Peptide toxins that selectively target insect Na(V) and Ca(V) channels

Numerous metazoans express venoms for the purpose of defense, competitor deterrence or prey capture. Peptide neurotoxins are particularly well represented in the venoms of arachnids, cnidarians and mollusks and these toxins often possess high affinity and specificity for particular classes of ion channels. Some of these toxins have become the defining pharmacology for certain vertebrate ion channel subtypes. Unfortunately, due to differences in the structure, pharmacology and ion selectivity of insect voltage-gated sodium (Na(V)) and calcium (Ca(V)) channels compared with their vertebrate counterparts, these peptide toxins have proven less useful for the characterization of insect ion channels. Despite these disparities in channel structure and function, the armament of peptide toxins that specifically modulate the activity of insect ion channels is slowly expanding. This review focuses on insect-selective peptide toxins and their utility for the study of insect Na(V) and Ca(V) channels. The high affinity and selectivity of some of these neurotoxins means that they have the potential to become the defining pharmacology for specific subtypes of insect ion channels. In addition, it might be possible to exploit the phyletic specificity of these toxins as the basis for rational development of novel classes of ion channel insecticides.     

Peptide toxins that selectively target insect NaV and CaV channels

Numerous metazoans express venoms for the purpose of defense, competitor deterrence, or prey capture. Peptide neurotoxins are particularly well represented in the venoms of arachnids, cnidarians and mollusks and these toxins often possess high affinity and specificity for particular classes of ion channels. Some of these toxins have become the defining pharmacology for certain vertebrate ion channel subtypes. Unfortunately, due to differences in the structure, pharmacology, and ion selectivity of insect voltage-gated sodium (Na_V) and calcium (Ca_V) channels compared with their vertebrate counterparts, these peptide toxins have proven less useful for the characterization of insect ion channels. Despite these disparities in channel structure and function, the armament of peptide toxins that specifically modulate the activity of insect ion channels is slowly expanding. This review focuses on insect-selective peptide toxins and their utility for the study of insect Na_V and Ca_V channels. The high affinity and selectivity of some of these neurotoxins means that they have the potential to become the defining pharmacology for specific subtypes of insect ion channels. In addition, it might be possible to exploit the phyletic specificity of these toxins as the basis for rational development of novel classes of ion channel insecticides.     

Structure-function studies of omega-atracotoxin, a potent antagonist of insect voltage-gated calcium channels.

The omega-atracotoxins are a family of 36 to 37-residue peptide neurotoxins that block insect but not mammalian voltage-gated calcium channels. The high phylogenetic specificity of these toxins recommends them as lead compounds for targeting insects that have developed resistance to chemical pesticides. We have begun to examine structure-function relationships in the omega-atracotoxins in order to explore the molecular basis of their activity and phylogenetic specificity. By probing the venom of the Blue Mountains funnel-web spider, Hadronyche versuta, for insecticidal toxins with masses close to that of omega-atracotoxin-Hv1a (omega-ACTX-Hv1a), we have isolated and sequenced five additional omega-atracotoxins. Five of the six omega-atracotoxins isolated from the venom of H. versuta (omega-ACTX-Hv1a to -Hv1e) differ from one another by only 1-3 residues and have similar insecticidal potencies. In contrast, omega-ACTX-Hv1f differs from the other toxins by up to 10 residues and it has markedly reduced insecticidal potency, thus providing information on key functional residues. The new atracotoxin sequences have revealed that the three N-terminal residues are highly conserved. Despite the fact that these residues are structurally disordered in solution we show here, by a series of N-terminal truncations, that they contribute significantly to insecticidal potency. However, loss of activity does not correlate with deletion of highly conserved residues, which leads us to propose that the disposition of the N-terminal charge, rather than the chemical properties of the N-terminal residues themselves, may be critical for the activity of omega-atracotoxin on insect calcium channels.     

omega-Aga-I: a presynaptic calcium channel antagonist from venom of the funnel web spider, Agelenopsis aperta

Spider venoms are proving to be important sources of specific ion channel toxins. Venom of Agelenopsis aperta, a funnel web spider, contains a class of polypeptide toxins which blocks neuromuscular synapses at nanomolar concentrations. Detailed physiological analyses of block caused by one of these toxins, omega-Aga-I, show that it suppresses transmitter release at insect and frog neuromuscular junctions and blocks calcium spikes in insect neuronal cell bodies. omega-Aga-I may define a binding site on neuronal calcium channels which is common to both vertebrates and invertebrates.     

ω-Aga-I: A presynaptic calcium channel antagonist from venom of the funnel web spider,Agelenopsis aperta

Spider venoms are proving to be important sources of specific ion channel toxins. Venom of Agelenopsis aperta, a funnel web spider, contains a class of polypeptide toxins which blocks neuromuscular synapses at nanomolar concentrations. Detailed physiological analyses of block caused by one of these toxins, ω-Aga-I show that it suppresses transmitter release at insect and frog neuromuscular junctions and blocks calcium spikes in insect neuronal cell bodies. ω-Aga-I may define a binding site on neuronal calcium channels which is common to both vertebrates and invertebrates.     

Functional significance of the beta hairpin in the insecticidal neurotoxin omega-atracotoxin-Hv1a

omega-Atracotoxin-Hv1a is an insect-specific neurotoxin whose phylogenetic specificity derives from its ability to antagonize insect, but not vertebrate, voltage-gated calcium channels. In order to help understand its mechanism of action and to enhance its utility as a lead compound for insecticide development, we used a combination of protein engineering and site-directed mutagenesis to probe the toxin for key functional regions. First, we constructed a Hairpinless mutant in which the C-terminal beta-hairpin, which is highly conserved in this family of neurotoxins, was excised without affecting the fold of the residual disulfide-rich core of the toxin. The Hairpinless mutant was devoid of insecticidal activity, indicating the functional importance of the hairpin. We subsequently developed a highly efficient system for production of recombinant toxin and then probed the hairpin for key functional residues using alanine-scanning mutagenesis followed by a second round of mutagenesis based on initial "hits" from the alanine scan. This revealed that two spatially proximal residues, Asn(27) and Arg(35), form a contiguous molecular surface that is essential for toxin activity. We propose that this surface of the beta-hairpin is a key site for interaction of the toxin with insect calcium channels.     

Scanning mutagenesis of omega-atracotoxin-Hv1a reveals a spatially restricted epitope that confers selective activity against insect calcium channels

We constructed a complete panel of alanine mutants of the insect-specific calcium channel blocker omega-atracotoxin-Hv1a. Lethality assays using these mutant toxins identified three spatially contiguous residues, Pro10, Asn27, and Arg35, that are critical for insecticidal activity against flies (Musca domestica) and crickets (Acheta domestica). Competitive binding assays using radiolabeled omega-atracotoxin-Hv1a and neuronal membranes prepared from the heads of American cockroaches (Periplaneta americana) confirmed the importance of these three residues for binding of the toxin to target calcium channels presumably expressed in the insect membranes. At concentrations up to 10 microm, omega-atracotoxin-Hv1a had no effect on heterologously expressed rat Cav2.1, Cav2.2, and Cav1.2 calcium channels, consistent with the previously reported insect selectivity of the toxin. 30 microm omega-atracotoxin-Hv1a inhibited rat Cav currents by 10-34%, depending on the channel subtype, and this low level of inhibition was essentially unchanged when Asn27 and Arg35, which appears to be critical for interaction of the toxin with insect Cav channels, were both mutated to alanine. We propose that the spatially contiguous epitope formed by Pro10, Asn27, and Arg35 confers specific binding to insect Cav channels and is largely responsible for the remarkable phyletic selectivity of omega-atracotoxin-Hv1a. This epitope provides a structural template for rational design of chemical insecticides that selectively target insect Cav channels.     

Interactions of δ-Conotoxins with Alkaloid Neurotoxins Reveal Differences Between the Silent and Effective Binding Sites on Voltage-Sensitive Sodium Channels

Abstract: The δ-conotoxin-TxVIA from Conus textile (δTxVIA) is a mollusk-specific conotoxin that slows sodium channel inactivation exclusively in mollusk neuronal membranes but reveals high-affinity binding to both mollusk (effective binding) and rat brain (silent binding) neuronal membranes, despite not having any toxic effect in vertebrates in vivo and in vitro. Using binding studies with radioactive δTxVIA we demonstrate that a different mollusk-specific conotoxin, δ-conotoxin-GmVIA from the venom of Conus gloriamaris , possesses "silent" and effective binding properties in rat brain and mollusk sodium channels, respectively. Binding studies and electrophysiological tests with both vertebrate muscle and insect neuronal preparations have indicated that the silent binding sites of δTxVIA are highly conserved in a wide range of distinct vertebrate and insect sodium channels. Direct probing of receptor site 2 by a tritiated derivative of batrachotoxin ([³H]BTX-B) revealed that [³H]BTX-B binding in mollusk sodium channels is of high affinity with no addition of enhancing ligands, unlike [³H]BTX-B binding in rat brain. In contrast to the negative allosteric modulation of δTxVIA binding by veratridine, δTxVIA is not able to affect the binding of [³H]BTX-B in mollusk neuronal membranes but reduces [³H]BTX-B binding in rat brain in the presence of α-scorpion toxins. The latter finding indicates the existence of a pharmacological distinction between the silent and effective binding sites of δTxVIA and points out possible functionally important structural differences between molluscan and rat brain sodium channels.     

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.     

蝎β型昆虫毒素的结构及其与钠通道作用机制

蝎毒素是蝎为防卫的需要而产生的一系列活性短肽.其中蝎昆虫特异性毒素可特异性结合并调控昆虫可兴奋细胞膜上的钠离子通道,是研究离子通道结构与功能的首选探针,并在转基因抗虫植物及生物杀虫剂研究方面具有潜在的应用价值.本文对蝎β型昆虫毒素的结构与功能及其对钠离子通道的作用方式和β毒素的电压传感器捕获(voltage sensor-trapping)模型做一综述,为进一步揭示蝎β毒素的结构与功能的关系和在农作物抗虫领域的应用提供依据.     

Peptides and genes coding for scorpion toxins that affect ion-channels

Most scorpion toxins are ligand peptides that recognize and bind to integral membrane proteins known as ion-channels. To date there are at least 202 distinct sequences described, obtained from 30 different species of scorpions, 27 from the family Buthidae and three from the family Scorpionidae. Toxins that recognize potassium and chloride channels are usually from 29 to 41 amino acids long, stabilized by three or four disulfide bridges, whereas those that recognize sodium channels are longer, 60 to 76 amino acid residues, compacted by four disulfide bridges. Toxins specific for calcium channels are scarcely known and have variable amino acid lengths. The entire repertoire of toxins, independently of their specificity, was analyzed together by computational programs and a phylogenetic tree was built showing two separate branches. The K(+) and Cl(-) channel specific toxins are clustered into 14 subfamilies, whereas those of Na(+) and Ca(2+) specific toxins comprise at least 12 subfamilies. There are clear similarities among them, both in terms of primary sequence and the main three-dimensional folding pattern. A dense core formed by a short alpha helix segment and several antiparallel beta-sheet stretches, maintained by disulfide pairing, seems to be a common structural feature present in all toxins. The physiological function of these peptides is manifested by a blockage of ion passage through the channels or by a modification of the gating mechanism that controls opening and closing of the ion pore.     

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.     

Omega-agatoxins: novel calcium channel antagonists of two subtypes from funnel web spider (Agelenopsis aperta) venom

A new series of polypeptide presynaptic antagonists ("omega-agatoxins") was purified from venom of the funnel web spider Agelenopsis aperta. Physiological data indicate that all of these peptides are antagonists of voltage-sensitive calcium channels. Although all three omega-agatoxins (Aga) described here (omega-Aga-IA, omega-Aga-IB, and omega-Aga-IIA) block insect neuromuscular transmission presynaptically, biochemical data permit their subclassification as Type I and Type II toxins. Type I toxins (omega-Aga-IA and -IB) are 7.5 kDa, have closely related amino acid sequences, and exhibit characteristic tryptophan-like UV absorbance spectra. Complete Edman sequencing of omega-Aga-IA reveals it to be a 66-amino acid polypeptide containing 9 cysteines and 5 tryptophan residues. omega-Aga-IIA, a Type II toxin, is 11 kDa, shows limited amino acid sequence similarity to the Type I toxins, and exhibits mixed tryptophan- and tyrosine-like absorbance. Nanomolar concentrations of omega-Aga-IIA inhibit the specific binding of 125I-labeled omega-conotoxin GVIA to chick synaptosomal membranes while omega-Aga-IA and -IB have no effect under identical conditions. The omega-agatoxins thus are defined as two subtypes of neuronal calcium channel toxins with different structural characteristics and calcium channel binding specificities.     

Jingzhaotoxin-I, a novel spider neurotoxin preferentially inhibiting cardiac sodium channel inactivation

Jingzhaotoxin-I (JZTX-I), a 33-residue polypeptide, is derived from the Chinese tarantula Chilobrachys jing-zhao venom based on its ability to evidently increase the strength and the rate of vertebrate heartbeats. The toxin has three disulfide bonds with the linkage of I-IV, II-V, and III-VI that is a typical pattern found in inhibitor cystine knot molecules. Its cDNA determined by rapid amplification of 3'- and 5'-cDNA ends encoded a 62-residue precursor with a small proregion of eight residues. Whole-cell configuration indicated that JZTX-I was a novel neurotoxin preferentially inhibiting cardiac sodium channel inactivation by binding to receptor site 3. Although JZTX-I also exhibits the interaction with channel isoforms expressing in mammalian and insect sensory neurons, its affinity for tetrodotoxin-resistant subtype in mammalian cardiac myocytes (IC50 = 31.6 nm) is approximately 30-fold higher than that for tetrodotoxin-sensitive subtypes in latter tissues. Not affecting outward delay-rectified potassium channels expressed in Xenopus laevis oocytes and tetrodotoxin-resistant sodium channels in mammal sensory neurons, JZTX-I hopefully represents a potent ligand to discriminate cardiac sodium channels from neuronal tetrodotoxin-resistant isoforms. Furthermore, different from any reported spider toxins, the toxin neither modifies the current-voltage relationships nor shifts the steady-state inactivation of sodium channels. Therefore, JZTX-I defines a new subclass of spider sodium channel toxins. JZTX-I is an alpha-like toxin first reported from spider venoms. The result provides an important witness for a convergent functional evolution between spider and other animal venoms.     

Isolation, synthesis and pharmacological characterization of delta-palutoxins IT, novel insecticidal toxins from the spider Paracoelotes luctuosus (Amaurobiidae).

Four novel insecticidal toxins were isolated from the venom of the spider Paracoelotes luctuosus (Araneae: Amaurobiidae) and named delta-palutoxins IT1 to IT4. The four toxins are homologous 36-37 amino acid peptides reticulated by four disulfide bridges and three have amidated C-terminal residues. The delta-palutoxins are highly homologous with the previously described mu-agatoxins and curtatoxins (77-97%). The four peptides demonstrated significant toxicity against larvae of the crop pest Spodoptera litura (Lepidoptera: Noctuidae) in a microinjection bioassay, with LD50 values in the 9-50 microg per g of insect range. This level of toxicity is equivalent to that of several of the most active scorpion toxins used in the development of recombinant baculoviruses, and the delta-palutoxins appear to be insect specific. Electrophysiological experiments demonstrated that delta-palutoxin IT1, the most active toxin acts by affecting insect sodium channel inactivation, resulting in the appearance of a late-maintained sodium current, in a similar fashion to insecticidal scorpion alpha and alpha-like toxins and is thus likely to bind to channel receptor site 3. However, delta-palutoxin IT1 was distinguished by its lack of effect on peak sodium conductance, on the early phase of sodium current inactivation and the absence of a shift in the activation voltage of the sodium channels. delta-Palutoxins are thus proposed as new insecticidal toxins related to the alpha and alpha-like scorpion toxins. They will be useful both in the development of recombinant baculoviruses in agrochemical applications and also as molecular probes for the investigation of molecular mechanisms of insect selectivity and structure and function of sodium channels.     

Invertebrate aquaporins: a review

Aquaporins (AQPs) or water channels render the lipid bilayer of cell membranes permeable to water. The numerous AQP subtypes present in any given species, the transport properties of each subtype and the variety of methods of their regulation allows different cell types to be transiently or permanently permeable to water or other solutes that AQPs are capable of transporting (e.g. urea or glycerol). AQPs have been well characterized in all vertebrate classes, other than reptilia. Here we review the current state of knowledge of invertebrate AQPs set in the context of the much more thoroughly studied vertebrate AQPs. By phylogenetic analysis of the total AQP complement of several completed insect genomes, we propose a classification system of insect AQPs including three sub-families (DRIP, BIB and PRIP) that have one representative from all the complete insect genomes. The physiological role of AQPs in invertebrates (insects, ticks and nematodes) is discussed, including their function in common invertebrate phenomena such as high-volume liquid diets, cryoprotection and anhydrobiosis.     

Sequence and electrophysiological characterization of two insect-selective excitatory toxins from the venom of the Chinese scorpion Buthus martensi

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.     

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

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

The sea anemone Bunodosoma granulifera contains surprisingly efficacious and potent insect-selective toxins

Two sodium channel toxins, BgII and BgIII, isolated from the sea anemone Bunodosoma granulifera, have been subjected to an elaborate electrophysiological and pharmacological comparison between five different cloned sodium channels expressed in Xenopus laevis oocytes in order to determine their efficacy, potency and selectivity. Our results reveal large differences in toxin-induced effect between the different sodium channels. These toxins possess the highest efficacy for the insect sodium channel (para). Our data also show that BgII, generally known as a neurotoxin, is especially potent on the insect sodium channel with an EC(50) value of 5.5+/-0.5 nM. Therefore, this toxin can be used as a template for further development of new insecticides. Based on our findings, an evolutionary relationship between crustaceans and insects is also discussed.