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Voltage-gated sodium channels are a major target for toxins and insecticides due to their central role in excitability, but due to the conservation of these channels in Animalia most insecticides do not distinguish between those of insects and mammals, thereby imposing risks to humans and livestock. Evidently, as long as modern agriculture depends heavily on the use of insecticides there is a great need for new substances capable of differentiating between sodium channel subtypes. Such substances exist in venomous animals, but ways for their exploitation have not yet been developed due to problems associated with manufacturing, degradation, and delivery to the target channels. Engineering of plants for expression of anti-insect toxins or use of natural vectors that express toxins near their target site (e.g. baculoviruses) are still problematic and raise public concern. In this problematic reality a rational approach might be to learn from nature how to design highly selective anti-insect compounds preferably in the form of peptidomimetics. This is a complex task that requires the elucidation of the face of interaction between insect-selective toxins and their sodium channel receptor sites. This review delineates current progress in: (i) elucidation of the bioactive surfaces of scorpion beta-toxins, especially the excitatory and depressant groups, which show high preference for insects and bind insect sodium channels with high affinity; (ii) studies of the mode of interaction of scorpion beta-toxins with receptor site-4 on voltage-gated sodium channels; and (iii) clarification of channel elements that constitute receptor site-4. This information may be useful in future attempts to mimic the bioactive surface of the toxins for the design of anti-insect selective peptidomimetics.

Interweaving strategies of electrophysiology, calcium imaging and immunocytochemistry bring new insights into the mode of action of the Brazilian scorpion Tityus serrulatusbeta-toxin VII. Pacemaker dorsal unpaired median neurons isolated from the cockroach central nervous system were used to study the effects of toxin VII. In current-clamp, 50 nm toxin VII produced a membrane depolarization and reduced spiking. At 200 nM, depolarization associated with multiphasic effects was seen. After artificial hyperpolarization, plateau potentials on which spontaneous electrical activity appeared were observed. In voltage clamp, toxin VII induced a negative shift of the voltage dependence of sodium current activation without significant effect on steady-state inactivation. In addition, toxin VII produced a permanent TTX-sensitive holding inward current, indicating that background sodium channels were targeted by beta-toxin. Cell-attached patch recordings indicated that these channels were switched from unclustered single openings to current fluctuating between distinct subconductance levels exhibiting increased open probability and open-time distribution. Toxin VII also produced a TTX-sensitive [Ca2+]i rise. Immunostaining with Cav2.2(alpha1b) antibodies and calcium imaging data obtained with omega-CgTx GVIA indicated that N-type high-voltage-activated calcium channels initiated calcium influx and were an essential intermediate in the pathway linking toxin VII-modified sodium channels to the activation of an additional route for calcium entry. By using inhibitors of (i) noncapacitative calcium entry (inhibitor LOE-908), (ii) NO-sensitive guanylyl cyclase (ODQ) and (iii) phosphodiesterase 2 (EHNA), together with cGMP antibodies, we demonstrated that noncapacitative calcium entry was the final step in a complex combination of events that was initiated by toxin VII-alteration of sodium channels and then involved successive activation of other membrane ion channels.

We isolated from the venom of the scorpion Leiurus quinquestriatus hebraeus an extremely active anti-insect selective depressant toxin, Lqh-dprIT(3). Cloning of Lqh-dprIT(3) revealed a gene family encoding eight putative polypeptide variants (a-h) differing at three positions (37A/G, 50D/E, and 58N/D). All eight toxin variants were expressed in a functional form, and their toxicity to blowfly larvae, binding affinity for cockroach neuronal membranes, and CD spectra were compared. This analysis links Asn-58, which appears in variants a-d, to a toxin conformation associated with high binding affinity for insect sodium channels. Variants e-h, bearing Asp-58, exhibit a different conformation and are less potent. The importance of Asn-58, which is conserved in other depressant toxins, was further validated by construction and analysis of an N58D mutant of the well-characterized depressant toxin, LqhIT(2). Current and voltage clamp assays using the cockroach giant axon have shown that despite the vast difference in potency, the two types of Lqh-dprIT(3) variants (represented by Lqh-dprIT(3)-a and Lqh-dprIT(3)-e) are capable of blocking the action potentials (manifested as flaccid paralysis in blowfly larvae) and shift the voltage dependence of activation to more negative values, which typify the action of beta-toxins. Moreover, the stronger and faster shift in voltage dependence of activation and lack of tail currents observed in the presence of Lqh-dprIT(3)-a suggest an extremely efficient trapping of the voltage sensor compared to that of Lqh-dprIT(3)-e. The current clamp assays revealed that repetitive firing of the axon, which is reflected in contraction paralysis of blowfly larvae, can be obtained with either the less potent Lqh-dprIT(3)-e or the highly potent Lqh-dprIT(3)-a at more negative membrane potentials. Thus, the contraction symptoms in flies are likely to be dominated by the resting potential of neuronal membranes. This study clarifies the electrophysiological basis of the complex symptoms induced by scorpion depressant toxins in insects, and highlights for the first time molecular features involved in their activity.

Two almost identical proteins with 70 amino acid residues each, closely packed by four disufide bridges, and molecular masses of 7899.5 and 7884.7 were isolated and sequenced from the venom of the scorpion Isometrus vittatus from Pakistan. They differ by an acidic amino acid residue (glutamic or aspartic) at the same position 55 of the peptide chain, however, they exhibit the same length, the same charge and are undistinguishable when separated by C(18) reverse phase HPLC. The mixture of the two proteins called IsomTx1 depolarizes the cockroach isolated axon; artificial repolarization is followed by sustained repetitive activity, artificial hyperpolarization facilitates bursting activity observed as an answer to rapid depolarization to -60 mV. The depolarization is antagonized by TTX. In voltage-clamp experiments IsomTx1 increases axonal sodium permeability which has a particular importance between resting and threshold potentials and moderately slows down the fast inactivation. These characteristics closely resemble those of other anti-insect scorpion toxins classified as contractive toxins from Androctonus and Buthotus venoms.

Scorpion alpha-neurotoxins can be classified into distinct subgroups according to their sequence and pharmacological properties. Using toxicity tests, binding studies, and electrophysiological recordings, BmK M1, a toxin from the Asian scorpion Buthus martensi Karsch, was experimentally identified as an alpha-like toxin. Being the first alpha-like toxin available in a recombinant form, BmK M1 was then modified by site-directed mutagenesis for investigation of the molecular basis of its activity. The results suggested a functional site which protrudes from the molecular scaffold as a unique tertiary arrangement, constituted by the five-residue reverse turn 8-12 and the C-terminal segment. The C-terminal basic residues Lys62 and His64 together with Lys8 in the turn, which are critical for the bioactivities, may directly interact with the receptor site on the sodium channel. Residues Asn11 and Arg58, indispensable for the activities, are mainly responsible for stabilizing the distinct conformation of the putative bioactive site. Among others, His10 and His64 seem to be involved in the preference of BmK M1 for phylogenetically distinct target sites. The comparison of BmK M1 with Aah2 (classical alpha-toxin) and Lqh(alpha)IT (alpha-insect toxin) showed that the specific orientation of the C-terminus mediated by the reverse turn might be relevant to the preference of alpha-toxin subgroups for phylogenetically distinct yet closely related receptor sites. The Y5G mutation indicated the "conserved hydrophobic surface" might be structurally important for stabilizing the beta-sheet in the alpha/beta-scaffold. The observations in this work shed light on the nature and roles of the residues possibly involved in the biological activity of a scorpion alpha-like toxin.

The two insecticidal peptides Bm32-VI and Bm33-I, isolated from the venom of the Chinese scorpion Buthus martensi induce paralytical symptoms typical of insect contractive toxins. They show, respectively, 74% and 77% homology with AaIT from Androctonus australis, comparable insecticidal activity and no vertebrate toxicity. Under voltage-clamp conditions, both toxins induced (1) an increased fast Na(+) current, (2) a shift in voltage dependence of Na(+) current activation, (3) the occurrence of a delayed current, and (4) a slow development of a holding current. Increased Na(+) conductance at negative potential values is responsible for axonal hyperexcitability and the contractive paralysis of insect prey.

A scorpion alpha-toxin-sensitive background sodium channel was characterized in short-term cultured adult cockroach dorsal unpaired median (DUM) neurons using the cell-attached patch-clamp configuration. Under control conditions, spontaneous sodium currents were recorded at different steady-state holding potentials, including the range of normal resting membrane potential. At -50 mV, the sodium current was observed as unclustered, single openings. For potentials more negative than -70 mV, investigated patches contained large unitary current steps appearing generally in bursts. These background channels were blocked by tetrodotoxin (TTX, 100 nm), and replacing sodium with TMA-Cl led to a complete loss of channel activity. The current-voltage relationship has a slope conductance of 36 pS. At -50 mV, the mean open time constant was 0.22 +/- 0.05 ms (n = 5). The curve of the open probability versus holding potentials was bell-shaped, with its maximum (0.008 +/- 0.004; n = 5) at -50 mV. LqhalphaIT (10-8 m) altered the background channel activity in a time-dependent manner. At -50 mV, the channel activity appeared in bursts. The linear current-voltage relationship of the LqhalphaIT-modified sodium current determined for the first three well-resolved open states gave three conductance levels: 34, 69 and 104 pS, and reversed at the same extrapolated reversal potential (+52 mV). LqhalphaIT increased the open probability but did not affect either the bell-shaped voltage dependence or the open time constant. Mammal toxin AaHII induced very similar effects on background sodium channels but at a concentration 100 x higher than LqhalphaIT. At 10-7 m, LqhalphaIT produced longer silence periods interrupted by bursts of increased channel activity. Whole-cell experiments suggested that background sodium channels can provide the depolarizing drive for DUM neurons essential to maintain beating pacemaker activity, and revealed that 10-7 m LqhalphaIT transformed a beating pacemaker activity into a rhythmic bursting.

alpha-Like toxins, a unique group designated among the scorpion alpha-toxin class that inhibit sodium channel inactivation, are highly toxic to mice but do not compete for alpha-toxin binding to receptor site 3 on rat brain sodium channels. We analysed the sequence of a new alpha-like toxin, which was also highly active on insects, and studied its action and binding on both mammalian and insect sodium channels. Action of the alpha-like toxin on isolated cockroach axon is similar to that of an alpha-toxin, and the radioactive toxin binds with a high affinity to insect sodium channels. Other sodium channel neurotoxins interact competitively or allosterically with the insect alpha-like toxin receptor site, similarly to alpha-toxins, suggesting that the alpha-like toxin receptor site is closely related to receptor site 3. Conversely, on rat brain sodium channels, specific binding of 125I-alpha-like toxin could not be detected, although at high concentration it inhibits sodium current inactivation on rat brain sodium channels. The difficulty in measuring binding to rat brain channels may be attributed to low-affinity binding due to the acidic properties of the alpha-like toxins that also impair the interaction with receptor site 3. The results suggest that alpha-like toxins bind to a distinct receptor site on sodium channels that is differentially related to receptor site 3 on mammalian and insect sodium channels.

Scorpion neurotoxins of the excitatory group show total specificity for insects and serve as invaluable probes for insect sodium channels. However, despite their significance and potential for application in insect-pest control, the structural basis for their bioactivity is still unknown. We isolated, characterized, and expressed an atypically long excitatory toxin, Bj-xtrIT, whose bioactive features resembled those of classical excitatory toxins, despite only 49% sequence identity. With the objective of clarifying the toxic site of this unique pharmacological group, Bj-xtrIT was employed in a genetic approach using point mutagenesis and biological and structural assays of the mutant products. A primary target for modification was the structurally unique C-terminal region. Sequential deletions of C-terminal residues suggested an inevitable significance of Ile73 and Ile74 for toxicity. Based on the bioactive role of the C-terminal region and a comparison of Bj-xtrIT with a Bj-xtrIT-based model of a classical excitatory toxin, AaHIT, a conserved surface comprising the C terminus is suggested to form the site of recognition with the sodium channel receptor.

NMR structures of a new toxin from the scorpion Leiurus quinquestriatus hebraeus (Lqh III) have been investigated in conjunction with its pharmacological properties. This toxin is proposed to belong to a new group of scorpion toxins, the alpha-like toxins that target voltage-gated sodium channels with specific properties compared with the classical alpha-scorpion toxins. Electrophysiological analysis showed that Lqh III inhibits a sodium current inactivation in the cockroach axon, but induces in addition a resting depolarization due to a slowly decaying tail current atypical to other alpha-toxin action. Binding studies indicated that radiolabeled Lqh III binds with a high degree of affinity (Ki=2.2 nM) on cockroach sodium channels and that the alpha-toxin from L quinquestriatus hebraeus highly active on insects (LqhalphaIT) and alpha-like toxins compete at low concentration for its receptor binding site, suggesting that the alpha-like toxin receptor site is partially overlapping with the receptor site 3. Conversely, in rat brain, Lqh III competes for binding of the most potent anti-mammal alpha-toxin from Androctonus australis Hector venom (AaH II) only at very high concentration. The NMR structures were used for the scrutiny of the similarities and differences with representative scorpion alpha-toxins targeting the voltage-gated sodium channels of either mammals or insects. Three turn regions involved in the functional binding site of the anti-insect LqhalphaIT toxin reveal significant differences in the Lqh III structure. The electrostatic charge distribution in the Lqh III toxin is also surprisingly different when compared with the anti-mammal alpha-toxin AaH II. Similarities in the electrostatic charge distribution are, however, recognized between alpha-toxins highly active on insects and the alpha-like toxin Lqh III. This affords additional important elements to the definition of the new alpha-like group of scorpion toxins and the mammal versus insect scorpion toxin selectivities., (Copyright 1999 Academic Press.)

Some toxins from scorpion venoms, much more toxic to insects than to other animal classes, possess high affinity to Na+ channels. These anti-insect scorpion toxins have been divided into: 1) alpha toxins which lack strict selectivity for insects, do not compete with following groups of anti-insect toxins, resemble other alpha scorpion toxins by their structure and their ability, as alpha anemone toxins, to prolong insect axonal action potential durations through a drastic slowing down of the Na+ current inactivation, 2) excitatory insect selective scorpion toxins which induce in blowfly larvae an immediate fast paralysis; in isolated cockroach axons, they depolarize and induce a sustained repetitive activity of short (normal) action potentials through a shift of Na+ activation mechanism towards more negative potentials and some decrease of inactivation at these potential values, 3) depressant insect selective neurotoxins which cause a slow progressive flaccid paralysis of larvae, depolarize insect axons and reduce or even suppress evoked action potentials; resting depolarizations which are antagonized by a post-application of TTX, are due to the opening of sodium channels at very negative potential values and to the suppression of their inactivation mechanism. The decrease of the maximal Na+ conductance following flaccid toxin action may be understood if toxin-modified channels opened at very negative potentials values remain open (or re-open) for much longer times than in control conditions and pass by substate less conductant states. Anti-insect scorpion toxins become of major interest into insect neurophysiology and also into insect pest control, due to their specific target sites and to the recent constructions of insecticidal baculovirus expressions of several of these toxins.

A toxin named babycurus-toxin 1 (mol. wt 8191), from telson extracts of the scorpion Babycurus centrurimorphus, was found to depolarize the cockroach giant axon. It progressively blocked the evoked action potentials after a short period of limited repetitive activity and after 30 min of toxin action it became impossible to evoke responses to current stimulations. Voltage-clamp experiments on the sodium current indicated that the toxin in micromolar concentrations progressively decreased the transient inward peak sodium current, but also slowed the activation phase of this sodium current and maintained an inward current during the voltage pulses, which deactivated slowly. The toxin also induced in the insect axon a slowly activating-deactivating component of the sodium current. This suggests that the toxin modifies both activation and inactivation mechanisms of sodium channels. Thus there is some similarity in the electrophysiological effects between BcTx1 and the beta-toxins active on mammals.

The selective toxicity of depressant scorpion neurotoxins to insects is useful in studying insect sodium channel gating and has an applied potential. In order to establish a genetic system enabling a structure-activity approach, the functional expression of such polypeptides is required. By engineering the cDNA encoding the depressant scorpion neurotoxin, LahIT2, behind the T7 promoter, large amounts of recombinant insoluble and nonactive toxin were obtained in Escherichia coli. Following denaturation and reduction, the recombinant protein, constructed with an additional N-terminal methionine residue, was subjected to renaturation. Optimal conditions for reconstitution of a functional toxin, having a dominant fold over many other possible isoforms, were established. The recombinant active toxin was purified by RP-HPLC and characterized. Toxicity (ED50) to insects, binding affinity (IC50) to an insect receptor site, and electrophysiological effect on an insect axonal preparation were found to be similar to those of the native toxin. Substitution of the C-terminal glycine by a Gly-Lys-Lys triplet did not abolish folding but affected toxicity (3.5-fold decrease) of LqhIT2. Apparently, this efficient bacterial expression system (500 micrograms HPLC-purified toxin/1 liter E. coli culture) provides the means for studying structure/ activity relationship and the molecular basis for the phylogenetic selectivity of scorpion depressant neurotoxins.

One contractive and two depressant toxins active on insect were purified by high-performance liquid chromatography from the venom of Buthus occitanus tunetanus (Bot). The two depressant toxins, BotIT4 and BotIT5, differ only at position 6 (Arg for Lys) and are equally toxic to insects (LD50 to Blatella germanica = 110 ng/100 mg body weight). They show a strong antigenic cross-reaction with a depressive toxin from Leiurus quinquestriatus quinquestriatus (LqqIT2). The two toxins are able to inhibit with high affinity (K0.5 between 2 and 3 nM) the specific binding of the radioiodinated excitatory insect toxin (125I-AaHIT) on its receptor site on Periplaneta americana synaptosomal membranes. These toxins depolarize the cockroach axon, irreversibly block the action potential, and slow down and very progressively block the transmembrane transient Na+ current. The contracturant toxin BotIT1 is highly toxic to B. germanica (LD50 = 60 ng/ 100 mg body weight) and barely toxic to mice (LD50 = 1 microgram/20 g body weight) when injected intracerebroventricularly. It does not compete with 125I-AaHIT for its receptor site on P. americana synaptosomal membranes. On cockroach axon, BotIT1 develops plateau potentials and slows down the inactivation mechanism of the Na+ channels. Thus, BotIT1 belongs to the group of alpha insect-selective toxins and shows a strong sequence identity (> 90%) with Lqh alpha IT and LqqIII, two insect alpha-toxins previously purified from the venom of L. q. hebraeus and L. q. quinquestriatus. respectively.

The effects of a recombinant depressant insect toxin from Leiurus quinquestriatus hebraeus, Lqh IT2-r, have been studied in current and voltage-clamp conditions on the isolated axonal and DUM neuron preparations of the cockroach Periplaneta americana. Lqh IT2-r depolarizes the axon, blocks the evoked action potentials, and modifies the amplitude and the kinetics of the sodium current. The inward transient peak current is greatly decreased and is followed by a maintained slow activating-deactivating sodium current. The slow component develops at membrane potentials more negative than the control, and has a time constant of activation of several tens of milliseconds. The flaccid properties of Lqh IT2-r do not correspond to a blockage of the Na+ channels, but may be attributed to modified Na+ channels which open at more negative potential, activate slowly and do not inactivate normally.

A new toxin, BotIT2, with a unique mode of action on the isolated giant axon of the cockroach Periplaneta americana and DUM (dorsal unpaired median) neurons, has been purified from the venom of the scorpion Buthus occitanus tunetanus. Its structural, antigenic and pharmacological properties are compared to those of three other groups of neurotoxins found in Buthidae scorpion venoms. Like excitatory, depressant and alpha-type insect-selective neurotoxins, BotIT2 is toxic to insects, but shows the following common and distinctive characteristics. (a) As alpha-type toxins, BotIT2 lack strict selectivity to insects; they have measurable but low toxicity to mice. (b) As depressant toxins and unlike alpha-type toxins, BotIT2 is able to displace iodinated AaHIT from its binding sites in insect neuronal membranes. This indicates that the binding site for BotIT2 is identical, contiguous or in allosteric interaction with that of AaHIT and depressant toxins. (c) The BotIT2 amino acid sequence shows strong similarity to depressant toxins. However, unexpectedly, despite this high sequence similarity, BotIT2 shares moderate cross-antigenic reactivity with depressant toxins. (d) Voltage and current-clamp studies show that BotIT2 induces limited depolarization concomitantly with the development of depolarizing after potential, repetitive activity and later plateau potentials terminated by bursts. Under voltage-clamp conditions, BotIT2 specifically acts on Na+ channels by decreasing the peak Na+ current and by simultaneously inducing a new current with very slow activation/deactivation kinetics. The voltage dependence of this slow current is not significantly different from that of the control current. These observations indicate that BotIT2 chiefly modifies the kinetics of axonal and DUM neuronal membrane Na(+)-channel activation.

Abstract: Arachnids have a venom apparatus and secrete a complex chemical mixture of low molecular mass organic molecules, enzymes and polypeptide neurotoxins designed to paralyze or kill their prey. Most of these toxins are specific for membrane voltage-gated sodium channels, although some may also target calcium or potassium channels and other membrane receptors. Scorpions and spiders have provided the greatest number of the neurotoxins studied so far, for which, a good number of primary and 3D structures have been obtained. Structural features, comprising a folding that determines a similar spatial distribution of charged and hydrophobic side chains of specific amino acids, are strikingly common among the toxins from spider and scorpion venoms. Such similarities are, in turn, the key feature to target and bind these proteins to ionic channels. The search for new insecticidal compounds, as well as the study of their modes of action, constitutes a current approach to rationally design novel insecticides. This goal tends to be more relevant if the resistance to the conventional chemical products is considered. A promising alternative seems to be the biotechnological approach using toxin-expressing recombinant baculovirus. Spider and scorpion toxins having insecticidal activity are reviewed here considering their structures, toxicities and action mechanisms in sodium channels of excitable membranes. [Copyright &y& Elsevier]