Insect Voltage-gated Sodium Research Articles

Insecticidal activity of the spider neurotoxin PPTX-04 through modulating insect voltage-gated sodium channel

Ion channels on cell membrane are molecular targets of more than half peptide neurotoxins from spiders. From Pardosa pseudoannulata, a predatory spider on a range of insect pests, we characterized a peptide neurotoxin PPTX-04 with an insecticidal activity. PPTX-04 showed high toxicity to Nilaparvata lugens, a main prey of P. pseudoannulata, and the toxicity was not affected by the resistance to etofenprox (IUPAC chemical name:1-ethoxy-4-[2-methyl-1-[(3-phenoxyphenyl)methoxy]propan-2-yl]benzene, purity: 99%). On N. lugens voltage-gated sodium channel NlNav1 expressed in Xenopus oocytes, PPTX-04 prolonged the channel opening and induced tail currents, which is similar to pyrethroid insecticides. However, PPTX-04 potency on NlNav1 was not affected by mutations conferring pyrethroid resistance in insects, which revealed that PPTX-04 and pyrethroids should act on different receptors in NlNav1. In contrast, two mutations at the extracellular site 4 significantly reduced PPTX-04 potency, which indicated that PPTX-04 would act on a potential receptor containing the site 4 in NlNav1. The result from the molecular docking supported the conclusion that the binding pocket of PPTX-04 in NlNav1 should contain the site 4. In summary, PPTX-04 had high insecticidal activity through acting on a distinct receptor site in insect Nav, and was a potential resource to control insect pests and manage resistance to pyrethroids.

A Globally Distributed Insecticide Resistance Allele Confers a Fitness Cost in the Absence of Insecticide in Aedes aegypti (Diptera: Culicidae), the Yellow Fever Mosquito.

The cosmopolitan mosquito Aedes aegypti is a vector of harmful arboviruses. Pyrethroid insecticides are used to reduce adult populations and prevent the spread of disease. Pyrethroids target the insect voltage-gated sodium channel (VGSC). Collectively, mutations in Vgsc that confer resistance are referred to as knock-down resistance or kdr. There are numerous kdr mutations found in A. aegypti Vgsc, and there is co-occurrence of some mutations. Full-length cDNA sequences have identified nine known kdr (e.g., 1534C) alleles. The 1534C allele is among the most common kdr alleles, but allele frequencies between populations vary considerably. We used the 1534C:RK strain, which has the 1534C (kdr) allele in the genetic background of the insecticide susceptible Rockefeller (ROCK) strain, and conducted population cage experiments to assess the potential intrinsic fitness cost of the 1534C allele relative to the susceptible allele (F1534) in the ROCK strain. Individuals were genotyped across generations using allele specific PCR. A fitness cost of the 1534C allele was detected across seven generations of mosquitos reared in the absence of insecticide selection pressure. The decrease in allele frequency was not due to drift. Comparison of our results to previous studies suggests that the magnitude of the fitness cost of kdr alleles in the absence of insecticide is disconnected from the level of resistance they confer, and that the fitness costs of different kdr alleles can be variable.

Derivative of Scorpion Neurotoxin BeM9 Is Selective for Insect Voltage-Gated Sodium Channels

Scorpion α-toxins are small proteins inhibiting the inactivation of voltage-gated sodium channels. They can selectively act on either mammalian (mammal toxins) or insect channels (insect toxins), or affect both types of channels (α-like toxins). Currently no model has been proposed that fully explains the dependence of selectivity upon amino acid sequence, but some patterns have already been established. Thus, most mammal toxins have an aspartic acid residue in position 8, which is involved in the formation of the nest motif, but it is still not clear whether this residue interacts directly with channels. The objective of our study was to obtain a derivative of the α-like toxin BeM9 with the replacement of lysine in position 8 by glutamate (K8E), changing the charge, but excluding the formation of the nest motif. In addition, we replaced the tyrosine in position 17 with glycine (Y17G), which is characteristic of mammal toxins. Surprisingly, the double-mutant derivative BeM9EG lost its activity on mammalian channels, becoming an insect toxin. To explain these changes, we constructed models of BeM9 and BeM9EG complexes with channels, and also performed molecular dynamics of isolated toxins. Analysis of intermolecular contacts in the complexes did not explain the reason for the selectivity change. Nevertheless, the structure of intramolecular contacts and data on molecular mobility indicate an important role of residues K8 and Y17 in stabilizing a certain conformation of BeM9 loops. We assume that the replacement of these residues allosterically affects the efficiency of toxin binding to channels.

The insecticidal activity of recombinant nemertide toxin α-1 from Lineus longissimus towards pests and beneficial species

The nemertide toxins from the phylum Nemertea are a little researched family of neurotoxins with potential for development as biopesticides. Here we report the recombinant production of nemertide α-1 (α-1), a 65-residue inhibitor cystine knot (ICK) peptide from Lineus longissimus, known to target insect voltage-gated sodium channels. The insecticidal activity of α-1 was assessed and compared with the well characterised ICK venom peptide, ω-atracotoxin/hexatoxin-Hv1a (Hv1a). α-1 elicited potent spastic paralysis when injected into cabbage moth (Mamestra brassicae) larvae; conferring an ED50 3.90 μg/larva (10.30 nmol/g larva), followed by mortality (60% within 48 h after 10 μg injection). By comparison, injection of M. brassicae larvae with recombinant Hv1a produced short-lived flaccid paralysis with an ED50 over 6 times greater than that of α-1 at 26.20 μg/larva (64.70 nmol/g larva). Oral toxicity of α-1 was demonstrated against two aphid species (Myzus persicae and Acyrthosiphon pisum), with respective LC50 values of 0.35 and 0.14 mg/mL, some 6-fold lower than those derived for recombinant Hv1a. When delivered orally to M. brassicae larvae, α-1 caused both paralysis (ED50 11.93 μg/larva, 31.5 nmol/g larva) and mortality. This contrasts with the lack of oral activity of Hv1a, which when fed to M. brassicae larvae had no effect on feeding or survival. Hv1a has previously been shown to be non-toxic by injection to the beneficial honeybee (Apis mellifera). By contrast, rapid paralysis and 100% mortality was observed following injection of α-1 (31.6 nmol/g insect). These results demonstrate the great potential of naturally occurring non-venomous peptides, such as α-1, for development as novel effective biopesticides, but equally highlights the importance of understanding the phyletic specificity of a given toxin at an early stage in the quest to discover and develop safe and sustainable pesticides.

Comparative characterization of two putative duplicated sodium channel genes in the red flour beetle, Tribolium castaneum

The insect voltage-gated sodium channel is the primary target of pyrethroids and novel efficient insecticides such as indoxacarb and metaflumizone. In this study, we cloned and characterized two putative sodium channel genes, TcNav1 and TcNav2, in Tribolium castaneum. The composite TcNav1 and TcNav2 encode a protein of 2045 and 2037 amino acid residues, sharing 76.1% and 75.5% amino acid identity with Drosophila para, respectively. Comparative analysis of genomic organization showed that TcNav1 and TcNav2 contain 26 and 27 exons, respectively. Analysis of the expression patterns showed that the mRNA levels of TcNav1 and TcNav2 were predominantly expressed in the head. RNAi-mediated knockdown of both TcNav1 and TcNav2 adversely affected adult emergence and significantly decreased sensitivity to deltamethrin. Significantly reduced pupation rate and sensitivity to beta-cypermethrin were observed after injection of siRNA targeting TcNav1 but not TcNav2. Taken together, we provide evidence that sodium channel gene has undergone duplication in T. castaneum, resulting in diversified developmental and toxicological functions.

Towards understanding transfluthrin efficacy in a pyrethroid-resistant strain of the malaria vector Anopheles funestus with special reference to cytochrome P450-mediated detoxification

Malaria vector control interventions rely heavily on the application of insecticides against anopheline mosquitoes, in particular the fast-acting pyrethroids that target insect voltage-gated sodium channels (VGSC). Frequent applications of pyrethroids have resulted in resistance development in the major malaria vectors including Anopheles funestus, where resistance is primarily metabolic and driven by the overexpression of microsomal cytochrome P450 monooxygenases (P450s). Here we examined the pattern of cross-resistance of the pyrethroid-resistant An. funestus strain FUMOZ-R towards transfluthrin and multi-halogenated benzyl derivatives, permethrin, cypermethrin and deltamethrin in comparison to the susceptible reference strain FANG. Transfluthrin and two multi-fluorinated derivatives exhibited micromolar potency - comparable to permethrin - to functionally expressed dipteran VGSC in a cell-based cation influx assay. The activity of transfluthrin and its derivatives on VGSC was strongly correlated with their contact efficacy against strain FUMOZ-R, although no such correlation was obtained for the other pyrethroids due to their rapid detoxification by the resistant strain. The low resistance levels for transfluthrin and derivatives in strain FUMOZ-R were only weakly synergized by known P450 inhibitors such as piperonyl butoxide (PBO), triflumizole and 1-aminobenzotriazole (1-ABT). In contrast, deltamethrin toxicity in FUMOZ-R was synergized > 100-fold by all three P450 inhibitors. The biochemical profiling of a range of fluorescent resorufin and coumarin compounds against FANG and FUMOZ-R microsomes identified 7-benzyloxymethoxy-4-trifluoromethylcoumarin (BOMFC) as a highly sensitive probe substrate for P450 activity. BOMFC was used to develop a fluorescence-based high-throughput screening assay to measure the P450 inhibitory action of potential synergists. Azole fungicides prochloraz and triflumizole were identified as extremely potent nanomolar inhibitors of microsomal P450s, strongly synergizing deltamethrin toxicity in An. funestus. Overall, the present study contributed to the understanding of transfluthrin efficacy at the molecular and organismal level and identified azole compounds with potential to synergize pyrethroid efficacy in malaria vectors.

How a Scorpion Toxin Selectively Captures a Prey Sodium Channel: The Molecular and Evolutionary Basis Uncovered.

The growing resistance of insects to chemical pesticides is reducing the effectiveness of conventional methods for pest control and thus, the development of novel insecticidal agents is imperative. Scorpion toxins specific for insect voltage-gated sodium channels (Navs) have been considered as one of the most promising insecticide alternatives due to their host specificity, rapidly evoked toxicity, biodegradability, and the lack of resistance. However, they have not been developed for uses in agriculture and public health, mainly because of a limited understanding of their molecular and evolutionary basis controlling their phylogenetic selectivity. Here, we show that the traditionally defined insect-selective scorpion toxin LqhIT2 specifically captures a prey Nav through a conserved trapping apparatus comprising a three-residue-formed cavity and a structurally adjacent leucine. The former serves as a detector to recognize and bind a highly exposed channel residue conserved in insects and spiders, two major prey items for scorpions; and the latter subsequently seizes the "moving" voltage sensor via hydrophobic interactions to reduce activation energy for channel opening, demonstrating its action in an enzyme-like manner. Based on the established toxin-channel interaction model in combination with toxicity assay, we enlarged the toxic spectrum of LqhIT2 to spiders and certain other arthropods. Furthermore, we found that genetic background-dependent cavity shapes determine the species selectivity of LqhIT2-related toxins. We expect that the discovery of the trapping apparatus will improve our understanding of the evolution and design principle of Nav-targeted toxins from a diversity of arthropod predators and accelerate their uses in pest control.

Molecular basis of selective resistance of the bumblebee BiNav1 sodium channel to tau-fluvalinate

Insecticides are widely used to control pests in agriculture and insect vectors that transmit human diseases. However, these chemicals can have a negative effect on nontarget, beneficial organisms including bees. Discovery and deployment of selective insecticides is a major mission of modern toxicology and pest management. Pyrethroids exert their toxic action by acting on insect voltage-gated sodium channels. Honeybees and bumblebees are highly sensitive to most pyrethroids, but are resistant to a particular pyrethroid, tau-fluvalinate (τ-FVL). Because of its unique selectivity, τ-FVL is widely used to control not only agricultural pests but also varroa mites, the principal ectoparasite of honeybees. However, the mechanism of bee resistance to τ-FVL largely remains elusive. In this study, we functionally characterized the sodium channel BiNav1-1 from the common eastern bumblebee (Bombus impatiens) in Xenopus oocytes and found that the BiNav1-1 channel is highly sensitive to six commonly used pyrethroids, but resistant to τ-FVL. Phylogenetic and mutational analyses revealed that three residues, which are conserved in sodium channels from 12 bee species, underlie resistance to τ-FVL or sensitivity to the other pyrethroids. Further computer modeling and mutagenesis uncovered four additional residues in the pyrethroid receptor sites that contribute to the unique selectivity of the bumblebee sodium channel to τ-FVL versus other pyrethroids. Our data contribute to understanding a long-standing enigma of selective pyrethroid toxicity in bees and may be used to guide future modification of pyrethroids to achieve highly selective control of pests with minimal effects on nontarget organisms.

Peptide-rich venom from the spider Heteropoda venatoria potently inhibits insect voltage-gated sodium channels

Heteropoda venatoria is a venomous spider species distributed worldwide and has a characteristic habit of feeding on insects. Reverse-phase high-performance liquid chromatography and matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry analyses revealed that H. venatoria venom contains hundreds of peptides with a predominant molecular weights of 3000–5000 Da. Intra-abdominal injection of the venom had severe toxic effects on cockroaches and caused death at higher concentrations. The LD50 was 28.18 μg/g of body weight in the cockroach. It was found that the venom had potent inhibitory effect on voltage-gated sodium channels (VGSCs) in Periplaneta americana cockroach dorsal unpaired median (DUM) neurons with an IC50 values of 6.25 ± 0.02 μg/mL. However, 100 μg/mL venom only partially blocked VGSC currents in rat dorsal root ganglion cells, a much lower inhibitory effect than that on DUM VGSCs. Our results indicate that the venom of H. venatoria contains diverse neurotoxins that might become new leads for bioinsecticides.

The insecticidal spider toxin SFI1 is a knottin peptide that blocks the pore of insect voltage-gated sodium channels via a large β-hairpin loop.

Spider venoms contain a plethora of insecticidal peptides that act on neuronal ion channels and receptors. Because of their high specificity, potency and stability, these peptides have attracted much attention as potential environmentally friendly insecticides. Although many insecticidal spider venom peptides have been isolated, the molecular target, mode of action and structure of only a small minority have been explored. Sf1a, a 46-residue peptide isolated from the venom of the tube-web spider Segesteria florentina, is insecticidal to a wide range of insects, but nontoxic to vertebrates. In order to investigate its structure and mode of action, we developed an efficient bacterial expression system for the production of Sf1a. We determined a high-resolution solution structure of Sf1a using multidimensional 3D/4D NMR spectroscopy. This revealed that Sf1a is a knottin peptide with an unusually large β-hairpin loop that accounts for a third of the peptide length. This loop is delimited by a fourth disulfide bond that is not commonly found in knottin peptides. We showed, through mutagenesis, that this large loop is functionally critical for insecticidal activity. Sf1a was further shown to be a selective inhibitor of insect voltage-gated sodium channels, consistent with its 'depressant' paralytic phenotype in insects. However, in contrast to the majority of spider-derived sodium channel toxins that function as gating modifiers via interaction with one or more of the voltage-sensor domains, Sf1a appears to act as a pore blocker.

Effect of insecticidal fusion proteins containing spider toxins targeting sodium and calcium ion channels on pyrethroid-resistant strains of peach-potato aphid (Myzus persicae).

The recombinant fusion proteins Pl1a/GNA and Hv1a/GNA contain the spider venom peptides δ-amaurobitoxin-PI1a or ω-hexatoxin-Hv1a respectively, linked to snowdrop lectin (GNA). Pl1a targets receptor site 4 of insect voltage-gated sodium channels (NaCh), while Hv1a targets voltage-gated calcium channels. Insecticide-resistant strains of peach-potato aphid (Myzus persicae) contain mutations in NaCh. The pyrethroid-resistant kdr (794J) and super-kdr (UKO) strains contain mutations at residues L1014 and M918 in the channel α-subunit respectively, while the kdr + super-kdr strain (4824J), insensitive to pyrethroids, contains mutations at both L1014 and M918. Pl1a/GNA and Hv1a/GNA fusion proteins have estimated LC50 values of 0.35 and 0.19 mg mL(-1) when fed to wild-type M. persicae. For insecticide-resistant aphids, LC50 for the Pl1a/GNA fusion protein increased by 2-6-fold, correlating with pyrethroid resistance (wild type < kdr < super-kdr < kdr + super-kdr strains). In contrast, LC50 for the Hv1a/GNA fusion protein showed limited correlation with pyrethroid resistance. Mutations in the sodium channel in pyrethroid-resistant aphids also protect against a fusion protein containing a sodium-channel-specific toxin, in spite of differences in ligand-channel interactions, but do not confer resistance to a fusion protein targeting calcium channels. The use of fusion proteins with differing targets could play a role in managing pesticide resistance.

A recombinant fusion protein containing a spider toxin specific for the insect voltage-gated sodium ion channel shows oral toxicity towards insects of different orders

Recombinant fusion protein technology allows specific insecticidal protein and peptide toxins to display activity in orally-delivered biopesticides. The spider venom peptide δ-amaurobitoxin-PI1a, which targets insect voltage-gated sodium channels, was fused to the “carrier” snowdrop lectin (GNA) to confer oral toxicity.The toxin itself (PI1a) and an amaurobitoxin/GNA fusion protein (PI1a/GNA) were produced using the yeast Pichia pastoris as expression host. Although both proteins caused mortality when injected into cabbage moth (Mamestra brassicae) larvae, the PI1a/GNA fusion was approximately 6 times as effective as recombinant PI1a on a molar basis. PI1a alone was not orally active against cabbage moth larvae, but a single 30 μg dose of the PI1a/GNA fusion protein caused 100% larval mortality within 6 days when fed to 3rd instar larvae, and caused significant reductions in survival, growth and feeding in 4th – 6th instar larvae. Transport of fusion protein from gut contents to the haemolymph of cabbage moth larvae, and binding to the nerve chord, was shown by Western blotting. The PI1a/GNA fusion protein also caused mortality when delivered orally to dipteran (Musca domestica; housefly) and hemipteran (Acyrthosiphon pisum; pea aphid) insects, making it a promising candidate for development as a biopesticide.

Intron Retention in mRNA Encoding Ancillary Subunit of Insect Voltage-Gated Sodium Channel Modulates Channel Expression, Gating Regulation and Drug Sensitivity

Insect voltage-gated sodium (Nav) channels are formed by a well-known pore-forming α-subunit encoded by para-like gene and ancillary subunits related to TipE from the mutation “temperature-induced-paralysis locus E.” The role of these ancillary subunits in the modulation of biophysical and pharmacological properties of Na+ currents are not enough documented. The unique neuronal ancillary subunit TipE-homologous protein 1 of Drosophila melanogaster (DmTEH1) strongly enhances the expression of insect Nav channels when heterologously expressed in Xenopus oocytes. Here we report the cloning and functional expression of two neuronal DmTEH1-homologs of the cockroach, Periplaneta americana, PaTEH1A and PaTEH1B, encoded by a single bicistronic gene. In PaTEH1B, the second exon encoding the last 11-amino-acid residues of PaTEH1A is shifted to 3′UTR by the retention of a 96-bp intron-containing coding-message, thus generating a new C-terminal end. We investigated the gating and pharmacological properties of the Drosophila Nav channel variant (DmNav1-1) co-expressed with DmTEH1, PaTEH1A, PaTEH1B or a truncated mutant PaTEH1Δ(270-280) in Xenopus oocytes. PaTEH1B caused a 2.2-fold current density decrease, concomitant with an equivalent α-subunit incorporation decrease in the plasma membrane, compared to PaTEH1A and PaTEH1Δ(270-280). PaTEH1B positively shifted the voltage-dependences of activation and slow inactivation of DmNav1-1 channels to more positive potentials compared to PaTEH1A, suggesting that the C-terminal end of both proteins may influence the function of the voltage-sensor and the pore of Nav channel. Interestingly, our findings showed that the sensitivity of DmNav1-1 channels to lidocaine and to the pyrazoline-type insecticide metabolite DCJW depends on associated TEH1-like subunits. In conclusion, our work demonstrates for the first time that density, gating and pharmacological properties of Nav channels expressed in Xenopus oocytes can be modulated by an intron retention process in the transcription of the neuronal TEH1-like ancillary subunits of P. americana.

The insecticidal neurotoxin Aps III is an atypical knottin peptide that potently blocks insect voltage-gated sodium channels

One of the most potent insecticidal venom peptides described to date is Aps III from the venom of the trapdoor spider Apomastus schlingeri. Aps III is highly neurotoxic to lepidopteran crop pests, making it a promising candidate for bioinsecticide development. However, its disulfide-connectivity, three-dimensional structure, and mode of action have not been determined. Here we show that recombinant Aps III (rAps III) is an atypical knottin peptide; three of the disulfide bridges form a classical inhibitor cystine knot motif while the fourth disulfide acts as a molecular staple that restricts the flexibility of an unusually large β hairpin loop that often houses the pharmacophore in this class of toxins. We demonstrate that the irreversible paralysis induced in insects by rAps III results from a potent block of insect voltage-gated sodium channels. Channel block by rAps III is voltage-independent insofar as it occurs without significant alteration in the voltage-dependence of channel activation or steady-state inactivation. Thus, rAps III appears to be a pore blocker that plugs the outer vestibule of insect voltage-gated sodium channels. This mechanism of action contrasts strikingly with virtually all other sodium channel modulators isolated from spider venoms that act as gating modifiers by interacting with one or more of the four voltage-sensing domains of the channel.

Allethrin Differentially Modulates Voltage-Gated Calcium Channel Subtypes in Rat PC12 Cells

Pyrethroid insecticides are one of the most widely used classes of insecticides. Previous studies revealed that pyrethroids potently affect the insect voltage-gated sodium (Na(+)) channel (VGSC), resulting in prolonged channel open time. However, recent findings have suggested that pyrethroids may affect targets other than the VGSC. In particular, several studies have shown that pyrethroids can modulate the activity of voltage-gated calcium (Ca(2+)) channels (VGCCs). However, these studies often reported conflicting results; some studies observed stimulatory effects, whereas others observed inhibitory effects of pyrethroids on VGCCs. This study investigated whether allethrin (AL), a well-characterized type I pyrethroid, altered VGCC characteristics measured by whole-cell recording in rat pheochromocytoma cells (PC12) differentiated with nerve growth factor (NGF). AL (5 microM) increased peak, end, and tail composite VGCC current independent of its effects on VGSCs. After blocking VGCC subtype-specific current with omega-conotoxin GVIA (GVIA, an N-type VGCC antagonist) or nimodipine (NIM, an L-type VGCC antagonist), our data further suggest that AL differentially affects VGCC subtypes. Thus, AL apparently stimulated GVIA-insensitive current while inhibiting NIM-insensitive current. AL also significantly altered the voltage dependency of activation and inactivation of L-type VGCCs. The differential modulation of VGCC subtypes by AL may explain some of the conflicting observations of other studies.

Synthesis, Solution Structure, and Phylum Selectivity of a Spider δ-Toxin That Slows Inactivation of Specific Voltage-gated Sodium Channel Subtypes

Magi 4, now renamed delta-hexatoxin-Mg1a, is a 43-residue neurotoxic peptide from the venom of the hexathelid Japanese funnel-web spider (Macrothele gigas) with homology to delta-hexatoxins from Australian funnel-web spiders. It binds with high affinity to receptor site 3 on insect voltage-gated sodium (Na(V)) channels but, unlike delta-hexatoxins, does not compete for the related site 3 in rat brain despite being previously shown to be lethal by intracranial injection. To elucidate differences in Na(V) channel selectivity, we have undertaken the first characterization of a peptide toxin on a broad range of mammalian and insect Na(V) channel subtypes showing that delta-hexatoxin-Mg1a selectively slows channel inactivation of mammalian Na(V)1.1, Na(V)1.3, and Na(V)1.6 but more importantly shows higher affinity for insect Na(V)1 (para) channels. Consequently, delta-hexatoxin-Mg1a induces tonic repetitive firing of nerve impulses in insect neurons accompanied by plateau potentials. In addition, we have chemically synthesized and folded delta-hexatoxin-Mg1a, ascertained the bonding pattern of the four disulfides, and determined its three-dimensional solution structure using NMR spectroscopy. Despite modest sequence homology, we show that key residues important for the activity of scorpion alpha-toxins and delta-hexatoxins are distributed in a topologically similar manner in delta-hexatoxin-Mg1a. However, subtle differences in the toxin surfaces are important for the novel selectivity of delta-hexatoxin-Mg1a for certain mammalian and insect Na(V) channel subtypes. As such, delta-hexatoxin-Mg1a provides us with a specific tool with which to study channel structure and function and determinants for phylum- and tissue-specific activity.

X-ray Structure and Mutagenesis of the Scorpion Depressant Toxin LqhIT2 Reveals Key Determinants Crucial for Activity and Anti-Insect Selectivity

Scorpion depressant β-toxins show high preference for insect voltage-gated sodium channels (Na vs) 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 quinquestriatus hebraeus), mutagenized its entire exterior, and determined its X-ray structure at 1.2 Å resolution. The toxin molecule is composed of a conserved cysteine-stabilized α/β-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 vs. The N-groove is flanked by Glu24 and Tyr28, which belong to the “pharmacophore” of scorpion β-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 β-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.

Solution structure of two insect‐specific spider toxins and their pharmacological interaction with the insect voltage‐gated Na+ channel

Delta-paluIT1 and delta-paluIT2 are toxins purified from the venom of the spider Paracoelotes luctuosus. Similar in sequence to mu-agatoxins from Agelenopsis aperta, their pharmacological target is the voltage-gated insect sodium channel, of which they alter the inactivation properties in a way similar to alpha-scorpion toxins, but they bind on site 4 in a way similar to beta-scorpion toxins. We determined the solution structure of the two toxins by use of two-dimensional nuclear magnetic resonance (NMR) techniques followed by distance geometry and molecular dynamics. The structures of delta-paluIT1 and delta-paluIT2 belong to the inhibitory cystine knot structural family, i.e. a compact disulfide-bonded core from which four loops emerge. Delta-paluIT1 and delta-paluIT2 contain respectively two- and three-stranded anti-parallel beta-sheets as unique secondary structure. We compare the structure and the electrostatic anisotropy of those peptides to other sodium and calcium channel toxins, analyze the topological juxtaposition of key functional residues, and conclude that the recognition of insect voltage-gated sodium channels by these toxins involves the beta-sheet, in addition to loops I and IV. Besides the position of culprit residues on the molecular surface, difference in dipolar moment orientation is another determinant of receptor binding and biological activity differences. We also demonstrate by electrophysiological experiments on the cloned insect voltage-gated sodium channel, para, heterologuously co-expressed with the tipE subunit in Xenopus laevis oocytes, that delta-paluIT1 and delta-paluIT2 procure an increase of Na+ current. delta-PaluIT1-OH seems to have less effect when the same concentrations are used.

The depressant scorpion neurotoxin LqqIT2 selectively modulates the insect voltage-gated sodium channel

LqqIT2 is a depressant neurotoxin present in the venom of the Leiurus quinquestriatus quinquestriatus scorpion, one of the world's most dangerous scorpions endemic to dry habitats in Africa and Asia. In order to determine its efficacy, potency and selectivity, LqqIT2 was subjected for the first time to an electrophysiological and pharmacological comparison between two different cloned sodium channels expressed in Xenopus laevis oocytes. Aside from typical β-toxin effects, LqqIT2 also affected the inactivation process and ion selectivity of the insect voltage-gated sodium channel. The most interesting feature of LqqIT2 is its total insect-selectivity. At a concentration of 1 μM, the insect-voltage-gated sodium channel, para, was profoundly modulated while its mammalian counterpart, the rat brain Na v1.2 channel, was not affected. This trait offers excellent prospects for the development of novel insecticides.

Synthesis and characterization of delta-atracotoxin-Ar1a, the lethal neurotoxin from venom of the Sydney funnel-web spider (Atrax robustus).

Delta-atracotoxin-Ar1a (delta-ACTX-Ar1a) is the major polypeptide neurotoxin isolated from the venom of the male Sydney funnel-web spider, Atrax robustus. This neurotoxin targets both insect and mammalian voltage-gated sodium channels, where it competes with scorpion alpha-toxins for neurotoxin receptor site-3 to slow sodium-channel inactivation. Progress in characterizing the structure and mechanism of action of this toxin has been hampered by the limited supply of pure toxin from natural sources. In this paper, we describe the first successful chemical synthesis and oxidative refolding of the four-disulfide bond containing delta-ACTX-Ar1a. This synthesis involved solid-phase Boc chemistry using double coupling, followed by oxidative folding of purified peptide using a buffer of 2 M GdnHCl and glutathione/glutathiol in a 1:1 mixture of 2-propanol (pH 8.5). Successful oxidation and refolding was confirmed using both chemical and pharmacological characterization. Ion spray mass spectrometry was employed to confirm the molecular weight. (1)H NMR analysis showed identical chemical shifts for native and synthetic toxins, indicating that the synthetic toxin adopts the native fold. Pharmacological studies employing whole-cell patch clamp recordings from rat dorsal root ganglion neurons confirmed that synthetic delta-ACTX-Ar1a produced a slowing of the sodium current inactivation and hyperpolarizing shifts in the voltage-dependence of activation and inactivation similar to native toxin. Under current clamp conditions, we show for the first time that delta-ACTX-Ar1a produces spontaneous repetitive plateau potentials underlying the clinical symptoms seen during envenomation. This successful oxidative refolding of synthetic delta-ACTX-Ar1a paves the way for future structure-activity studies to determine the toxin pharmacophore.