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.     

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.     

Functional duality and structural uniqueness of depressant insect-selective neurotoxins

Depressant insect-selective neurotoxins derived from scorpion venoms (a) induce in blowfly larvae a short, transient phase of contraction similar to that induced by excitatory neurotoxins followed by a prolonged flaccid paralysis and (b) displace excitatory toxins from their binding sites on insect neuronal membranes. The present study was undertaken in order to examine the basis of these similarities by comparing the primary structures and neuromuscular effects of depressant and excitatory toxins. A new depressant toxin (LqhIT2) was purified from the venom of the Israeli yellow scorpion. The effects of this toxin on a prepupal housefly neuromuscular preparation mimic the effects on the intact animal; i.e., a brief period of repetitive bursts of junction potentials is followed by suppression of their amplitude and finally by a block of neuromuscular transmission. Loose patch clamp recordings indicate that the repetitive activity has a presynaptic origin in the motor nerve and closely resembles the effect of the excitatory toxin AaIT. The final synaptic block is attributed to neuronal membrane depolarization, which results in an increase in spontaneous transmitter release; this effect is not induced by excitatory toxin. The amino acid sequences of three depressant toxins were determined by automatic Edman degradation. The depressant toxins comprise a well-defined family of polypeptides with a high degree of sequence conservation. This group differs considerably in primary structure from the excitatory toxin, with which it shares identical or related binding sites, and from the two groups of scorpion toxins that affect sodium conductance in mammals. The two opposing pharmacological effects of depressant toxins are discussed in light of the above data.     

Spider toxins selectively block calcium currents in Drosophila

Toxins from spider venom, originally purified for their ability to block synaptic transmission in Drosophila, are potent and specific blockers of Ca2+ currents measured in cultured embryonic Drosophila neurons using the whole-cell, patch-clamp technique. Differential actions of toxins from two species of spiders indicate that different types of Drosophila neuronal Ca2+ currents can be pharmacologically distinguished. Hololena toxin preferentially blocks a non-inactivating component of the current, whereas Plectreurys toxin blocks both inactivating and non-inactivating components. These results suggest that block of a non-inactivating Ca2+ current is sufficient to block neurotransmitter release at Drosophila neuromuscular junction.     

Synthesis and biological characterization of a series of analogues of ω-conotoxin gvia

The 27-residue polypeptide ω-conotoxin GVIA (ω-CgTx), from the venom of the cone shell Conus geographus, blocks N-type neuronal calcium channels. It contains three disulphide bridges. We reporte here the synthesis and biological characterization of a seires of analogues in which one disulphide has been replaced by substitution of appropriate Cys residues with Ser, viz. [Ser^1,16]-ω-CgTx, [Ser^8,19]-ω-CgTx, [Ser^15,26]-ω-CgTx, [Ser¹⁶]-ω-CgTx_8-27 and [Ser¹⁵]-ω-CgTx_1-19. All syntheses were conducted manually using either Boc or Fmoc methodology. Deprotected peptides were oxidized to their bridged forms using either aerial oxidation or aqueous dimethyl sulphoxide. Peptides were purified using RP-HPLC, and their purity and identity were checked by RP-HPLC, capillary electrophoresis and mass spectrometry. Inhibition of neuronal N-type calcium channels was assessed as the inhibition of the twitch responses of rat vas deferens stimualted with single electrical pulses at 20 second intervals. None of these analogues was biologically active, suggesting that the disulphides play an important role in maintaining biological activity.     

Action of different toxins from the scorpion Androctonus australis on a locust nerve-muscle preparation

The neuromuscular effects of four purified toxins and crude venom from the scorpion Androctonus australis were investigated in the extensor tibiae nerve-muscle preparation of the locust Locusta migratoria. Insect and crustacean toxin and the mammal toxins I and II which have previously been shown to act on fly larvae, isopods, and mice all paralyse locust larvae. The paralytic potencies decrease in the following order: insect toxin → mammal toxin I → crustacean toxin → mammal toxin II.The toxins and crude venom cause repetitive activity of the motor axons. This leads to long spontaneous trains of junction potentials in the case of crude venom and insect toxin. The other toxins chiefly cause short bursts of action and junction potentials following single stimuli.The ‘slow’ excitatory motor axon invariably is affected sooner than the inhibitory or the ‘fast’ excitatory one. The minimal doses of toxins required to affect the ‘slow’ motor axon decrease in an order somewhat different from that established for their paralytic potencies: insect toxin → crustacean toxin → mammal toxin I → mammal toxin II.Crude venom depolarises and destabilises the muscle membrane potential at low doses. At high doses it decreases the membrane resistance, whereas insect toxin leads to an increase.Crude venom and insect toxin enhance the frequency of mejps, whereas mammal toxin I leads to the occurrence of ‘giant’ mejps.The pattern of axonal activities indicates that the various peripheral branches of the motor nerve are the primary target of the toxins.The time course of nerve action potentials is affected by mammal toxin I and crustacean toxin which cause anomalous shapes and prolongations not caused by insect toxin.The results with other animals suggest that only the insect toxin is selective in its activity. The way it affects the axon might be quite different from that previously reported for scorpion venoms or toxins.     

受体介导的鳞翅目昆虫对Bt毒素抗性机制进展

苏云金芽孢杆菌作为一种对人畜安全、环境友好型绿色杀虫剂在全球被广泛使用。Bt毒素与昆虫中肠上特定毒素受体结合并发挥作用,形成毒素穿孔导致昆虫死亡是其重要的杀虫机制之一,靶标害虫对Bt毒素产生抗性是制约转Bt作物长期有效种植和Bt毒素持续使用的重要因素。文中从鳞翅目昆虫中肠细胞Bt毒素重要受体的研究阐述昆虫对Bt的抗性机制,为Bt抗性机制的深入研究和对害虫的防控与治理提供了一定的理论参考。     

Neosurugatoxin blocks an α-bungarotoxin-sensitive neuronal nicotinic acetylcholine receptor

Neosurugatoxin (NSTX), a neurotoxin isolated from the Japanese ivory mollusc Babylonia japonica, is a potent neuronal nicotinic acetylcholine receptor (nAChR) antagonist. Hitherto, NSTX has been found to block only neuronal nAChRs that are insensitive to α-Bgt. Here, we report for the first time that NSTX blocks an α-Bgt-sensitive nAChR on an identified insect motor neurone. Bath-applied NSTX at a concentration of 10 nM and above reversibly blocks the nicotine-induced depolarizations recorded from the cockroach (Periplaneta americana) fast coxal depressor motor neurone (D_f) and is without effect on GABA-induced responses detected on the same cell. NSTX is among the most potent blockers tested to date on nAChRs of motor neurone D_f. © 1993 Wiley-Liss, Inc.     

Activity spectra of Bacillus Thuringiensis δ-endotoxins against eight insect cell lines

Summary Eight continuous insect cell lines were tested for susceptibility to the δ-endotoxins of several lepidopteran-active strains and cloned-gene products of Bacillus thuringiensis. The assays were performed on cells suspended in agarose gel, which allowed the toxins activated at pH 10.5 to be applied directly in a high-pH buffer without causing solvent toxicity to the cells. The responses of the cell lines to the various toxins produced activity spectra that were used to identify functionally similar and dissimilar toxin proteins. IPRI-CF-1 and FPMI-MS-5, derived from neonate larvae of Choristoneura fumiferana and Manduca sexta, respectively, exhibited the greatest sensitivity to the toxins tested, whereas B. thuringiensis subsp. entomocidus had the broadest in vitro host range. Analysis of activity spectra led to the identification of the particular Cry protein that was responsible for the broad toxicity of this subspecies and demonstrated a distinct difference in toxin composition between two strains of subsp. sotto. The identical spectra observed for subsp. kurstaki HD-1 and NRD-12 is consistent with insect bioassay data obtained previously by other workers and supports the conclusion that there is virtually no difference in activity between these two strains. The in vitro assay system, referred to as the “lawn assay” and used to test B. thuringiensis activated toxins against insect cell lines, is particularly useful in mode-of-action studies and as a rapid, preliminary test for the presence of specific cytolytic proteins, rather than as a method for screening toxins of wild-type strains for insecticidal activity. The response of cells in vitro to B. thuringiensis toxins is often very different from that of the insect from which the cells were derived.     

Discovery and structure of a potent and highly specific blocker of insect calcium channels

We have isolated a novel family of insect-selective neurotoxins that appear to be the most potent blockers of insect voltage-gated calcium channels reported to date. These toxins display exceptional phylogenetic specificity, with at least a 10,000-fold preference for insect versus vertebrate calcium channels. The structure of one of the toxins reveals a highly structured, disulfide-rich core and a structurally disordered C-terminal extension that is essential for channel blocking activity. Weak structural/functional homology with omega-agatoxin-IVA/B, the prototypic inhibitor of vertebrate P-type calcium channels, suggests that these two toxin families might share a similar mechanism of action despite their vastly different phylogenetic specificities.     

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.     

NGP1-01 is a Brain-permeable Dual Blocker of Neuronal Voltage- and Ligand-operated Calcium Channels

Calcium overload of neurons leads to cell death and is a key feature in neurodegenerative diseases. The polycyclic amine NGP1-01 blocks L-type voltage operated calcium channels in cardiomyocytes. Here, we tested whether NGP1-01 blocks neuronal calcium channels. NGP1-01 (1 μM) inhibited depolarization-induced calcium influx by 78% in cortical neurons preloaded with fura-2 AM, with a potency similar to nimodipine. NGP1-01 (1 μM) also inhibited N-methyl-d-aspartate (NMDA)-induced (1 mM) calcium influx by 52%, only slightly less potent than memantine. Using in vivo-microdialysis, we monitored choline release during NMDA infusion as a measure of excitotoxic membrane breakdown. Intraperitoneal injection of NGP1-01 (40 mg/kg) reduced NMDA-induced membrane breakdown by 31% (P<0.01) while memantine (10 mg/kg) reduced choline release by 40%. Our results demonstrate that NGP1-01 simultaneously blocks both major neuronal calcium channels and is sufficiently brain-permeable. We conclude that NGP1-01 is a promising lead structure for a new class of dual-mechanism neuroprotective agents.     

Identification of a neuronal calcium sensor (NCS-1) possibly involved in the regulation of receptor phosphorylation

Abstract

Persistent stimulation of G protein-coupled receptors by agonists leads rapidly to reduced responses, a phenomenon described as desensitization. It involves primarily the phosphorylation of receptor sites by specific kinases of the G protein-coupled receptor kinase (GRK) family. The β-adrenergic receptor kinase 1 (GRK2) desensitizes agonist-activated β₂-adrenergic receptors, whereas rhodopsin kinase (GRK1) phosphorylates and inactivates photon-activated rhodopsin. Little is known about the role of calcium in desensitization. Here we report the characterization of a novel neuronal calcium sensor (NCS) named NCS-1 possibly involved in the regulation of receptor phosphorylation. NCS-1 is a new member of the EF-hand superfamily, which includes calmodulin, troponin C, parvalbumin, and recoverins. By Northern analysis and in situ hybridization, we discovered that NCS-1 is specifically expressed in the central and peripheral nervous systems. Chick NCS-1 has 72% of amino acid identity with Drosophila frequenin, a protein found in the nervous system and at the motor nerve terminals of neuromuscular junctions. By analogy with the reported function for two other members of the NCS family, we discuss whether G protein-coupled receptors or GRKs are the targets of neuronal calcium sensors.     

delta-Philanthotoxin, a semi-irreversible blocker of ion-channels

1. The digger wasp, Philanthus triangulum, which preys on honeybees, produces a paralysing venom possessing a wide variety of activities. 2. In insects, the venom has a central as well as a peripheral effect; the latter effect consists of a presynaptic as well as a postsynaptic block of the skeletal neuromuscular transmission. 3. The presynaptic block is probably caused by an inhibition of the re-uptake of the transmitter. The postsynaptic effect probably consists of a block of open ion channels. 4. The venom contains at least four active toxins called alpha-, beta-, gamma- and delta-philanthotoxin (PTX). alpha-PTX blocks transmission in the cockroach CNS. The other three toxins block neuromuscular transmission. delta-PTX being the most active toxin in blocking glutamate evoked postsynaptic depolarizations. 5. In the junctional, as well as in the extrajunctional, muscle fibre membrane delta-PTX blocks ion channels in a use-dependent manner. Once the channel has been blocked, unblocking seems to be channels in a use-dependent manner. Once the channel has been blocked, unblocking seems to be semi-irreversible when agonist activation is low (spontaneous release of transmitter and/or leak of glutamate from the pipette). 6. The time constant of blocking is roughly estimated to be in the order of 10 msec, that of unblocking seems to be several hundreds of msec.     

Successful refolding and NMR structure of rMagi3: A disulfide‐rich insecticidal spider toxin

The need for molecules with high specificity against noxious insects leads the search towards spider venoms that have evolved highly selective toxins for insect preys. In this respect, spiders as a highly diversified group of almost exclusive insect predators appear to possess infinite potential for the discovery of novel insect‐selective toxins. In 2003, a group of toxins was isolated from the spider Macrothele gigas and the amino acid sequence was reported. We obtained, by molecular biology techniques in a heterologous system, one of these toxins. Purification process was optimized by chromatographic methods to determine the three‐dimensional structure by nuclear magnetic resonance in solution, and, finally, their biological activity was tested. rMagi3 resulted to be a specific insect toxin with no effect on mice.     

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.     

Curtatoxins. Neurotoxic insecticidal polypeptides isolated from the funnel-web spider Hololena curta

Three polypeptide neurotoxins (curtatoxins) were isolated from the venom of the spider Hololena curta by reverse-phase high performance liquid chromatography, gel permeation, and ion-exchange chromatography. The purified toxins induced an immediate paralysis in the cricket Acheta domestica that resulted in desiccation and death of the insect within 24-48 h (LD50 congruent to 4-20 micrograms/g); this toxic effect is consistent with irreversible presynaptic neuromuscular blockade. Curtatoxins are a class of sequence-related, cysteine-rich, carboxyl-terminal amidated polypeptides of 36 to 38 amino acid residues. The cysteine residues are conserved at identical sequence positions among these polypeptides and form 4 intramolecular disulfide bonds. Hydropathy calculations show that the toxins have an internal hydrophobic region flanked by hydrophilic and oppositely charged amino- and carboxyl-terminal ends. By analogy to other cysteine-rich arthropod venom proteins, the folded structure of the curtatoxins is likely important for their target specificity and mode of action at the neuromuscular junction.     

Spider Venom Toxin Protects Plants from Insect Attack

Many of the toxin proteins, that have been heterogeneously expressed in agricultural crops to provide resistance to insect pests, are too specific or are only mildly effective against the major insect pests. Spider venoms are a complex cocktail of toxins that have evolved specifically to kill insects. Here we show that the ω-ACTX-Hv1a toxin (Hvt), a component of the venom of the Australian funnel web spider (Hadronyche versuta) that is a calcium channel antagonist, retains its biological activity when expressed in a heterologous system. Expressed as a fusion protein in E. coli, the purified toxin fusion immobilized and killed Helicoverpa armigera and Spodoptera littoralis caterpillars when applied topically. Transgenic expression of Hvt in tobacco effectively protected the plants from H. armigera and S. littoralis larvae, with 100% mortality within 48 h. We conclude that the Hvt is an attractive and effective molecule for the transgenic protection of plants from herbivorous insects which should be evaluated further for possible application in agriculture. The authors Sher Afzal Khan and Zahid Mukhtar contributed equally to this work.     

Characterization of Bacillus thuringiensis Cry toxin binding novel GPI anchored aminopeptidase from fat body of the moth Spodoptera litura

Aminopeptidase N (APN) isoforms were identified as candidate receptors for Bacillus thuringiensis Cry toxins from the midgut of several insect species. In this study a partial cDNA encoding aminopeptidase (slfbAPN) was cloned from fat body of the moth Spodoptera litura. In the deduced amino acid sequence the characteristic metallopeptidase sequences, HEXXHX₁₈E and GAMENWG were conserved but the sequence showed only 33–39% identity to other insect APNs, which were also reported to be Cry toxin receptors. The presence of a putative GPI anchor signal sequence at the C-terminus indicated that it is a membrane-anchored protein. The slfbAPN expression was restricted to the fat body as suggested by northern blot analysis of different tissues. Biochemical analyses including immunoblotting, ligand blotting and lectin blotting, demonstrated that slfbAPN is a membrane-anchored glycoprotein in the fat body and it binds to Cry toxins. The nucleotide sequence shown here has been submitted to the GenBank sequence data bank and is available under accession number EF372603.     

Evolution of Bacillus thuringiensis Cry toxins insecticidal activity

Insecticidal Cry proteins produced by Bacillus thuringiensis are use worldwide in transgenic crops for efficient pest control. Among the family of Cry toxins, the three domain Cry family is the better characterized regarding their natural evolution leading to a large number of Cry proteins with similar structure, mode of action but different insect specificity. Also, this group is the better characterized regarding the study of their mode of action and the molecular basis of insect specificity. In this review we discuss how Cry toxins have evolved insect specificity in nature and analyse several cases of improvement of Cry toxin action by genetic engineering, some of these examples are currently used in transgenic crops. We believe that the success in the improvement of insecticidal activity by genetic evolution of Cry toxins will depend on the knowledge of the rate‐limiting steps of Cry toxicity in different insect pests, the mapping of the specificity binding regions in the Cry toxins, as well as the improvement of mutagenesis strategies and selection procedures.