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At least 451 records · Page 25Linked to original sources
PubMed2002

Changes in protease activity and Cry3Aa toxin binding in the Colorado potato beetle: implications for insect resistance to Bacillus thuringiensis toxins.

Widespread commercial use of Bacillus thuringiensis Cry toxins to control pest insects has increased the likelihood for development of insect resistance to this entomopathogen. In this study, we investigated protease activity profiles and toxin-binding capacities in the midgut of a strain of Colorado potato beetle (CPB) that has developed resistance to the Cry3Aa toxin of B. thuringiensis subsp. tenebrionis. Histological examination revealed that the structural integrity of the midgut tissue in the toxin-resistant (R) insect was retained whereas the same tissue was devastated by toxin action in the susceptible (S) strain. Function-based activity profiling using zymographic gels showed specific proteolytic bands present in midgut extracts and brush border membrane vesicles (BBMV) of the R strain not apparent in the S strain. Aminopeptidase activity associated with insect midgut was higher in the R strain than in the S strain. Enzymatic processing of toxin did not differ in either strain and, apparently, is not a factor in resistance. BBMV from the R strain bound approximately 60% less toxin than BBMV from the S strain, whereas the kinetics of toxin saturation of BBMV was 30 times less in the R strain than in the S strain. However, homologous competition inhibition binding of (125)I-Cry3Aa to BBMV did not reveal any differences in binding affinity (K(d) approximately 0.1 microM) between the S and R strains. The results indicate that resistance by the CPB to the Cry3Aa toxin correlates with specific alterations in protease activity in the midgut as well as with decreased toxin binding. We believe that these features reflect adaptive responses that render the insect refractory to toxin action, making this insect an ideal model to study host innate responses and adaptive changes brought on by bacterial toxin interaction.

Aminopeptidases↗
PubMed1997

Insect chitinases: molecular biology and potential use as biopesticides.

Chitin, an insoluble structural polysaccharide that occurs in the exoskeletal and gut linings of insects, is a metabolic target of selective pest control agents. One potential biopesticide is the insect molting enzyme, chitinase, which degrades chitin to low molecular weight, soluble and insoluble oligosaccharides. For several years, our laboratories have been characterizing this enzyme and its gene. Most recently, we have been developing chitinase for use as a biopesticide to control insect and also fungal pests. Chitinases have been isolated from the tobacco hornworm, Manduca sexta, and several other insect species, and some of their chemical, physical, and kinetic properties have been determined. Also, cDNA and genomic clones for the chitinase from the hornworm have been isolated and characterized. Transgenic plants that express hornworm chitinase constitutively have been generated and found to exhibit host plant resistance. A transformed entomopathogenic virus that produces the enzyme displayed enhanced insecticidal activity. Chitinase also potentiated the efficacy of the toxin from the microbial insecticide, Bacillus thuringiensis. Insect chitinase and its gene are now available for biopesticidal applications in integrated pest management programs. Current knowledge regarding the molecular biology and biopesticidal action of insect and several other types of chitinases is described in this mini-review.

Amino Acid Sequence↗
PubMed1999

Insect pheromones--an overview of biosynthesis and endocrine regulation.

This overview describes, compares, and attempts to unify major themes related to the biosynthetic pathways and endocrine regulation of insect pheromone production. Rather than developing and dedicating an entirely unique set of enzymes for pheromone biosynthesis, insects appear to have evolved to add one or a few tissue-specific auxiliary or modified enzymes that transform the products of "normal" metabolism to pheromone compounds of high stereochemical and quantitative specificity. This general understanding is derived from research on model species from one exopterygote insect order (Blattodea) and three endopterygote insect orders (Coleoptera, Diptera, and Lepidoptera). For instance, the ketone hydrocarbon contact sex pheromone of the female German cockroach, Blattella germanica, derives its origins from fatty acid biosynthesis, arising from elongation of a methyl-branched fatty acyl-CoA moiety followed by decarboxylation, hydroxylation, and oxidation. Coleopteran sex and aggregation pheromones also arise from modifications of fatty acid biosynthesis or other biosynthetic pathways, such as the isoprenoid pathway (e.g. Cucujidae, Curculionidae, and Scolytidae), or from simple transformations of amino acids or other highly elaborated host precursors (e.g. Scarabaeidae and Scolytidae). Like the sex pheromone of B. germanica, female-produced dipteran (e.g. Drosophilidae and Muscidae) sex pheromone components originate from elongation of fatty acyl-CoA moieties followed by loss of the carbonyl carbon and the formation of the corresponding hydrocarbon. Female-produced lepidopteran sex pheromones are also derived from fatty acids, but many moths utilize a species-specific combination of desaturation and chain-shortening reactions followed by reductive modification of the carbonyl carbon. Carbon skeletons derived from amino acids can also be used as chain initiating units and elongated to lepidopteran pheromones by this pathway (e.g. Arctiidae and Noctuidae). Insects utilize at least three hormonal messengers to regulate pheromone biosynthesis. Blattodean and coleopteran pheromone production is induced by juvenile hormone III (JH III). In the female common house fly, Musca domestica, and possibly other species of Diptera, it appears that during hydrocarbon sex pheromone biosynthesis, ovarian-produced ecdysteroids regulate synthesis by affecting the activities of one or more fatty acyl-CoA elongation enzyme(s) (elongases). Lepidopteran sex pheromone biosynthesis is often mediated by a 33 or 34 amino acid pheromone biosynthesis activating neuropeptide (PBAN) through alteration of enzyme activities at one or more steps prior to or during fatty acid synthesis or during modification of the carbonyl group. Although a molecular level understanding of the regulation of insect pheromone biosynthesis is in its infancy, in the male California fivespined ips, Ips paraconfusus (Coleoptera: Scolytidae), JH III acts at the transcriptional level by increasing the abundance of mRNA for 3-hydroxy-3-methylglutaryl-CoA reductase, a key enzyme in de novo isoprenoid aggregation pheromone biosynthesis.

Amino Acid Sequence↗
PubMed2003

Cytotoxicity against insect cells of entomopathogenic fungi of the genera Hypocrella (anamorph Aschersonia): possible agents for biological control.

Extracts from entomopathogenic fungi of the genus Hypocrella (7 species) and its anamorph Aschersonia (11 species) were screened for cytotoxicity to Sf9 and C6/36 insect cells and L929, BHK(21)C13 and HepG2 mammalian cells. Cytotoxic extracts to insect cells (ID50's < or = 10 microg ml(-1)) but not to mammalian cells (ID50's > or = 10 microg ml(-1)) conformed to the criteria of the project and were considered 'lead' extracts for further investigation. 'Leads' were found in two of the Hypocrella species: H. discoidea, and H. tamurai and in three of the Aschersonia species: A. samoensis, A. badia, and A. tamurai. Bioassay-guided fractionation of the cell extract of the fungus A. samoensis BCC 1393 led to the identification of two known anthraquinone dimers, (+)rugulosin (1) and skyrin (2) which showed selective toxicity towards insect cells. (+)Rugulosin (1) and skyrin (2) exhibited strong cytotoxic activity against the insect cell line Sf9 with respective ID50 values of 1.2 and 9.6 microg ml(-1), but showed weak activity toward mammalian cells. This first report of (+)rugulosin (1) and skyrin (2) in A. samoensis is confirmed and demonstrated in another four strains of A. samoensis isolated in Thailand. The preferential cytotoxicity against Sf9 insect cells gives evidence that these insect-pathogenic fungi of the Hypocrella/Aschersonia group might be useful as an agent for pest control.

Aedes↗
PubMed1992

Localization of receptor sites for insect-selective toxins on sodium channels by site-directed antibodies.

Site-directed antibodies corresponding to conserved putative extracellular segments of sodium channels, coupled with binding studies of radiolabeled insect-selective scorpion neurotoxins, were employed to clarify the relationship between the toxins' receptor sites and the insect sodium channel. (1) The depressant insect toxin LqhIT2 was shown to possess two noninteracting binding sites in locust neuronal membranes: a high-affinity (KD1 = 0.9 +/- 0.6 nM) and low-capacity (Bmax1 = 0.1 +/- 0.07 pmol/mg) binding site as well as a low-affinity (KD2 = 185 +/- 13 nM) and high-capacity (Bmax2 = 10.0 +/- 0.6 pmol/mg) binding site. (2) The high-affinity site serves as a target for binding competition by the excitatory insect toxin AaIT. (3) The binding of LqhIT2 was significantly inhibited in a dose-dependent manner by each of four site-directed antibodies. The binding inhibition resulted from reduction in the number of binding sites. (4) The antibody-mediated inhibition of [125I]AaIT binding differs from that of LqhIT2: three out of the four antibodies which inhibited LqhIT2 binding only partially affected AaIT binding. Two antibodies, one corresponding to extracellular and one to intracellular segments of the channel, did not affect the binding of either toxin. These data suggest that the receptors to the depressant and excitatory insect toxins (a) comprise an integral part of the insect sodium channel, (b) are formed by segments of external loops in domains I, III, and IV of the sodium channel, and (c) are localized in close proximity but are not identical in spite of the competitive interaction between these toxins.

Animals↗
PubMed1990

A scorpion venom neurotoxin paralytic to insects that affects sodium current inactivation: purification, primary structure, and mode of action.

A new toxin, Lqh alpha IT, which caused a unique mode of paralysis of blowfly larvae, was purified from the venom of the scorpion Leiurus quinquestriatus hebraeus, and its structural and pharmacological properties were compared to those of three other groups of neurotoxins found in Buthinae scorpion venoms. Like the excitatory and depressant insect-selective neurotoxins, Lqh alpha IT was highly toxic to insects, but it differed from these toxins in two important characteristics: (a) Lqh alpha IT lacked strict selectivity for insects; it was highly toxic to crustaceans and had a measurable but low toxicity to mice. (b) It did not displace an excitatory insect toxin, 125I-AaIT, from its binding sites in the insect neuronal membrane; this indicates that the binding sites for Lqh alpha IT are different from those shared by the excitatory and depressant toxins. However, in its primary structure and its effect on excitable tissues, Lqh alpha IT strongly resembled the well-characterized alpha scorpion toxins, which affect mammals. The amino acid sequence was identical with alpha toxin sequences in 55%-75% of positions. This degree of similarity is comparable to that seen among the alpha toxins themselves. Voltage- and current-clamp studies showed that Lqh alpha IT caused an extreme prolongation of the action potential in both cockroach giant axon and rat skeletal muscle preparations as a result of the slowing and incomplete inactivation of the sodium currents. These observations indicate that Lqh alpha IT is an alpha toxin which acts on insect sodium channels.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2003

Native fluorescence from juvenile stages of common food storage insects.

Insect infestation of stored foods has significant economic and health consequences; the development of novel methods of detection thus presents considerable opportunities. The fluorescence from nine species of storage insects (beetles and moths) was studied; the juvenile stages of all nine species exhibited fluorescence under long-wave (365 nm) UV light; none of the adult insects emit fluorescence, so the fluorophore(s) might be a compound(s) associated with the unsclerotized cuticle. The spectra of larval stages of Ephestia kuehniella, Oryzaephilus surinamensis, Corcyra cepahlonica, Tribolium castaneum, and Tribolium confusum exhibited excitation maxima in the range from 345 to 350 nm and emission maxima in the range from 421 to 427 nm, suggesting that fluorescence arises from a common chromophore; similarities in fluorescence properties implicate one of the many pteridine ring-containing compounds (pterins) commonly found in insects. Larvae and even eggs were readily imaged on foods using fluorescence under 365-nm excitation. Fluorescence thus appears to be ubiquitous in immature food storage insects, and fluorescence detection may be useful as a general method to detect insects in foods and agricultural commodities during storage or processing.

Animals↗
PubMed2003

A novel medium for expression of proteins selectively labeled with 15N-amino acids in Spodoptera frugiperda (Sf9) insect cells.

Whereas bacterial expression systems are widely used for production of uniformly or selectively (15)N-labeled proteins the usage of the baculovirus expression system for labeling is limited to very few examples in the literature. Here we present the complete formulations of the two insect media, IML406 and 455, for the high-yield production of selectively (15)N-labeled proteins in insect cells. The quantities of (15)N-amino acids utilized in the production of labeled GST were similar in the case of bacterial and viral expression. For the most studied amino acids essential for insect cells the (15)N-HSQC spectra, recorded with GST labeled in insect cells, showed no cross labeling and provided therefore spectra of better quality compared to NMR spectra of GST expressed in E. coli. Also in the case of amino acids not essential for Sf9 cells we were able to label a defined number of amino acid species. Therefore the selective labeling using the baculovirus expression vector system represents a complement or even an alternative to the bacterial expression system. Based on these findings we can provide a first simple overview of the network of the amino acid metabolism in E. coli and insect cells focused on nitrogen. For some amino acids the expression of labeled proteins in insect cells can replace the cell-free protein expression.

Amino Acid Sequence↗
PubMed2005

Insects breathe discontinuously to avoid oxygen toxicity.

The respiratory organs of terrestrial insects consist of tracheal tubes with external spiracular valves that control gas exchange. Despite their relatively high metabolic rate, many insects have highly discontinuous patterns of gas exchange, including long periods when the spiracles are fully closed. Two explanations have previously been put forward to explain this behaviour: first, that this pattern serves to reduce respiratory water loss, and second, that the pattern may have initially evolved in underground insects as a way of dealing with hypoxic or hypercapnic conditions. Here we propose a third possible explanation based on the idea that oxygen is necessary for oxidative metabolism but also acts as a toxic chemical that can cause oxidative damage of tissues even at relatively low concentrations. At physiologically normal partial pressures of CO2, the rate of CO2 diffusion out of the insect respiratory system is slower than the rate of O2 entry; this leads to a build-up of intratracheal CO2. The spiracles must therefore be opened at intervals to rid the insect of accumulated CO2, a process that exposes the tissues to dangerously high levels of O2. We suggest that the cyclical pattern of open and closed spiracles observed in resting insects is a necessary consequence of the need to rid the respiratory system of accumulated CO2, followed by the need to reduce oxygen toxicity.

Animals↗
PubMed2002

Recombinant scorpion insectotoxin AaIT kills specifically insect cells but not human cells.

The nucleotide sequence deduced from the amino acid sequence of the scorpion insectotoxin AaIT was chemically synthesized and was expressed in Escherichia coli. The authenticity of this in vitro expressed peptide was confirmed by N-terminal peptide sequencing. Two groups of bioassays, artificial diet incorporation assay and contact insecticidal effect assay, were carried out separately to verify the toxicity of this recombinant toxin. At the end of a 24 h experimental period, more than 60% of the testing diamondback moth (Plutella xylostella) larvae were killed in both groups with LC50 value of 18.4 microM and 0.70 microM respectively. Cytotoxicity assay using cultured Sf9 insect cells and MCF-7 human cells demonstrated that the toxin AaIT had specific toxicity against insect cells but not human cells. Only 0.13 microM recombinant toxin was needed to kill 50% of cultured insect cells while as much as 1.3 microM toxin had absolutely no effect on human cells. Insect cells produced obvious intrusions from their plasma membrane before broken up. We infer that toxin AaIT bind to a putative sodium channel in these insect cells and open the channel persistently, which would result in Na+ influx and finally cause destruction of insect cells.

Amino Acid Sequence↗
PubMed2000

Detection and distribution patterns of telomerase activity in insects.

Telomeres of most insects consist of pentanucleotide (TTAGG)n repeats, although the repeats are absent in Diptera and some other insect species, where the telomere regions are perhaps maintained without telomerase. To understand various and unusual telomere formation in insects, we have studied the characteristic features of a putative insect telomerase that has not been previously described. Using a modified telomeric repeat amplification protocol (TRAP), we first detected the telomerase activity in crickets, cockroaches and two Lepidopteran insects. The telomerase from crickets and cockroaches required dATP, dGTP and dTTP but not dCTP as a substrate and sequence analyses of the products of TRAP revealed that the (TTAGG)n repeats are synthesized by telomerase. The cockroach telomerase was detected both in somatic (fat body, muscle and neural tissues) and germ line (testis) cells, suggesting that expression of this enzyme is not regulated in a tissue-specific manner at an adult stage. While we detected high levels of telomerase activity in crickets and cockroaches, we could not detect activity in all tissues and cell cultures of the silkworm, Bombyx mori and in two Drosophila and one Sarcophaga cell lines. This supports the theory that Dipteran insects maintain their telomeres without telomerase.

Animals↗
PubMed2004

Insect aquaplaning: Nepenthes pitcher plants capture prey with the peristome, a fully wettable water-lubricated anisotropic surface.

Pitcher plants of the genus Nepenthes have highly specialized leaves adapted to attract, capture, retain, and digest arthropod prey. Several mechanisms have been proposed for the capture of insects, ranging from slippery epicuticular wax crystals to downward-pointing lunate cells and alkaloid secretions that anesthetize insects. Here we report that perhaps the most important capture mechanism has thus far remained overlooked. It is based on special surface properties of the pitcher rim (peristome) and insect "aquaplaning." The peristome is characterized by a regular microstructure with radial ridges of smooth overlapping epidermal cells, which form a series of steps toward the pitcher inside. This surface is completely wettable by nectar secreted at the inner margin of the peristome and by rain water, so that homogenous liquid films cover the surface under humid weather conditions. Only when wet, the peristome surface is slippery for insects, so that most ant visitors become trapped. By measuring friction forces of weaver ants (Oecophylla smaragdina) on the peristome surface of Nepenthes bicalcarata, we demonstrate that the two factors preventing insect attachment to the peristome, i.e., water lubrication and anisotropic surface topography, are effective against different attachment structures of the insect tarsus. Peristome water films disrupt attachment only for the soft adhesive pads but not for the claws, whereas surface topography leads to anisotropic friction only for the claws but not for the adhesive pads. Experiments on Nepenthes alata show that the trapping mechanism of the peristome is also essential in Nepenthes species with waxy inner pitcher walls.

Animals↗
PubMed2002

A single Photorhabdus gene, makes caterpillars floppy (mcf), allows Escherichia coli to persist within and kill insects.

Photorhabdus luminescens, a bacterium with alternate pathogenic and symbiotic phases of its lifestyle, represents a source of novel genes associated with both virulence and symbiosis. This entomopathogen lives in a "symbiosis of pathogens" with nematodes that invade insects. Thus the bacteria are symbiotic with entomopathogenic nematodes but become pathogenic on release from the nematode into the insect blood system. Within the insect, the bacteria need to both avoid the peptide- and cellular- (hemocyte) mediated immune response and also to kill the host, which then acts as a reservoir for bacterial and nematode reproduction. However, the mechanisms whereby Photorhabdus evades the insect immune system and kills the host are unclear. Here we show that a single large Photorhabdus gene, makes caterpillars floppy (mcf), is sufficient to allow Esherichia coli both to persist within and kill an insect. The predicted high molecular weight Mcf toxin has little similarity to other known protein sequences but carries a BH3 domain and triggers apoptosis in both insect hemocytes and the midgut epithelium.

Amino Acid Sequence↗
PubMed1995

Activation of mammalian retinoid X receptors by the insect growth regulator methoprene.

We report that methoprene and its derivatives can stimulate gene transcription in vertebrates by acting through the retinoic acid-responsive transcription factors, the retinoid X receptors (RXRs). Methoprene is an insect growth regulator in domestic and agricultural use as a pesticide. At least one metabolite of methoprene, methoprene acid, directly binds to RXR and is a transcriptional activator in both insect and mammalian cells. Unlike the endogenous RXR ligand, 9-cis-retinoic acid, this activity is RXR-specific; the methoprene derivatives do not activate the retinoic acid receptor pathway. Methoprene is a juvenile hormone analog that acts to retain juvenile characteristics during insect growth, preventing metamorphosis into an adult, and it has been shown to have ovicidal properties in some insects. Thus, a pesticide that mimics the action of juvenile hormone in insects can also activate a mammalian retinoid-responsive pathway. This finding provides a basis through which the potential bioactivity of substances exposed to the environment may be reexamined and points the way for discovery of new receptor ligands in both insects and vertebrates.

Animals↗
PubMed1996

Differential fatty acid selection during biosynthetic S-acylation of a transmembrane protein (HEF) and other proteins in insect cells (Sf9) and in mammalian cells (CV1).

The transmembrane glycoprotein HEF and its acylation deficient mutant M1 were expressed in Sf9 insect cells infected with recombinant baculovirus and in CV1 mammalian cells using the vaccinia T7 system. In insect cells (Sf9), both wild type HEF and HEF(M1) are synthesized in their precursor form HEF0, which appears as a double band in SDS gels. Digestion with glycopeptidase F and endoglycosidase H reveals that the larger 84-kDa form is modified by the attachment of unprocessed carbohydrates of the high mannose type whereas the smaller 76-kDa form is non-glycosylated. As revealed by in vitro labeling experiments with palmitic acid another modification of HEF is the attachment of a long chain fatty acid to cysteine residue Cys-652 which is located at the internal border of the cytoplasmic membrane. After labeling with [3H]palmitic acid in both systems only HEF(WT) is acylated, whereas HEF(M1) is not. High performance liquid chromatography analysis of the fatty acids bound to HEF(WT) expressed in Sf9 insect cells reveals nearly 80% of palmitic acid. In contrast to this finding, the acylation pattern of HEF expressed in CV1 cells shows nearly the same amounts of stearic and palmitic acid (40%). Since the interconversion of the input [3H]palmitic acid to stearic acid is even lower in CV1 cells than in insect cells, it follows that only HEF expressed in mammalian, but not in insect cells selects for stearic acid during its biosynthetic acylation. We extended our study to acylation of endogenous proteins in Sf9 cells. In finding only palmitate linked to protein we present evidence that, in contrast to mammalian cells, insect cells (Sf9) cannot transfer stearic acid to polypeptide. This finding favors the hypothesis of enzymatic acylation over non-enzymatic mechanisms of acyl transfer to protein.

Acylation↗
PubMed2001

Sialylation of N-glycans on the recombinant proteins expressed by a baculovirus-insect cell system under beta-N-acetylglucosaminidase inhibition.

We investigated the ability of a baculovirus-insect cell system to produce sialylated glycoproteins. Despite the presence of enzymes for synthesizing complex-type N-glycans, the most frequent structure of insect N-glycan is the paucimannosidic type, Man(3)GlcNAc(2)(+/-Fuc). The reason for the overwhelming assembly of paucimannosidic N-glycans is not yet well understood. We hypothesized that this predominance might be due to insect-specific, Golgi-associated beta-N-acetylglucosaminidase (GlcNAcase)-mediated removal of N-acetylglucosamine residues from the precursor N-glycan, thereby preventing its galactosylation and terminal sialylation. As we expected, the suppression of intrinsic GlcNAcase activity with a specific inhibitor, 2-acetamido-1,2-dideoxynojirimycin, allowed the accumulation of sialylated glycoproteins in the supernatants of insect cell cultures after baculoviral infection. Our observation indicates that GlcNAcase-dependent depletion of N-acetylglucosamine residues from intermediate N-glycans is critical for the assembly of paucimannosidic N-glycans in insect cells and, more importantly, that insect cells (under specific conditions) retain the ability to construct sialylated N-glycans like those in mammalian cells.

Acetylglucosaminidase↗
PubMed2004

A novel secreted protein toxin from the insect pathogenic bacterium Xenorhabdus nematophila.

The bacterium Xenorhabdus nematophila is an insect pathogen that produces several proteins that enable it to kill insects. Screening of a cosmid library constructed from X. nematophila strain A24 identified a gene that encoded a novel protein that was toxic to insects. The 42-kDa protein encoded by the toxin gene was expressed and purified from a recombinant system, and was shown to kill the larvae of insects such as Galleria mellonella and Helicoverpa armigera when injected at doses of around 30-40 ng/g larvae. Sequencing and bioinformatic analysis suggested that the toxin was a novel protein, and that it was likely to be part of a genomic island involved in pathogenicity. When the native bacteria were grown under laboratory conditions, a soluble form of the 42-kDa toxin was secreted only by bacteria in the phase II state. Preliminary histological analysis of larvae injected with recombinant protein suggested that the toxin primarily acted on the midgut of the insect. Finally, some of the common strategies used by the bacterial pathogens of insects, animals, and plants are discussed.

Amino Acid Sequence↗
PubMed2004

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.

Amino Acid Substitution↗