A spider toxin that induces a typical effect of scorpion alpha-toxins but competes with beta-toxins on binding to insect sodium channels

Delta-palutoxins from the spider Paracoelotes luctuosus (Araneae: Amaurobiidae) are 36-37 residue long peptides that show preference for insect sodium channels (NaChs) and modulate their function. Although they slow NaCh inactivation in a fashion similar to that of receptor site 3 modifiers, such as scorpion alpha-toxins, they actually bind with high affinity to the topologically distinct receptor site 4 of scorpion beta-toxins. To resolve this riddle, we scanned by Ala mutagenesis the surface of delta-PaluIT2, a delta-palutoxin variant with the highest affinity for insect NaChs, and compared it to the bioactive surface of a scorpion beta-toxin. We found three regions on the surface of delta-PaluIT2 important for activity: the first consists of Tyr-22 and Tyr-30 (aromatic), Ser-24 and Met-28 (polar), and Arg-8, Arg-26, Arg-32, and Arg-34 (basic) residues; the second is made of Trp-12; and the third is made of Asp-19, whose substitution by Ala uncoupled the binding from toxicity to lepidopteran larvae. Although spider delta-palutoxins and scorpion beta-toxins have developed from different ancestors, they show some commonality in their bioactive surfaces, which may explain their ability to compete for an identical receptor (site 4) on voltage-gated NaChs. Yet, their different mode of channel modulation provides a novel perspective about the structural relatedness of receptor sites 3 and 4, which until now have been considered topologically distinct.     

Common features in the functional surface of scorpion beta-toxins and elements that confer specificity for insect and mammalian voltage-gated sodium channels

Scorpion beta-toxins that affect the activation of mammalian voltage-gated sodium channels (Navs) have been studied extensively, but little is known about their functional surface and mode of interaction with the channel receptor. To enable a molecular approach to this question, we have established a successful expression system for the anti-mammalian scorpion beta-toxin, Css4, whose effects on rat brain Navs have been well characterized. A recombinant toxin, His-Css4, was obtained when fused to a His tag and a thrombin cleavage site and had similar binding affinity for and effect on Na currents of rat brain sodium channels as those of the native toxin isolated from the scorpion venom. Molecular dissection of His-Css4 elucidated a functional surface of 1245 A2 composed of the following: 1) a cluster of residues associated with the alpha-helix, which includes a putative "hot spot" (this cluster is conserved among scorpion beta-toxins and contains their "pharmacophore"); 2) a hydrophobic cluster associated mainly with the beta2 and beta3 strands, which is likely to confer the specificity for mammalian Navs; 3) a single bioactive residue (Trp-58) in the C-tail; and 4) a negatively charged residue (Glu-15) involved in voltage sensor trapping as inferred from our ability to uncouple toxin binding from activity upon its substitution. This study expands our understanding about the mode of action of scorpion beta-toxins and illuminates differences in the functional surfaces that may dictate their specificities for mammalian versus insect sodium channels.     

Variations in receptor site-3 on rat brain and insect sodium channels highlighted by binding of a funnel-web spider delta-atracotoxin.

Delta-atracotoxins (delta-ACTXs) from Australian funnel-web spiders differ structurally from scorpion alpha-toxins (Sc(alpha)Tx) but similarly slow sodium current inactivation and compete for their binding to sodium channels at receptor site-3. Characterization of the binding of 125I-labelled delta-ACTX-Hv1a to various sodium channels reveals a decrease in affinity for depolarized (0 mV; Kd=6.5 +/- 1.4 nm) vs.polarized (-55 mV; Kd=0.6 +/- 0.2 nm) rat brain synaptosomes. The increased Kd under depolarized conditions correlates with a 4.3-fold reduction in the association rate and a 1.8-increase in the dissociation rate. In comparison, Sc(alpha)Tx binding affinity decreased 33-fold under depolarized conditions due to a 48-fold reduction in the association rate. The binding of 125I-labelled delta-ACTX-Hv1a to rat brain synaptosomes is inhibited competitively by classical Sc(alpha)Txs and allosterically by brevetoxin-1, similar to Sc(alpha)Tx binding. However, in contrast with classical Sc(alpha)Txs, 125I-labelled delta-ACTX-Hv1a binds with high affinity to cockroach Na+ channels (Kd=0.42 +/- 0.1 nm) and is displaced by the Sc(alpha)Tx, Lqh(alpha)IT, a well-defined ligand of insect sodium channel receptor site-3. However, delta-ACTX-Hv1a exhibits a surprisingly low binding affinity to locust sodium channels. Thus, unlike Sc(alpha)Txs, which are capable of differentiating between mammalian and insect sodium channels, delta-ACTXs differentiate between various insect sodium channels but bind with similar high affinity to rat brain and cockroach channels. Structural comparison of delta-ACTX-Hv1a to Sc(alpha)Txs suggests a similar putative bioactive surface but a 'slimmer' overall shape of the spider toxin. A slimmer shape may ease the interaction with the cockroach and mammalian receptor site-3 and facilitate its association with different conformations of the rat brain receptor, correlated with closed/open and slow-inactivated channel states.     

Study of the binding residues between ANEPII and insect sodium channel receptor

The present study aimed at determining the functional characteristics of anti-neuroexcitation peptide II (ANEPII). The depressant insect toxin ANEPII from the Chinese scorpion Buthus martensii Karsch had an effect on insect sodium channels. Previous studies showed that scorpion depressant toxins induce insect flaccid paralysis upon binding to receptor site-4, so we tried to predict the functional residues involved using computational techniques. In this study, three-dimensional structure modeling of ANEPII and site-4 of the insect sodium channel were carried out by homology modeling, and these models were used as the starting point for nanosecond-duration molecular dynamics simulations. Docking studies of ANEPII in the sodium channel homology model were conducted, and likely ANEPII binding loci were investigated. Based on these analyses, the residues Tyr34, Trp36, Gly39, Leu40, Trp53, Asn58, Gly61 and Gly62 were predicted to interact with sodium channel receptor and to act as functional residues.     

X-ray structure and mutagenesis of the scorpion depressant toxin LqhIT2 reveals key determinants crucial for activity and anti-insect selectivity

Scorpion depressant beta-toxins show high preference for insect voltage-gated sodium channels (Na(v)s) and modulate their activation. Although their pharmacological and physiological effects were described, their three-dimensional structure and bioactive surface have never been determined. We utilized an efficient system for expression of the depressant toxin LqhIT2 (from Leiurus quinquestriatushebraeus), mutagenized its entire exterior, and determined its X-ray structure at 1.2 A resolution. The toxin molecule is composed of a conserved cysteine-stabilized alpha/beta-core (core-globule), and perpendicular to it an entity constituted from the N and C-terminal regions (NC-globule). The surface topology and overall hydrophobicity of the groove between the core and NC-globules (N-groove) is important for toxin activity and plays a role in selectivity to insect Na(v)s. The N-groove is flanked by Glu24 and Tyr28, which belong to the "pharmacophore" of scorpion beta-toxins, and by the side-chains of Trp53 and Asn58 that are important for receptor site recognition. Substitution of Ala13 by Trp in the N-groove uncoupled activity from binding, suggesting that this region of the molecule is also involved in "voltage-sensor trapping", the mode of action that typifies scorpion beta-toxins. The involvement of the N-groove in recognition of the receptor site, which seems to require a defined topology, as well as in sensor trapping, which involves interaction with a moving channel region, is puzzling. On the basis of the mutagenesis studies we hypothesize that following binding to the receptor site, the toxin undergoes a conformational change at the N-groove region that facilitates the trapping of the voltage-sensor in its activated position.     

Dissection of the functional surface of an anti-insect excitatory toxin illuminates a putative "hot spot" common to all scorpion beta-toxins affecting Na+ channels

Scorpion beta-toxins affect the activation of voltage-sensitive sodium channels (NaChs). Although these toxins have been instrumental in the study of channel gating and architecture, little is known about their active sites. By using an efficient system for the production of recombinant toxins, we analyzed by point mutagenesis the entire surface of the beta-toxin, Bj-xtrIT, an anti-insect selective excitatory toxin from the scorpion Buthotus judaicus. Each toxin mutant was purified and analyzed using toxicity and binding assays, as well as by circular dichroism spectroscopy to discern the differences among mutations that caused structural changes and those that specifically affected bioactivity. This analysis highlighted a functional discontinuous surface of 1405 A(2), which was composed of a number of non-polar and three charged amino acids clustered around the main alpha-helical motif and the C-tail. Among the charged residues, Glu(30) is a center of a putative "hot spot" in the toxin-receptor binding-interface and is shielded from bulk solvent by a hydrophobic "gasket" (Tyr(26) and Val(34)). Comparison of the Bj-xtrIT structure with that of other beta-toxins that are active on mammals suggests that the hot spot and an adjacent non-polar region are spatially conserved. These results highlight for the first time structural elements that constitute a putative "pharmacophore" involved in the interaction of beta-toxins with receptor site-4 on NaChs. Furthermore, the unique structure of the C-terminal region most likely determines the specificity of excitatory toxins for insect NaChs.     

muO conotoxins inhibit NaV channels by interfering with their voltage sensors in domain-2

The muO-conotoxins MrVIA and MrVIB are 31-residue peptides from Conus marmoreus, belonging to the O-superfamily of conotoxins with three disulfide bridges. They have attracted attention because they are inhibitors of tetrodotoxin-insensitive voltage-gated sodium channels (Na(V)1.8) and could therefore serve as lead structure for novel analgesics. The aim of this study was to elucidate the molecular mechanism by which muO-conotoxins affect Na(V) channels. Rat Na(V)1.4 channels and mutants thereof were expressed in mammalian cells and were assayed with the whole-cell patch-clamp method. Unlike for the M-superfamily mu-conotoxin GIIIA from Conus geographus, channel block by MrVIA was strongly diminished after activating the Na(V) channels by depolarizing voltage steps. Searching for the source of this voltage dependence, the gating charges in all four-voltage sensors were reduced by site-directed mutagenesis showing that alterations of the voltage sensor in domain-2 have the strongest impact on MrVIA action. These results, together with previous findings that the effect of MrVIA depends on the structure of the pore-loop in domain-3, suggest a functional similarity with scorpion beta-toxins. In fact, MrVIA functionally competed with the scorpion beta-toxin Ts1 from Tityus serrulatus, while it did not show competition with mu-GIIIA. Ts1 and mu-GIIIA did not compete either. Thus, similar to scorpion beta-toxins, muO-conotoxins are voltage-sensor toxins targeting receptor site-4 on Na(V) channels. They "block" Na(+) flow most likely by hindering the voltage sensor in domain-2 from activating and, hence, the channel from opening.     

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.     

Comparative pharmacology and cloning of two novel arachnid sodium channels: Exploring the adaptive insensitivity of scorpion to its toxins

Scorpion toxins have been found lacking effect on Na(+) current of its own sodium channel, whereas the molecular mechanism remains mystery. In this study, the binding affinity of pharmacologically distinct scorpion toxins was found much weaker to scorpion (Buthus martensii) nerve synaptosomes than to spider (Ornithoctonus huwena) ones. The sodium channel cDNA from these two species were further cloned. The deduced proteins contain 1871 and 1987 amino acids respectively. Several key amino acid substitutions, i.e., A1610V, I1611L and S1617K, are found in IVS3-S4 constituting receptor site-3, and for receptor site-4, two residues (Leu-Pro) are inserted near IIS4 of scorpion sodium channel.     

Solution structure and alanine scan of a spider toxin that affects the activation of mammalian voltage-gated sodium channels

Magi 5, from the hexathelid spider Macrothele gigas, is a 29-residue polypeptide containing three disulfide bridges. It binds specifically to receptor site 4 on mammalian voltage-gated sodium channels and competes with scorpion beta-toxins, such as Css IV from Centruroides suffusus suffusus. As a consequence, Magi 5 shifts the activation voltage of the mammalian rNav1.2a channel to more hyperpolarized voltages, whereas the insect channel, DmNav1, is not affected. To gain insight into toxin-channel interactions, Magi 5 and 23 analogues were synthesized. The three-dimensional structure of Magi 5 in aqueous solution was determined, and its voltage-gated sodium channel-binding surfaces were mapped onto this structure using data from electrophysiological measurements on a series of Ala-substituted analogues. The structure clearly resembles the inhibitor cystine knot structural motif, although the triple-stranded beta-sheet typically found in that motif is partially distorted in Magi 5. The interactive surface of Magi 5 toward voltage-gated sodium channels resembles in some respects the Janus-faced atracotoxins, with functionally important charged residues on one face of the toxin and hydrophobic residues on the other. Magi 5 also resembles the scorpion beta-toxin Css IV, which has distinct nonpolar and charged surfaces that are critical for channel binding and has a key Glu involved in voltage sensor trapping. These two distinct classes of toxin, with different amino acid sequences and different structures, may utilize similar groups of residues on their surface to achieve the common end of modifying voltage-gated sodium channel function.     

Direct evidence that receptor site-4 of sodium channel gating modifiers is not dipped in the phospholipid bilayer of neuronal membranes

In a recent note to Nature, R. MacKinnon has raised the possibility that potassium channel gating modifiers are able to partition in the phospholipid bilayer of neuronal membranes and that by increasing their partial concentration adjacent to their receptor, they affect channel function with apparent high affinity (Lee and MacKinnon (2004) Nature 430, 232-235). This suggestion was adopted by Smith et al. (Smith, J. J., Alphy, S., Seibert, A. L., and Blumenthal, K. M. (2005) J. Biol. Chem. 280, 11127-11133), who analyzed the partitioning of sodium channel modifiers in liposomes. They found that certain modifiers were able to partition in these artificial membranes, and on this basis, they have extrapolated that scorpion beta-toxins interact with their channel receptor in a similar mechanism as that proposed by MacKinnon. Since this hypothesis has actually raised a new conception, we examined it in binding assays using a number of pharmacologically distinct scorpion beta-toxins and insect and mammalian neuronal membrane preparations, as well as by analyzing the rate by which the toxin effect on gating of Drosophila DmNa(v)1 and rat brain rNa(v)1.2a develops. We show that in general, scorpion beta-toxins do not partition in neuronal membranes and that in the case in which a depressant beta-toxin partitions in insect neuronal membranes, this partitioning is unrelated to its interaction with the receptor site and the effect on the gating properties of the sodium channel. These results negate the hypothesis that the high affinity of beta-toxins for sodium channels is gained by their ability to partition in the phospholipid bilayer and clearly indicate that the receptor site for scorpion beta-toxins is accessible to the extracellular solvent.     

NMR solution structure of BmK-betaIT, an excitatory scorpion beta-toxin without a 'hot spot' at the relevant position

BmK-betaIT (previously named as Bm32-VI in the literature), an excitatory scorpion beta-toxin, is purified from the venom of the Chinese scorpion Buthus martensii Karsch. It features a primary sequence typical of the excitatory anti-insect toxins: two contiguous Cys residues (Cys37-Cys38) and a shifted location of the fourth disulfide bridges (Cys38-Cys64), and demonstrates bioactivity characteristic of the excitatory beta-toxins. However, it is noteworthy that BmK-betaIT is not conserved with a glutamate residue at the preceding position of the third Cys residue, and is the first example having a non-glutamate residue at the relevant position in the excitatory scorpion beta-toxin subfamily. The 3D structure of BmK-betaIT is determined with 2D NMR spectroscopy and molecular modeling. The solution structure of BmK-betaIT is closely similar to those of BmK IT-AP and Bj-xtrIT, only distinct from the latter by lack of an alpha(0)-helix. The surface functional patch comparison with those of BmK IT-AP and Bj-xtrIT reveals their striking similarity in the spatial arrangement. These results infer that the functional surface of beta-toxins is composed of two binding regions and a functional site. The main binding site is consisted of hydrophobic residues surrounding the alpha(1)-helix and its preceding loop, which is common to all beta-type scorpion toxins affecting Na(+) channels. The second binding site, which determines the specificity of the toxin, locates at the C-terminus for excitatory insect beta-toxin, while rests at the beta-sheet and its linking loop for anti-mammal toxins. The functional site involved in the voltage sensor-trapping model, which characterizes the function of all beta-toxins, is the negatively charged residue Glu15.     

Crystal structure of neurotoxin Ts1 from Tityus serrulatus provides insights into the specificity and toxicity of scorpion toxins.

The crystal structure of neurotoxin Ts1, a major component of the venom of the Brazilian scorpion Tityus serrulatus, has been determined at 1.7 A resolution. It is the first X-ray structure of a highly toxic anti-mammalian beta-toxin. The folding of the polypeptide chain of Ts1 is similar to that of other scorpion toxins. A cysteine-stabilised alpha-helix/beta-sheet motif forms the core of the flattened molecule. All residues identified as functionally important by chemical modification and site-directed mutagenesis are located on one side of the molecule, which is therefore considered as the Na+channel recognition site. The distribution of charged and non-polar residues over this surface determines the specificity of the toxin-channel interaction. Comparison to other scorpion toxins shows that positively charged groups at positions 1 and 12 as well as a negative charge at position 2 are likely determinants of the specificity of beta-toxins. In contrast, the contribution of the conserved aromatic cluster to the interaction might be relatively small. Comparison of Ts1 to weak beta-toxins from Centruroides sculpturatus Ewing reveals that a number of basic amino acid residues located on the face of the molecule opposite to the binding surface may account for the high toxicity of Ts1.     

Exploring the obscure profiles of pharmacological binding sites on voltage-gated sodium channels by BmK neurotoxins

Diverse subtypes of voltage-gated sodium channels (VGSCs) have been found throughout tissues of the brain, muscles and the heart. Neurotoxins extracted from the venom of the Asian scorpion Buthus martensi Karsch (BmK) act as sodium channel-specific modulators and have therefore been widely used to study VGSCs. α-type neurotoxins, named BmK I, BmK αIV and BmK abT, bind to receptor site-3 on VGSCs and can strongly prolong the inactivation phase of VGSCs. In contrast, β-type neurotoxins, named BmK AS, BmK AS-1, BmK IT and BmK IT2, occupy receptor site-4 on VGSCs and can suppress peak currents and hyperpolarize the activation kinetics of sodium channels. Accumulating evidence from binding assays of scorpion neurotoxins on VGSCs, however, indicate that pharmacological sensitivity of VGSC subtypes to different modulators is much more complex than that suggested by the simple α-type and β-type neurotoxin distinction. Exploring the mechanisms of possible dynamic interactions between site 3-/4-specific modulators and region- and/or species-specific subtypes of VGSCs would therefore greatly expand our understanding of the physiological and pharmacological properties of diverse VGSCs. In this review, we discuss the pharmacological and structural diversity of VGSCs as revealed by studies exploring the binding properties and cross-competitive binding of site 3- or site 4-specific modulators in VGSC subtypes in synaptosomes from distinct tissues of diverse species.     

Depolarization Differentially Affects Allosteric Modulation by Neurotoxins of Scorpion α-Toxin Binding on Voltage-Gated Sodium Channels

Abstract: Voltage-gated sodium channels serve as a target for many neurotoxins that bind to several distinct, allosterically interacting receptor sites. We examined the effect of membrane potentials (incited by increasing external K⁺ concentrations) on the binding modulation by veratridine, brevetoxin, and tetrodotoxin of the scorpion α-toxin AaH II to receptor site 3 on sodium channels of rat brain synaptosomes. Depolarization is shown to differentially modulate neurotoxin effects on AaH II binding: Veratridine increase is potentiated, brevetoxin's inhibitory effect is reduced, and tetrodotoxin enhancement is evident mainly at resting membrane potential (5 m M K⁺). Both tetrodotoxin and veratridine apparently reverse the inhibition of AaH II binding by brevetoxin at resting membrane potential, but only veratridine is able to partially restore AaH II binding at 0 mV (135 m M K⁺). Thus, the allosteric interactions are grouped into two categories, depending on the membrane potential. Under depolarized conditions, the cooperative effects among veratridine and brevetoxin on AaH II binding fit the previously described two-state conformational model. At resting membrane potential, additional interactions are revealed, which may be explained by assuming that toxin binding induces conformational changes on the channel structure, in addition to being state-dependent. Our results provide a new insight into neurotoxin action and the complex dynamic changes underlying allosteric coupling of neurotoxin receptor sites, which may be related to channel gating.     

A new approach to insect-pest control—combination of neurotoxins interacting with voltage sensitive sodium channels to increase selectivity and specificity

Voltage-sensitive sodium channels are responsible for the generation of electrical signals in most excitable tissues and serve as specific targets for many neurotoxins. At least seven distinct classes of neurotoxins have been designated on the basis of physiological activity and competitive binding studies. Although the characterization of the neurotoxin receptor sites was predominantly performed using vertebrate excitable preparations, insect neuronal membranes were shown to possess similar receptor sites. We have demonstrated that the two mutually competing antiinsect excitatory and depressant scorpion toxins, previously suggested to occupy the same receptor site, bind to two distinct receptors on insect sodium channels. The latter provides a new approach to their combined use in insect control strategy. Although the sodium channel receptor sites are topologically separated, there are strong allosteric interactions among them. We have shown that the lipid-soluble sodium channel activators, veratridine and brevetoxin, reveal divergent allosteric modulation on scorpion α-toxins binding at homologous receptor sites on mammalian and insect sodium channels. The differences suggest a functionally important structural distinction between these channel subtypes. The differential allosteric modulation may provide a new approach to increase selective activity of pesticides on target organisms by simultaneous application of allosterically interacting drugs, designed on the basis of the selective toxins. Thus, a comparative study of neurotoxin receptor sites on mammalian and invertebrate sodium channels may elucidate the structural features involved in the binding and activity of the various neurotoxins, and may offer new targets and approaches to the development of highly selective pesticides.     

Molecular characterization of a possible progenitor sodium channel toxin from the Old World scorpion Mesobuthus martensii

Toxins affecting sodium channels widely exist in the venoms of scorpions throughout the world. These molecules comprise an evolutionarily related peptide family with three shared features including conserved three-dimensional structure and gene organization, and similar function. Based on different pharmacological profiles and binding properties, scorpion sodium channel toxins are divided into alpha- and beta-groups. However, their evolutionary relationship is not yet established. Here, we report a gene isolated from the venom gland of scorpion Mesobuthus martensii which encodes a novel sodium channel toxin-like peptide of 64 amino acids, named Mesotoxin. The Mesotoxin gene is organized into three exons and two introns with the second intron location conserved across the family. This peptide is unusual in that it has only three disulfides and a long cysteine-free tail with loop size and structural characteristics close to beta-toxins. Evolutionary analysis favors its basal position in the origin of scorpion sodium channel toxins as a progenitor. The discovery of Mesotoxin will assist investigations into the key issue regarding the origin and evolution of scorpion toxins.     

NMR analysis of interaction of LqhalphaIT scorpion toxin with a peptide corresponding to the D4/S3-S4 loop of insect para voltage-gated sodium channel

Voltage-gated sodium channels (Navs) are large transmembrane proteins that initiate action potential in electrically excitable cells. This central role in the nervous system has made them a primary target for a large number of neurotoxins. Scorpion alpha-neurotoxins bind to Navs with high affinity and slow their inactivation, causing a prolonged action potential. Despite the similarity in their mode of action and three-dimensional structure, alpha-toxins exhibit great variations in selectivity toward insect and mammalian Navs, suggesting differences in the binding surfaces of the toxins and the channels. The scorpion alpha-toxin binding site, termed neurotoxin receptor site 3, has been shown to involve the extracellular S3-S4 loop in domain 4 of the alpha-subunit of voltage-gated sodium channels (D4/S3-S4). In this study, the binding site for peptides corresponding to the D4/S3-S4 loop of the para insect Nav was mapped on the highly insecticidal alpha-neurotoxin, LqhalphaIT, from the scorpion Leiurus quinquestriatus hebraeus, by following changes in the toxin amide 1H and 15N chemical shifts upon binding. This analysis suggests that the five-residue turn (residues LqK8-LqC12) of LqhalphaIT and those residues in its vicinity interact with the D4/S3-S4 loop of Nav. Residues LqR18, LqW38, and LqA39 could also form a patch contributing to the interaction with D4/S3-S4. Moreover, a new bioactive residue, LqV13, was identified as being important for Nav binding and specifically for the interaction with the D4/S3-S4 loop. The contribution of LqV13 to NaV binding was further verified by mutagenesis. Future studies involving other extracellular regions of Navs are required for further characterization of the structure of the LqhalphaIT-Navs binding site.     

Jingzhaotoxin-III, a novel spider toxin inhibiting activation of voltage-gated sodium channel in rat cardiac myocytes

We have isolated a cardiotoxin, denoted jingzhaotoxin-III (JZTX-III), from the venom of the Chinese spider Chilobrachys jingzhao. The toxin contains 36 residues stabilized by three intracellular disulfide bridges (I-IV, II-V, and III-VI), assigned by a chemical strategy of partial reduction and sequence analysis. Cloned and sequenced using 3'-rapid amplification of cDNA ends and 5'-rapid amplification of cDNA ends, the full-length cDNA encoded a 63-residue precursor of JZTX-III. Different from other spider peptides, it contains an uncommon endoproteolytic site (-X-Ser-) anterior to mature protein and the intervening regions of 5 residues, which is the smallest in spider toxin cDNAs identified to date. Under whole cell recording, JZTX-III showed no effects on voltage-gated sodium channels (VGSCs) or calcium channels in dorsal root ganglion neurons, whereas it significantly inhibited tetrodotoxin-resistant VGSCs with an IC(50) value of 0.38 microm in rat cardiac myocytes. Different from scorpion beta-toxins, it caused a 10-mV depolarizing shift in the channel activation threshold. The binding site for JZTX-III on VGSCs is further suggested to be site 4 with a simple competitive assay, which at 10 microm eliminated the slowing currents induced by Buthus martensi Karsch I (BMK-I, scorpion alpha-like toxin) completely. JZTX-III shows higher selectivity for VGSC isoforms than other spider toxins affecting VGSCs, and the toxin hopefully represents an important ligand for discriminating cardiac VGSC subtype.     

Binding characteristics of BmK I, an alpha-like scorpion neurotoxic polypeptide, on cockroach nerve cord synaptosomes.

In this study, the binding characteristics of BmK I, an alpha-like neurotoxic polypeptide purified from the venom of the Chinese scorpion Buthus martensi Karsch, were investigated on rat brain and cockroach nerve cord synaptosomes. The results showed that BmK I can bind to a single class of noninteracting binding sites on cockroach nerve cord synaptosomes with medium affinity (Kd = 16.5 +/ - 4.4 nM) and low binding capacity (Bmax = 1.05 +/- 0.23 pmol/mg protein), but lacks specific binding on rat brain synaptosomes. BmK AS, BmK AS-1 (two novel sodium channel-blocking ligands), BmK IT (an excitatory insect-selective toxin) and BmK IT2 (a depressant insect-selective toxin) from the same venom were found to be capable of depressing BmK I binding in cockroach nerve cord synaptosomes, which might be attributed to either allosteric modulation of voltage-gated Na+ channels by these toxic polypeptides or partial overlapping between the receptor binding sites of BmK I and these toxins. This thus supported the notion that alpha-like scorpion neurotoxic polypeptides bind to a distinct receptor site on sodium channels, which might be similar to the binding receptor site of alpha-type insect toxins, and also related to those of BmK AS type and insect-selective scorpion toxins on insect sodium channels.