P.N.R. Usherwood

DDT, pyrethrins, pyrethroids and insect sodium channels

The long term use of many insecticides is continually threatened by the ability of insects to evolve resistance mechanisms that render the chemicals ineffective. Such resistance poses a serious threat to insect pest control both in the UK and worldwide. Resistance may result from either an increase in the ability of the insect to detoxify the insecticide or by changes in the target protein with which the insecticide interacts. DDT, the pyrethrins and the synthetic pyrethroids (the latter currently accounting for around 17% of the world insecticide market), act on the voltage‐gated sodium channel proteins found in insect nerve cell membranes. The correct functioning of these channels is essential for normal transmission of nerve impulses and this process is disrupted by binding of the insecticides, leading to paralysis and eventual death. Some insect pest populations have evolved modifications of the sodium channel protein which prevent the binding of the insecticide and result in the insect developing resistance. Here we review some of the work (done at Rothamsted Research and elsewhere) that has led to the identification of specific residues on the sodium channel that may constitute the DDT and pyrethroid binding sites. IUBMB Life, 59: 151‐162, 2007

Spider toxins as tools for dissecting elements of excitatory amino acid transmission

Abstract During millions of years of evolution spiders have refined toxins which either paralyse or kill their prey, but it is becoming increasingly clear that many of these chemicals are neurotoxins which affect prey and non-prey alike. Their widespread action throughout the animal kingdom is hardly surprising given the common properties that characterize the excitable systems of most animals, and because of this neuroscientists have been alerted to the potential of these compounds as probes of central and peripheral nervous function. The vast majority of spider species belong to the Suborder Labidognatha which contains the aerial web-weaving spiders. The black widow and red-back spiders ( Latrodectus spp., Family Theridiidae), which are members of this Suborder, are familiar to most neuroscientists by virtue of the powerful proteinaceous, presynaptic toxins that they manufacture, but these are complex chemicals of high molecular weight which suggests that spider toxins offer few immediate opportunities for the chemical industry. However, the recent discovery of neurotoxins in venoms of orb-web spiders (Family Araneidue, Suborder Labidognatha) of low molecular weight ( 1–4 has dramatically altered this perspective. These toxins are potent antagonists of transmitter receptors, which, because they seemingly exhibit a high specificity for quisqualate-sensitive l-glutamate receptors, are potentially attractive propositions as lead structures for pharmaceutical and pesticide industries.

Block of open channels of recombinant AMPA receptors and native AMPA/kainate receptors by Adamantane derivatives

1 The effects of two adamantane derivatives, 1‐trimethylammonio‐5‐(l‐adamantane‐methylammoniopentane dibromide) (IEM‐1460) and 1‐ammonio‐5‐(l‐adamantane‐methylammoniopentane dibromide) (IEM‐1754) on kainate‐induced currents were studied in Xenopus oocytes expressing recombinant ionotropic glutamate receptors and in freshly isolated neurones from rat hippocampal slices. 2 The adamantane derivatives caused use‐and voltage‐dependent block of open channels of recombinant AMPA receptors. This antagonism was dependent on receptor subunit composition; channels gated by recombinant, homomeric GluRl and GluR3 receptors exhibited a higher sensitivity to block than those gated by receptors containing edited GluR2 subunits. In the former cases, IEM‐1460 had an IC50 of 1.6 μm at a holding potential (Vh) of −80 mV and IEM‐1754 was 3.8 times less potent than IEM‐1460. In contrast, 100 μm IEM‐1460 inhibited responses to 100 μm kainate of receptors containing edited GluR2 subunits by only 7.8 ± 2.4% (n= 5 oocytes) at a Vh of −80 mV. 3 Native AMPA/kainate receptors in isolated hippocampal cells were inhibited by adamantane derivatives in a use‐ and voltage‐dependent manner. This antagonism was dependent on cell type: pyramidal neurones were less sensitive to IEM‐1460 (IC50= 1617 μm at Vh=−80mV) than interneurones (IC50= 1.6 μm at Vh=−80 mV). IEM‐1460 and IEM‐1754 were equipotent when applied to pyramidal neurones, but IEM‐1754 was less potent (∼3 times) than IEM‐1460 when applied to interneurones. 4 It is concluded that the presence of the edited GluR2 subunit in recombinant AMPA receptors and native AMPA/kainate receptors inhibits channel block by organic cations and that adamantane derivatives are potentially valuable tools for identifying classes of AMPA/kainate receptors and their roles in synaptic transmission.

Spider toxins affecting glutamate receptors : polyamines in therapeutic neurochemistry

Polyamine amide toxins obtained from venous of spiders and wasps interact selectively with ionotropic glutamate receptors (GLU-R) of vertebrate central nervous systems. The sites and modes of action of these polyamine amide toxins are reviewed with particular reference to their structure-activity relationships. Qualitatively, their effects on GLU-R are identical to those exerted by polyamines such as spermine, but the polyamine amides are more potent. These compounds (a) potentiate and (b) antagonize GLU-R, the latter arising through open channel block. For the N-methyl-D-aspartate receptor this non-competitive antagonism probably arises through binding of toxin to the Mg2+ site(s) located in the channel gated by this receptor. Similarities and differences between GLU-R in vertebrates and in invertebrates with respect to their interactions with polyamines and polyamine amide toxins are discussed. In both groups the low specificity of these compounds is illustrated by their antagonism at nicotinic acetylcholine receptors in addition to GLU-R. Electrophysiological studies, including those employing Xenopus oocytes, are reviewed and future prospects for the use of polyamine amides in therapy are discussed.

Spermine and philanthotoxin potentiate excitatory amino acid responses of Xenopus oocytes injected with rat and chick brain RNA

The effects of spermine and a synthetic analogue (PhTX-343) of the polyamine amide toxin, delta-philanthotoxin, on the responses of Xenopus oocytes to application of amino acids were examined using voltage clamp. The oocytes were injected with either total rat brain RNA or chick cerebrum, poly(A+)RNA. The responses to N-methyl-D-aspartate and L-kainate were potentiated by low concentrations (10(-11)-10(-7) M) of PhTX-343 and by 10(-5)-10(-4) M spermine. There was variability between oocytes in terms of their responsiveness to these compounds and recovery from their effects was slow and often incomplete. Prolonged or repeated applications of PhTX-343 and spermine eventually resulted in inhibition. Higher concentrations of these compounds always inhibited the responses to acidic amino acids. Low concentrations of PhTX-343 and spermine also potentiated the responses to nicotine and gamma-aminobutyric acid. These results are discussed in terms of the postulated polyamine binding site on the N-methyl-D-aspartate receptor.

Block of locust muscle glutamate receptors by δ-philathotoxin occurs after receptor activations

Abstract One component (δ-philathottoxin (δ-PTX)) of the venom from the wasp ( Philanthus triangulum blocks transmission postsynaptically at excitatory synapses on locust muscle. δ-PTX depresses both the iontophoretic glutamate potential and the excitatory junctional current (e.j.c.) in a glutamate receptor activation-dependent manner. The rate of recovery from the effects of the toxin is reduced following either prolonged application of l -glutamate or repetitive iontophoretic application of this amino acid or high frequency neural stimulation of the muscle in the presence of δ-PTX. The decay phase of the e.j.c. is shortened by δ-PTX. The effects of δ-PTX on the e.j.c. are not voltage dependent. The open-close kinetics of glutamate channels in extrajunctional muscle membrane are modified by δ-PTX as shown by patch clamp analysis. The mean life time of the glutamate channel is reduced, whilst the mean interval between single opening events is increased with the events often occurring in bursts. These data are consistent with glutamate channel blocking by this toxin. It is proposed that the toxin blocks open channels gated by both junctional and extrajunctional glutamate receptors on locust muscle. It is further proposed that δ-PTX enters a compartment of the muscle through the glutamate open channels and that it can also block the open channels from this site.

A comparative study of voltage-gated sodium channels in the Insecta: implications for pyrethroid resistance in Anopheline and other Neopteran species

We report the complete cDNA sequence of the Anopheles gambiae voltage‐gated sodium channel (VGSC) α‐subunit isolated from mature adult mosquitoes. The genomic DNA contains 35 deduced exons with a predicted translation of ≤ 2139 amino acid cDNAs. The transcription of the gene is, however, complex, alternate splicing being evident for at least five optional exons (or exon segments) and two sets of mutually exclusive exons. Overall gene organization was also compared with that of other VGSCs within the Insecta. Several insecticides used in mosquito control (including DDT and synthetic pyrethroids) target the VGSC. Isolation of the sodium channel cDNA for An. gambiae: (1) allows prediction of likely single nucleotide polymorphisms that may arise at residue L1014 to cause resistance to insecticides; (2) defines An. gambiae exon usage in key areas of the VGSC protein that are known (from previous studies in a range of different pest species) to have roles in altering insecticide susceptibility and in generating resistance; and (3) is a critical first step towards development of refined malarial control strategies and of new diagnostics for resistance monitoring.

Mutations in DIIS5 and the DIIS4-S5 linker of Drosophila melanogaster sodium channel define binding domains for pyrethroids and DDT.

Mutations in the DIIS4–S5 linker and DIIS5 have identified hotspots of pyrethroid and DDT interaction with the Drosophila para sodium channel. Wild‐type and mutant channels were expressed in Xenopus oocytes and subjected to voltage‐clamp analysis. Substitutions L914I, M918T, L925I, T929I and C933A decreased deltamethrin potency, M918T, L925I and T929I decreased permethrin potency and T929I, L925I and I936V decreased fenfluthrin potency. DDT potency was unaffected by M918T, but abolished by T929I and reduced by L925I, L932F and I936V, suggesting that DIIS5 contains at least part of the DDT binding domain. The data support a computer model of pyrethroid and DDT binding.

Postsynaptic block of a glutamatergic synapse by low molecular weight fractions of spider venom.

Fractions of low molecular weight (ca. 600-1000 dalton) isolated by high performance liquid chromatography (HPLC) and thin layer chromatography (TLC) from the venoms of the New-World spiders, Argiope trifasciata and Araneus gemma block transmission at glutamatergic nerve-muscle junctions in the locust, Schistocerca gregaria. These fractions are probably small peptides containing phenolic or indolic residues. Their effects on the neurally evoked twitch contraction, the junctional potential to glutamate ionophoresis and the voltage-clamped excitatory postsynaptic current of locust muscle indicate uncompetitive antagonism of the glutamate receptor channel complex through open channel block. In view of their slow reversibility of action they should make useful tools for future biochemical studies of locust glutamate receptors.

Markov, fractal, diffusion, and related models of ion channel gating. A comparison with experimental data from two ion channels

The gating kinetics of single-ion channels are generally modeled in terms of Markov processes with relatively small numbers of channel states. More recently, fractal (Liebovitch et al. 1987. Math. Biosci. 84:37-68) and diffusion (Millhauser et al. 1988. Proc. Natl. Acad. Sci. USA. 85:1502-1507) models of channel gating have been proposed. These models propose the existence of many similar conformational substrates of the channel protein, all of which contribute to the observed gating kinetics. It is important to determine whether or not Markov models provide the most accurate description of channel kinetics if progress is to be made in understanding the molecular events of channel gating. In this study six alternative classes of gating model are tested against experimental single-channel data. The single-channel data employed are from (a) delayed rectifier K+ channels of NG 108-15 cells and (b) locust muscle glutamate receptor channels. The models tested are (a) Markov, (b) fractal, (c) one-dimensional diffusion, (d) three-dimensional diffusion, (e) stretched exponential, and (f) expo-exponential. The models are compared by fitting the predicted distributions of channel open and closed times to those observed experimentally. The models are ranked in order of goodness-of-fit using a boot-strap resampling procedure. The results suggest that Markov models provide a markedly better description of the observed open and closed time distributions for both types of channel. This provides justification for the continued use of Markov models to explore channel gating mechanisms.