Na+ channels. Many of them active on insects were purified from the venom of Buthus occitanus tunetanus. Using amino acid sequences of BotIT2 and RACE-PCR amplification (Rapid amplification of cDNA ends) technique, we isolated, identified and sequenced the nucleotide sequence from the venom glands of the scorpion Buthus occitanus tunetanus. The cDNA encodes a precursor of an insect toxin of 60 amino acid residues. The deduced nucleotide sequence toxin was identical to the determined amino acid sequence of BotIT2. BotIT2 is more similar to the excitatory toxins in its mode of action and to the depressant toxins in its primary structure.     

Function and solution structure of hainantoxin-I, a novel insect sodium channel inhibitor from the Chinese bird spider Selenocosmia hainana

Hainantoxin-I is a novel peptide toxin, purified from the venom of the Chinese bird spider Selenocosmia hainana (=Ornithoctonus hainana). It includes 33 amino acid residues with a disulfide linkage of I-IV, II-V and III-VI, assigned by partial reduction and sequence analysis. Under two-electrode voltage-clamp conditions, hainantoxin-I can block rNa(v)1.2/beta(1) and the insect sodium channel para/tipE expressed in Xenopus laevis oocytes with IC(50) values of 68+/-6 microM and 4.3+/-0.3 microM respectively. The three-dimensional solution structure of hainantoxin-I belongs to the inhibitor cystine knot structural family determined by two-dimensional (1)H nuclear magnetic resonance techniques. Structural comparison of hainantoxin-I with those of other toxins suggests that the combination of the charged residues and a vicinal hydrophobic patch should be responsible for ligand binding. This is the first report of an insect sodium channel blocker from spider venom and it provides useful information for the structure-function relationship studies of insect sodium channels.     

Molecular interactions of the gating modifier toxin ProTx-II with NaV 1.5: implied existence of a novel toxin binding site coupled to activation

Voltage-gated Na(+) channels are critical components in the generation of action potentials in excitable cells, but despite numerous structure-function studies on these proteins, their gating mechanism remains unclear. Peptide toxins often modify channel gating, thereby providing a great deal of information about these channels. ProTx-II is a 30-amino acid peptide toxin from the venom of the tarantula, Thrixopelma pruriens, that conforms to the inhibitory cystine knot motif and which modifies activation kinetics of Na(v) and Ca(v), but not K(v), channels. ProTx-II inhibits current by shifting the voltage dependence of activation to more depolarized potentials and, therefore, differs from the classic site 4 toxins that shift voltage dependence of activation in the opposite direction. Despite this difference in functional effects, ProTx-II has been proposed to bind to neurotoxin site 4 because it modifies activation. Here, we investigate the bioactive surface of ProTx-II by alanine-scanning the toxin and analyzing the interactions of each mutant with the cardiac isoform, Na(v)1.5. The active face of the toxin is largely composed of hydrophobic and cationic residues, joining a growing group of predominantly K(v) channel gating modifier toxins that are thought to interact with the lipid environment. In addition, we performed extensive mutagenesis of Na(v)1.5 to locate the receptor site with which ProTx-II interacts. Our data establish that, contrary to prior assumptions, ProTx-II does not bind to the previously characterized neurotoxin site 4, thus making it a novel probe of activation gating in Na(v) channels with potential to shed new light on this process.     

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.     

Purification, subunit structure and pharmacological effects on cardiac and smooth muscle cells of a polypeptide toxin isolated from the marine snail Conus tessulatus

The most active component in smooth muscle contraction, isolated from the whole venom of the marine snail Conus tessulatus, has a molecular mass of about 55 kDa. The toxin protein, tessulatus toxin, appeared to be constituted by two distinct polypeptide bands of 26 kDa and 29 kDa. The pure toxin caused a marked contraction of both guinea-pig ileum and rabbit aorta at nanomolar concentrations. Tessulatus-toxin-induced contraction was indirectly prevented by classical inhibitors of the voltage-dependent Ca2+ channel. Tessulatus toxin caused a large increase in the initial rate of 45Ca2+ uptake by cardiac cells. This uptake was insensitive to Ca2+ channel blockers at concentrations 100-1000 fold higher than those known to block voltage-dependent Ca2+ channels in these cells. Voltage clamp experiments have confirmed that tessulatus toxin was not directly active on the Ca2+ current. Tessulatus-toxin-stimulated 45Ca2+ influx was inhibited by dichlorobenzamil and suppressed when Na+ was substituted by Li+, indicating that the toxin acted via activation of the Na+/Ca2+ exchange system in cardiac cells. Activation by tessulatus toxin of the Na+/Ca2+ exchange system occurred via a toxin-stimulated Na+ entry into cardiac cells and was observed in the same range of toxin concentration which produced 45Ca2+ entry. The Na+ entry system that was activated by tessulatus toxin was insensitive to classic inhibitors of known Na+ entry systems in cardiac cells. Possible mechanisms by which tessulatus toxin induced Na+ entry into cardiac cells and contractions in smooth muscles are discussed. Tessulatus toxin is cytotoxic when used at high concentrations.