Kinetics of sodium channel modification as the basis for the variation in the nerve membrane effects of pyrethroids and DDT analogs

The effects of a wide range of pyrethroids and DDT analogs on the membrane potential and membrane sodium currents were studied in crayfish giant axons. Compounds differed greatly in their ability to produce depolarizing afterpotentials, repetitive firing, and membrane depolarization. The differences observed at the membrane potential level could be explained by differences in the kinetics with which the insecticides interact with the nerve membrane sodium channel. The compounds containing a cyano group at the α position retain sodium channels in a modified open state persistently, depolarize the membrane, and block the action potential without causing repetitive firing. The pyrethroids without an α-cyano group and DDT analogs retain sodium channels in a modified open state only transiently, cause large depolarizing afterpotentials, and evoke repetitive firing with minimal effect on the resting potential. The effects of the phenoxybenzyl pyrethroids were found to be intermediate between these two extremes suggesting that a continuous variation exists in kinetics with which pyrethroids and DDT analogs modify sodium channels. It was not necessary to assume a second site of action to account for the variability observed. The implications of these results to the construction of quantitative structure-activity relationships is discussed.     

State-dependent modification of voltage-gated sodium channels by pyrethroids

Pyrethroids disrupt nerve function by altering the rapid kinetic transitions between conducting and nonconducting states of voltage-gated sodium channels that underlie the generation of nerve action potentials. Recent studies of pyrethroid action on cloned insect and mammalian sodium channel isoforms expressed in Xenopus laevis oocytes show that in some cases pyrethroid modification is either absolutely dependent on or significantly enhanced by repeated channel activation. These use-dependent effects have been interpreted as evidence of preferential binding of at least some pyrethroids to the open, rather than resting, state of the sodium channel. This paper reviews the evidence for state-dependent modification of insect and mammalian sodium channels expressed in oocytes by pyrethroids and considers the implications of state-dependent effects for understanding the molecular mechanism of pyrethroid action and the development and testing of models of the pyrethroid receptor.     

主要农业害虫对茚虫威的抗性现状及其治理策略

茚虫威属于噁二嗪类杀虫剂，与大多数杀虫剂不同的是其进入害虫体内需要经活化代谢转变成N-去甲氧羰基代谢物（decarbomethoxylated metabolite，DCJW）后不可逆地阻断钠通道，进而发挥杀虫活性。茚虫威由于其作用机制不同于常见的使钠离子通道延迟关闭的菊酯类药剂而被广泛用于鳞翅目和一些同翅目、鞘翅目害虫的防治。抗药性是任何杀虫药剂使用后面临的问题，茚虫威也不例外，许多害虫对其产生了不同程度的抗性。昆虫对茚虫威产生抗性的机制包括酯酶活性、谷胱甘肽S-转移酶（glutathione S-transferase，GST）和P450活性的增加以及分子靶标F1845Y、V1848I、L1014P的突变，这些对茚虫威抗性机制的研究基本都是基于抗性种群和敏感种群开展的，需要进一步验证其对抗性研究的贡献度。针对我国田间害虫种群对茚虫威的抗性现状，及时实施对茚虫威有效的抗性治理是迫切的。对于茚虫威的抗性治理除了传统的杀虫药剂轮用、混用外，需要利用其作用机制特点开展抗性治理策略研究。一是充分利用其活化代谢的特点，开展组合药剂的研究应用；二是菊酯类药剂和茚虫威的作用机制均与钠离子通道有关，但是前者是使钠离子通道关闭延迟，而后者是阻断钠离子通道，开展相关基础研究，使菊酯类药剂与茚虫威合理地用于抗性治理中。本文综述了茚虫威的抗性现状、抗性机制与交互抗性、茚虫威的抗性风险评价，针对茚虫威的抗性特点提出了抗性治理策略。     

Modulation of sodium channels by the oxadiazine insecticide indoxacarb and its N-decarbomethoxylated metabolite in rat dorsal root ganglion neurons

The effects of the oxadiazine insecticide indoxacarb and its N-decarbomethoxylated metabolite (DCJW) on tetrodotoxin-resistant (TTX-R) voltage-gated sodium channels in rat dorsal ganglion neurons were studied using the whole-cell patch clamp technique. Indoxacarb and DCJW suppressed the peak amplitude of action potentials, and DCJW exhibited a faster time course and higher potency than indoxacarb in the blocking effects. In voltage-clamp experiments, indoxacarb and DCJW suppressed TTX-R sodium currents in a time-dependent manner without a steady-state level of suppression. IC50 values for indoxacarb and DCJW on TTX-R sodium currents were estimated to be 10.7 and 0.8 microM after 25 min of bath application, respectively. DCJW was about 10 times more potent than indoxacarb in blocking TTX-R sodium currents. Although the suppressive effects of indoxacarb were partially reversible after washout with drug-free external solution, no recovery of sodium current was observed in DCJW treated neurons after prolonged washout. In current-voltage relationships, both indoxacarb and DCJW blocked the sodium currents to the same degree in the entire range of membrane potentials. The sodium conductance-voltage curve was not shifted along the voltage axis by indoxacarb and DCJW at 10 microM. In contrast, the steady-state inactivation curves were shifted in the hyperpolarizing direction by indoxacarb as well as by DCJW. Based on these results, it was concluded that indoxacarb and DCJW potently blocked the TTX-R sodium channel in rat DRG neurons with hyperpolarizing shifts of the steady-state inactivation curves, suggesting preferential association of the insecticides to the inactivated state of sodium channels. The small structural variation between indoxacarb and DCJW resulted in clear differences in potency for blocking sodium channels and reversibility after washout.     

Pyrethroids, knockdown resistance and sodium channels

Knockdown resistance to DDT and the pyrethrins was first described in 1951 in the housefly (Musca domestica L.). This trait, which confers reduced neuronal sensitivity to these insecticides, was subsequently shown to confer cross-resistance to all synthetic pyrethroid insecticides that have been examined to date. As a consequence, the worldwide commercial development of pyrethroids as a major insecticide class over the past three decades has required constant awareness that pyrethroid overuse has the potential to reselect this powerful resistance mechanism in populations that previously were resistant to DDT. Demonstration of tight genetic linkage between knockdown resistance and the housefly gene encoding voltage-sensitive sodium channels spurred efforts to identify gene mutations associated with knockdown resistance and understand how these mutations confer a reduction in the sensitivity of the pyrethroid target site. This paper summarizes progress in understanding pyrethroid resistance at the molecular level, with particular emphasis on studies in the housefly.     

Knockdown resistance to DDT and pyrethroids: from target-site mutations to molecular modelling

Naturally derived insecticides such as pyrethrum and man-made insecticides such as DDT and the synthetic pyrethroids act on the voltage-gated sodium channel proteins found in insect nerve-cell membranes. The correct functioning of these channels is essential for the 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 that inhibit the binding of the insecticide and result in the insect developing resistance. This perspective outlines the current understanding of the molecular processes underlying target-site resistance to these insecticides (termed kdr and super-kdr), and how this knowledge may in future contribute to the design of novel insecticidal compounds.     

Mutations in the sodium channel associated with pyrethroid resistance in the greenhouse whitefly, Trialeurodes vaporariorum

BACKGROUND: Trialeurodes vaporariorum Westwood is an important pest of protected crops in temperate regions of the world. Resistance to pyrethroid insecticides is long established in this species, but the molecular basis of the mechanism(s) responsible has not previously been disclosed. RESULTS: Mortality rates of three European strains of T. vaporariorum to the pyrethroid bifenthrin were calculated, and each possessed significant resistance (up to 662‐fold) when compared with a susceptible reference strain. Direct sequencing revealed three amino acid substitutions in the para‐type voltage‐gated sodium channel (the pyrethroid and DDT target site) of bifenthrin‐resistant T. vaporariorum at positions previously implicated with pyrethroid or DDT resistance (M918L, L925I and T929I) in other related species. CONCLUSION: This study indicates that resistance to bifenthrin in T. vaporariorum is associated with target‐site insensitivity, and that the specific mutations in the sodium channel causing resistance may differ between localities. Copyright © 2012 Society of Chemical Industry     

Structure-Activity Relationships for the Action of Dihydropyrazole Insecticides on Mouse Brain Sodium Channels

Assays of radiosodium uptake into mouse brain vesicles and the binding of [³H]batrachotoxinin A 20-α-benzoate (BTX-B) were used to compare the actions of six dihydropyrazole (3-aryl-1-arylcarbamoyl-2-pyrazoline) insecticides on mouse brain sodium channels. The relative potencies of the six dihydropyrazoles as inhibitors of either pumiliotoxin B-stimulated sodium uptake measured in the presence of scorpion (Leiurus quinquestriatus) venom or veratridine-stimulated sodium uptake were closely correlated with the relative potencies of these compounds as inhibitors of the binding of BTX-B to mouse brain sodium channels. A comparison of the enantiomers of the most potent dihydropyrazole, RH 3421, as inhibitors of radiosodium uptake showed that the (−) enantiomer of RH 3421 was approximately six-fold more potent than the (+) enantiomer. The potencies of these dihydropyrazoles in these assays and the stereoselectivity observed in the action of enantiomers of RH 3421 are in good agreement with available information on insecticidal activity in this group of compounds. Assays of the combined effects of RH 3421 and dibucaine as inhibitors of BTX-B binding revealed mutually competitive interactions between these compounds. This finding is consistent with the existence of a common site of action for dihydropyrazoles and local anesthetics on the sodium channel. The results of these studies provide further evidence for the toxicological relevance of the effects of dihydropyrazoles on sodium channels.     

Can selective peptides be combined efficiently with agrochemicals? A new approach to insect control

Sodium channels have been a major target for the development of insecticides such as synthetic pyrethroids. However, insecticides currently available induce resistance and present limited selectivity to insect pests. Molecular and biochemical studies, as well as binding experiments using radiolabelled neurotoxins, have shown that sodium channels expressed in various insect orders must be structurally and pharmacologically different. At least three groups of peptide neurotoxins derived from scorpion venom are highly active on insects and very weakly or practically inactive on mammals. It is proposed that various insecticides are examined for possible cooperative interactions with the peptide toxins highly active on insects, and pairs of ligands are identified that will increase the selectivity not only between mammals and insects but also between different pest and non-pest insects. This is feasible on the basis of the differential allosteric modulations observed between LqhαIT, an α-toxin highly active on insects, and brevetoxin on locust versus cockroach and rat brain sodium channels. Moreover, combination of LqhαIT with the pyrethroid deltamethrin increased the binding of [¹²⁵I] LqhαIT by more than 1.8-fold, and the combined presence of brevetoxin further increased the binding. Such allosteric modulation may provide a new approach to increase the selective activity of pesticides on target organisms by simultaneous application of allosterically interacting drugs, designed on the basis of the selective peptide toxins.     

Actions of pyrethroid insecticides on insect axonal sodium channels

The actions of pyrethroid insecticides were tested on isolated giant axons of the cockroach Periplaneta americana, using oil-gap, single-fibre recording techniques. Current-clamp and voltage-clamp experiments were used to determine the actions of pyrethroids on axonal membrane potentials and ionic currents. Treatment with deltamethrin at micromolar concentrations caused gradual depolarisation of the axon accompanied by a reduction in amplitude of the action potential. This depolarisation was enhanced by an increase in stimulation frequency. Other synthetic pyrethroids: 3,4,5,6-tetrahydrophthalimidomethyl (1RS)-cis-3-[(RS)-2,2-dimethylcyclopropyl]-2,2-dimethylcyclopropanecarboxylate, biopermethrin and its (1S)-enantiomer, (1R)-tetramethrin, S-bioallethrin, bioresmethrin and its (1S)-enantiomer, cismethrin, and 5-benzyl-3-furylmethyl (E)-(1R)-cis-2,2-dimethyl-3-(2-oxothiolan-3-ylidenemethyl)cyclopropanecarboxylate (RU-15525, ‘Kadethrin’) were investigated. The (1S)-enantiomers were inactive, but all the other pyrethroids tested, apart from deltamethrin, induced prolonged negative (depolarising) after-potentials. All the treatments with the active pyrethroids resulted in the appearance of a voltage and time-dependent ‘maintained’ sodium conductance. The duration of this ‘slow’ conductance varied considerably depending on the pyrethroid under test. Clearly, the effectiveness of pyrethroids on whole insects is not determined only by the degree to which they directly modify the properties of sodium channels. Nevertheless, voltage-clamp experiments on isolated axons readily permit direct comparison of the actions of different pyrethroids on the sodium channels of insect neurones.     

Action of pyrazoline-type insecticides at neuronal target sites

Pyrazoline-type insecticides were discovered in the early 1970s, but did not readily yield commercial insecticide products due to their unfavorable toxicity profiles and environmental properties. The recent registration of indoxacarb, a compound derived from pyrazolines, has drawn renewed attention to this insecticide class. This review summarizes the development of the pyrazoline-type insecticides and examines the mechanism of action of these compounds based on available electrophysiological, pharmacological, and toxicological information.     

The action of pyrethroids on the voltage-sensitive calcium channel of Paramecium tetraurelia

Calcium regulation is an important event in synaptic transmission and neuronal function, which is governed by a very intricate signal transduction system which is not completely understood. Using a variety of pharmacological assays, we have characterized the action of deltamethrin on the ciliary voltage-sensitive calcium channel and on phospholipase C activity of Paramecium tetraurelia Sonneborn, an organism that does not possess a voltage-sensitive sodium channel. In fura-2 fluorometric assays, which examined whole cells and ciliary membrane vesicles enriched with calcium channels, deltamethrin stimulated Ca²⁺ uptake. We also determined that the phospholipase C activity of the ciliary membrane vesicles is regulated by the βγ-subunit from heterotrimeric G-proteins. Subsequent treatment with deltamethrin resulted in a substantial and highly significant increase in phospholipase C activity. These results provide evidence that the molecular mode of action of pyrethroids on the voltage-sensitive calcium channel is distinct from the action of this insecticide on the voltage-sensitive sodium channel and may be dependent, in part, upon an interaction with the βγ-subunit of heterotrimeric G-protein.     

线粒体复合体Ⅰ呼吸抑制剂的研究

线粒体复合体Ⅰ呼吸抑制剂不仅在医药上有着重要的研究价值,在农药方面也有着特殊的意义。这类药剂的作用机制比较特殊,害虫不易产生抗性,是一类非常有前途的杀虫药剂。从植物,微生物等生物体中发现了许多线粒体复合体Ⅰ呼吸抑制剂,如鱼藤酮、粉蝶霉素A、辣椒碱,番荔枝内酯、myxalamid等,它们可以作为农药的先导化合物进行药物合成。根据不同的作用方式,线粒体复合体Ⅰ呼吸抑制剂可分3种类型,分别以粉蝶霉素A、鱼藤酮、辣椒碱为代表。     

Structure-related effects of pyrethroid insecticides on the lateral-line sense organ and on peripheral nerves of the clawed frog, Xenopus laevis

The effects of seven different pyrethroid insecticides on the lateral-line sense organ and on peripheral nerves of the clawed frog, Xenopus laevis, were investigated by means of electrophysiological methods. The results show that two classes of pyrethroid can be clearly distinguished. (i) Pyrethroids without an α-cyano group (permethrin, cismethrin, and bioresmethrin). These noncyano pyrethroids induce short trains of nerve impulses in the lateral-line sense organ. In peripheral nerve branches they induce a depolarizing afterpotential and repetitive firing. These effects are very similar to those previously reported for allethrin. (ii) Pyrethroids with an α-cyano-3-phenoxybenzyl alcohol (cypermethrin, fenpropathrin, deltamethrin, and fenvalerate). In the lateral-line sense organ these α-cyano pyrethroids induce very long trains of nerve impulses which may last for seconds and may contain hundreds or even thousands of impulses. The α-cyano compounds do not cause repetitive activity in peripheral nerves. Instead they induce a quickly reversible, stimulus frequency-dependent suppression of the action potential. Since the chemical structure of cypermethrin differs from that of permethrin only in the α-cyano group and because all four α-cyano compounds act in a very similar way, it is concluded that the α-cyano substituent is responsible for the large differences in neurotoxic effects. In the lateral-line sense organ the duration of nerve impulse trains induced by the noncyano as well as the α-cyano pyrethroids increases dramatically when the temperature is lowered. Further, in sensory fibers the effects of both classes of pyrethroid on the nerve action potential are more pronounced compared to their effects on motor fibers. It is argued that the different neurotoxic effects reported here originate from a common mechanism of action of pyrethroids, which is a prolongation of the transient increase of sodium permeability of the nerve membrane associated with excitation.It is concluded that the sodium channel in the nerve membrane is the major target site of noncyano and α-cyano pyrethroids.     

氟啶虫酰胺作用靶标—内向整流钾离子通道研究进展

氟啶虫酰胺是一种高选择性杀虫剂，主要用于防治刺吸式口器害虫。有关该杀虫剂的分子靶标长期未知。近年来随着研究的逐渐深入，相关作用靶标已逐步得到明确。早期研究表明，氟啶虫酰胺通过抑制剌吸式口器害虫的取食，造成害虫因饥饿而死亡；最新研究发现，其作用靶标是内向整流钾离子(Kir)通道，纳摩尔浓度级的氟啶虫酰胺即可阻断褐飞虱的Kir通道。氟啶虫酰胺通过抑制昆虫Kir通道，干扰昆虫细胞的离子稳态与平衡电位，尤其是破坏马氏管与唾液腺的正常分泌功能，从而影响昆虫的取食与排泄过程，最终导致害虫死亡。文章对氟啶虫酰胺的作用靶标、作用机制及应用等方面的研究进展进行了综述，总结分析了昆虫Kir通道的结构及其所参与的生理功能，分析了氟啶虫酰胺通过抑制该离子通道致使害虫死亡的具体作用机制，并介绍了针对靶向Kir通道药物的高通量筛选方法与研究进展，可为杀虫剂新靶标挖掘与靶向此类新靶标药剂的创制提供研究思路。     

杀虫剂fluralaner及其衍生物的生物效应和毒理学研究进展

新型异唑啉类杀虫剂fluralaner通过干扰γ-氨基丁酸(GABA)门控氯离子通道发挥作用。作为新型高效GABA门控氯离子通道干扰剂,与环戊二烯类、苯基吡唑类和大环内酯类杀虫剂相比,fluralaner在分子结构、作用位点、选择性以及交互抗性等方面均有显著差异。本文综述了fluralaner及其衍生物的研发、毒性、防治范围以及作用机理等,并预测了其应用前景。     

抑制性谷氨酸受体(IGluRs)通道及其相关杀虫剂的作用

抑制性谷氨酸受体(IGluRs)属于半胱氨酸环配体门控离子通道超家族,主要介导神经和肌肉细胞中抑制性的神经传递,目前仅在无脊椎动物中发现,在脊椎动物中尚未发现,因此是高选择性杀虫剂的理想靶标。IGluRs主要分布在无脊椎动物的神经和肌肉组织中,对控制吞咽、运动、感知和保幼激素的生物合成等可能起关键作用。人们对IGluRs的了解大多来自于对线虫和模式昆虫的研究,目前在线虫中共发现了4种α亚基和1种β亚基,是否有一种新的亚基类型如γ亚基尚不确定,从昆虫体内仅克隆了α亚基。就生理功能和药理特性而言,IGluRs与γ-氨基丁酸(GABA)受体最为类似,但其氨基酸序列却与甘氨酸受体相似性最高。作用于IGluRs的杀虫剂包括阿维菌素/美倍霉素类、苯基吡唑类杀虫剂氟虫腈以及吲哚二萜类化合物Nodulisporic acid等。对IGluRs的生理功能、分子特性、药理性质及相关杀虫剂的作用机理等的研究进展进行了综述。     

Cloning of a sodium channel gene and identification of mutations putatively associated with fenpropathrin resistance in Tetranychus urticae

Tetranychus urticae Koch is the most serious mite pest to various orchard trees and garden plants. Biochemical and molecular analyses were conducted to elucidate resistance mechanisms in a fenpropathrin-resistant mite strain (FenR). No significant differences were found in the activities of carboxylesterase and glutathione-S-transferase between the susceptible (UD and PyriF) and FenR strains. Cytochrome P450 activity was highest in PyriF, followed by FenR and UD. Analysis of detoxification enzyme assays, therefore, suggested that metabolic detoxification plays little role, if any, in fenpropathrin resistance. However, the FenR strain showed approximately 104- and 33.3-fold slower knockdown responses than UD and PyriF strains, respectively, suggestive of sodium channel insensitivity as a major resistance mechanism. We cloned cDNA fragments of the para-homologous sodium channel α-subunit gene (Tuvssc) and determined its full-length nucleotide sequences. The complete open reading frame of Tuvssc was 6627 nucleotides, encoding 2209 amino acids. The amino acid sequences of Tuvssc were 47.5% and 51.2% identical to the fruit fly and varroa mite, respectively. Amino acid sequence comparison between the three strains revealed two mutations (L1022V and A1376D) and one deletion (HisDel^1278-1280) found only in FenR mites, among which the L1022V mutation was proposed to play a major role in knockdown resistance to fenpropathrin.     

Recent studies on the effects of DDT and pyrethroid insecticides on nervous activity in cockroaches

A summary is given of the results of three sets of electrophysiological experiments on the American cockroach (Periplaneta americana). The effects of DDT and S-bioallethrin [bioallethrin (S)-cyclopentenyl isomer] on single giant axons were studied using the voltage-clamp technique. Whereas both molecules induced long tails of inward (sodium ion) current, the voltage and time-dependency of these tails differed. With DDT, the tail was proportional to the activation of the peak current and decreased with the duration of the pulse, suggesting that the molecules were bound to open sodium channels and delayed their closing. With S-bioallethrin, the voltage dependency of the tail was different from that of the peak current, and the tail current increased exponentially with the duration of the depolarisation, suggesting that the pyrethroid insecticide modified resting (or silent) sodium channels into slowly activating channels. Modified action potentials, mimicking those produced by the two molecules, were computed on the basis of these results. Deltamethrin, one of the most potent pyrethroid insecticides, was applied topically on a leg mechanoreceptor and was found to have little effect on the local ‘receptor potential’ but to inhibit action potential production. The effects of topical applications of deltamethrin on the dorsal part of the abdomen, on nervous activity in the abdominal connectives, were studied under different experimental conditions. The results suggest that the insecticide molecules diffused rapidly through the cuticle, were concentrated in the haemolymph, and eventually reached the central nervous system, where they inhibited nerve activity.     

Molecular studies of knockdown resistance to pyrethroids: cloning of domain II sodium channel gene sequences from insects

Knockdown resistance (kdr) is a target-site resistance mechanism that confers nerve insensitivity to DDT and pyrethroid insecticides. In the housefly, Musca domestica, molecular cloning of the para-type sodium channel gene has revealed two amino acid mutations that are associated with kdr and super-kdr resistance phenotypes. Both mutations are located in the domain II region of the channel; Leu1014 to Phe in the hydrophobic segment IIS6 and Met918 to Thr in the IIS4-IIS5 linker. To investigate whether these mutations also occur in other insects, we have designed degenerate primers based on conserved sequences in the domain II region of the sodium channel and used these to PCR amplify this region from insecticide-susceptible strains of eight diverse insect species representing four different insect Orders: Helicoverpa armigera, Plutella xylostella, Spodoptera littoralis (Lepidoptera), Blattella germanica (Dictyoptera), Tribolium castaneum (Coleoptera), Myzus persicae, Aphis gossypii and Phorodon humuli (Hemiptera). The primers amplified closely related para-type sodium channel sequences from each insect with a minimum of 85% amino acid identity between species. All of the sequences contained ‘susceptible’ Leu and Met residues at the positions associated with kdr and super-kdr resistance in the housefly. Recent results detailing the presence of a kdr-type Leu to Phe mutation in pyrethroid-resistant strains of two important agricultural pests, P. xylostella and M. persicae, are discussed. ©1997 SCI