昆虫谷氨酸门控氯离子通道研究进展

作者:未知

  摘要 谷氨酸门控氯离子通道(GluCls)介导快速抑制性神经传导,目前只发现于无脊椎动物中,是开发新型杀虫剂的理想作用靶标。GluCls属于半胱氨酸环超家族的配体门控离子通道,在昆虫中只发现有1个α亚基,但可以通过选择性剪接生成多种亚基剪接变体并且能够形成功能性受体。除了典型的神经传导功能外,GluCls还参与调控昆虫保幼激素合成及生长发育等生理功能。GluCls的氨基酸突变和表达量变化是导致昆虫对杀虫剂产生抗药性的部分原因。本文主要从GluCls的分子特征、选择性剪接、药理学性质、生理功能和昆虫的抗药性5个方面对昆虫GluCls的研究进展作一综述,为新型杀虫剂的研发提供理论基础。
  关键词 谷氨酸门控氯离子通道; 分子特征; 选择性剪接; 药理学性质; 生理学功能; 昆虫抗药性
  中图分类号: Q 966
   文献标识码: A
   DOI: 10.16688/j.zwbh.2019106
  Research advances in insect glutamate-gated chloride channels
  MENG Xiangkun, YANG Xuemei, GE Huichen, WANG Jianjun
  (College of Horticulture and Plant Protection, Yangzhou University, Yangzhou 225009, China)
  Abstract
  Glutamate-gated chloride channels (GluCls) mediate fast inhibitory neurotransmission in invertebrate nervous systems, and are of considerable interest in insecticide discovery. GluCls belong to the ligand-gated ion channels (LGICs) superfamily. Although only one α subunit was found in insects, a number of variants are generated by the alternative splicing of GluCl α subunit and form the functional GluCls receptors. In addition to the classical neurotransmission function, GluCls have been demonstrated to regulate the biosynthesis of juvenile hormone and the growth and development of insects. The mutation and changes of expression level of GluCls contributed to insecticide resistance in insects. This review introduced the research status of insect GluCls,including the molecular characteristics, alternative splicing, pharmacological properties, physiological function, relationships with insect resistance, and provide the basis for the development of new insecticides.
  Key words
  GluCls; molecular characteristic; alternative splicing; pharmacological property; physiological function; insect resistance
  谷氨酸是脊椎動物和无脊椎动物神经系统中主要的神经传递递质,作用于细胞膜上的谷氨酸受体,在脊椎动物中通过门控阳离子通道介导兴奋性传递。而在无脊椎动物中谷氨酸既是兴奋性的神经递质,又是抑制性的神经递质[1]。哺乳动物中,谷氨酸受体可分为离子型和代谢型两种类型。其中离子型受体包括N-甲基-D-天冬氨酸受体(NMDAR)、海人藻酸受体(KAR)和α-氨基-3羟基-5甲基-4异恶唑受体(AMPAR),它们与离子通道偶联形成受体通道复合物,介导信号传导[2]。代谢型谷氨酸受体属于G蛋白偶联受体,这类受体被激活后通过G蛋白效应酶、第二信使等组成的信号转导系统起作用,产生相应的生理反应[3]。此外,在无脊椎动物中还发现一种离子型抑制性谷氨酸受体(inhibitory glutamate receptors,IGluRs),谷氨酸作为抑制性的神经递质与此类受体结合进一步开启氯离子通道,因此这类受体也被称为谷氨酸门控氯离子通道(glutamate-gated chloride channels,GluCls)[1]。在昆虫神经系统的杀虫剂靶标中,GluCls只在线虫、昆虫等无脊椎动物神经和肌肉细胞中被发现,在脊椎动物中尚未发现。因此,昆虫GluCls是开发高选择性杀虫剂的一个理想作用靶标[45]。目前作用于昆虫GluCls的杀虫剂主要有大环内酯类杀虫剂伊维菌素、阿维菌素和苯基吡唑类杀虫剂氟虫腈等。
  1 GluCls的分子特征
  GluCls与烟碱型乙酰胆碱受体(nAChRs)、5-羟色胺(5-HT)受体及γ-氨基丁酸(GABA)受体均是属于半胱氨酸环超家族的配体门控离子通道,它们具有相似的结构特征,都是由5个亚基组成的五聚体跨膜蛋白,蛋白中包括激动剂/竞争性抑制剂结合位点和跨膜通道等结构[6]。每个亚基从N端到C端可以分为4个区域:即包含配体结合区的N端亲水区、3个跨膜片段(TM1~TM3)组成的疏水区、长度可变的胞内亲水大环和包括第4个跨膜片段TM4在内的C端疏水区[7]。其中,5个亚基的第2个跨膜片段TM2共同组成受体的离子通道。根据受体亚基组成的异同,可分为5个亚基相同的同型五聚体和亚基不同的异型五聚体。GluCls与其他配体门控氯离子通道具有密切关系,GluCls与γ-氨基丁酸受体在生理功能和药理特性上最为类似,但其氨基酸序列却与甘氨酸受体相似性最高[89]。   GluCl受体基因最早于秀丽隐杆线虫Caenorhabditis elegans中被克隆发现,在线虫中共克隆到6个GluCl受体亚基,包括4个α亚基,1个β亚基以及1个可能的γ亚基[1011]。但目前在昆虫中只发现1个GluCl受体亚基,即α亚基。首个昆虫GluCl受体亚基在黑腹果蝇Drosophila melanogaster中被克隆,与秀丽隐杆线虫的GluCl α和β亚基具有很高相似性,在核苷酸水平相似性高达67%和62%,因此被命名为DmGluCl α亚基[12]。随后GluCl α亚基在其他一些昆虫,如赤拟谷盗Tribolium castaneum、家蝇Musca domestica、意大利蜜蜂Apis mellifera中被鉴定发现,它们同DmGluCl α亚基的氨基酸相似性高达80%~90%[13]。昆虫GluCl α亚基在4个重要的TM跨膜区氨基酸序列一致性极高,主要的序列变异区域在TM3和TM4间的胞内环中。胞内环中包含若干不同的蛋白激酶磷酸化位点,在决定亚基的功能上起重要作用[14]。
  2 GluCl的选择性剪接
  选择性剪接是指从一个mRNA前体通过不同的剪接方式(选择不同的剪接位点)产生不同的mRNA剪接变体的过程。目前,已发现在黑腹果蝇、赤拟谷盗、家蚕Bombyx mori、意大利蜜蜂、家蝇、灰飞虱Laodelphax striatellus、小菜蛾Plutella xylostella和西花蓟马Frankliniella occidentalis等多种昆虫中均存在不同的GluCl α亚基剪接变体[4,6,1520]。昆虫GluCl α亚基由10个外显子组成,可通过氨基酸缺失、互斥外显子、外显子跳跃、3′选择性剪接和内含子保留5种方式生成不同的亚基剪接变体,而发生在外显子3和9中的选择性剪接则是昆虫GluCl的研究热点[20]。外显子3选择性剪接普遍存在于昆虫GluCl中,编码部分N端区域,紧邻配体结合区Loop D上游[21]。GluCl外显子3选择性剪接可生成3种不同的GluCl剪接变体(GluCl 3A、GluCl 3B和GluCl 3C);此外在家蚕和小菜蛾中还发现一种完全缺失外显子3的GluCl剪接变体[20,22]。相对于外显子3,目前关于昆虫GluCl外显子9选择性剪接的研究较少。GluCl外显子9选择性剪接位于TM3和TM4之间的胞内大环中,其选择性剪接造成不同数目的氨基酸缺失,从而产生不同的GluCl剪接变体(GluCl 9A、GluCl 9B和GluCl 9C)[1820,2325]。GluCl选择性剪接不但可以单独发生在外显子3或外显子9中,还可以同时在2个外显子中发生,进一步丰富昆虫GluCl亚基的多样性[19,21]。
  对昆虫GluCl不同选择性剪接变体的表达定位分析发现,不同GluCl剪接变体在昆虫不同部位的表达丰度存在差异[19,21]。通过精细免疫定位对意大利蜜蜂中GluCl α亚基剪接变体在脑中的分布进行分析,发现3个亚基在蜜蜂脑的不同部位均有表达,其中GluCl 3A主要分布于神经纤维网,GluCl 3B则主要分布于细胞体[15]。不同亚基在一些部位的共同表达说明它们可能组成异型五聚体,而在一些部位的差异表达也预示着它们可能具有不同的生理功能。对家蝇的3个GluCl亚基剪接变体研究发现,它们在家蝇不同组织及不同发育龄期的表达量各不相同,其中GluCl 3A和GluCl 3B主要在家蠅成虫的头部组织中表达,而GluCl 3C则主要在成虫的足等外周组织中表达[17]。目前在灰飞虱中已发现由外显子3和外显子9选择性剪接产生的6种GluCl亚基,这些剪接变体都在灰飞虱头部组织中具有最高的表达量并且随着灰飞虱的生长发育剪接变体的表达量均逐渐上升[19]。在鳞翅目昆虫二化螟Chilo suppressalis中,3个外显子3剪接变体均在神经索和脑组织中高表达,相对于GluCl 3A和GluCl 3C,GluCl 3B在二化螟中枢神经组织中的表达量更高[21]。不同于在灰飞虱中的发现,随着二化螟的生长发育,3个外显子3剪接变体的表达量在幼虫期间均逐渐下降,在蛹期间逐渐增加。此外,GluCl 3C在二化螟表皮和肠道组织中同样具有很高的表达量,并且在二化螟蛹后期表达量显著增加[21]。这些结果表明,昆虫GluCl剪接变体主要在神经组织中表达,在一些非神经组织如足、表皮和肠道中也具有较高的表达量,但在不同昆虫中其表达模式可能不同。
  3 GluCls的药理学性质
  利用电生理技术对昆虫神经元细胞中的GluCls研究发现,昆虫中至少存在2种药理学性质不同的GluCls受体[2629]。例如,在美洲大蠊Periplaneta americana神经元中发现2种GluCls,它们对激动剂鹅膏蕈氨酸及阻断剂木防己苦毒素表现出不同的敏感性[2829]。在意大利蜜蜂和飞蝗Locusta migratoria的神经元细胞中同样发现2种能被谷氨酸引发不同电流的GluCls,并且氟虫腈对2种受体的抑制效果也不同[2627,30]。此外,对意大利蜜蜂触角叶细胞的研究发现,谷氨酸能够引发一种短暂的和一种持续的反应电流;GluCls能被氟虫腈和木防己苦毒素抑制,并且受体对木防己苦毒素表现出不同的反应电流,这也说明在意大利蜜蜂触角叶细胞中同样存在2种不同的GluCls[26,31]。对线虫的研究表明,亚基组成不同可导致受体药理学性质的差异[3233],但目前在昆虫中只发现1个GluCl亚基,推测昆虫的GluCls可能由GluCl α通过选择性剪接产生的不同亚基组成,从而具有不同的药理学性质[24]。
  利用体外表达及电生理学的方法,对昆虫GluCl不同剪接变体的药理学性质测定发现,不同GluCl剪接变体组成的受体对激动剂或抑制剂的敏感性存在差异。单独或共表达3个外显子3剪接变体组成的家蝇GluCls对激动剂谷氨酸和伊维菌素的敏感性相同,但对阻断剂氟虫腈和木防己苦毒素的敏感性却不同,说明家蝇3个GluCl亚基剪接变体具有不同的药理学性质[17]。对小菜蛾GluCls的研究发现,单独或共同表达3个外显子9剪接变体组成的GluCls对谷氨酸的敏感性几乎相同,但对阿维菌素和氟虫腈的敏感性却不同[20]。这些研究结果说明昆虫GluCl的外显子3和外显子9选择性剪接都能够生成功能性的GluCls,但可能具有不同的药理学性质。但对灰飞虱GluCl外显子9剪接变体的研究发现,分别表达的2个剪接变体组成的GluCls对谷氨酸、氟虫腈和木防己苦毒素具有相似的EC50和IC50值,这可能是由于2个剪接变体的配体结合区域具有相似的氨基酸[19]。此外,由于单个亚基剪接变体体外表达就能组成功能性GluCls,目前尚不明确昆虫体内GluCls是同聚体还是由α亚基的不同剪接变体形成的异聚体。   4 GluCls的生理功能
  GluCls主要分布于无脊椎动物的中枢神经和神经肌肉连接处。对线虫的研究发现,GluCls除了具有典型的神经兴奋传递功能外,还能够调节线虫的运动、取食、信号感知和繁殖[11,34]。昆虫中,GluCls作为杀虫剂靶标的离子通道功能已被广泛研究,同时其他的生理功能也逐渐被发现。对意大利蜜蜂的研究中,通过注射GluCls抑制剂能够影响蜜蜂的嗅觉记忆和对刺激的感知,使用伊维菌素和氟虫腈处理蜜蜂则能够破坏蜜蜂的长期记忆[31,3537]。意大利蜜蜂GluCl α 亚基被干扰后,能够破坏其嗅觉记忆的恢复[38]。当分别沉默不同的GluCl亚基剪接变体后,则能够影响蜜蜂对不同气味的嗅觉记忆恢复[39]。对太平洋折翅蠊Diploptera punctata咽侧体的研究发现,当使用GluCls激动剂鹅膏蕈氨酸、伊维菌素或谷氨酸處理腺体后,能够降低保幼激素的合成量;继续使用GluCls阻断剂木防己苦毒素处理,则能够使保幼激素含量恢复到正常水平;不同浓度的激动剂或阻断剂对保幼激素合成的影响也各不相同[40]。鉴于兴奋性的离子型谷氨酸受体如N-甲基-D-天冬氨酸受体、海人藻酸受体等同样能够影响太平洋折翅蠊咽侧体中保幼激素的合成,推测不管是抑制性还是兴奋性的谷氨酸受体,都可能是通过改变咽侧体细胞内钙离子的浓度来进一步影响保幼激素的合成[4142]。另一方面,不同龄期的蜜蜂体内保幼激素含量的不同能够影响蜜蜂嗅觉记忆和行为,说明GluCls在生物体内的生理功能可能存在内在联系[4344]。
  此外,研究还发现GluCls能够调控飞蝗和果蝇的飞行、静止和唤醒行为,调节果蝇对光的逃避和嗅觉行为[30,4547]。昆虫GluCl被干扰后,谷实夜蛾Helicoverpa zea卵的孵化率降低,二化螟幼虫体重和化蛹率显著下降,说明GluCls在昆虫的生长发育过程中具有重要功能[21,48]。不同GluCl剪接变体在昆虫不同组织中的差异表达,也可能预示着GluCl剪接变体具有不同的生理功能[21]。这些研究说明,昆虫GluCls具有功能多样性,但不同GluCl剪接变体的具体功能,GluCls在昆虫生理功能之间的联系以及如何影响保幼激素的合成进而影响昆虫的生长发育还有待进一步探索。
  5 GluCls与昆虫抗药性
  目前,关于杀虫剂作用靶标GluCls与昆虫抗药性关系的研究主要集中在大环内酯类杀虫剂阿维菌素和伊维菌素。已经明确GluCls突变是导致昆虫、螨类对阿维菌素和伊维菌素产生抗药性的主要原因。DmGluCl的P299S突变造成果蝇对伊维菌素产生3倍的抗药性[49]。位于TM3跨膜区的TuGluCl1 G323D和TuGluCl3 G326E突变分别导致二斑叶螨Tetranychus urticae对阿维菌素产生了18倍和2 000倍的抗药性[5052]。同样位于GluCl TM3跨膜区的A309V突变则导致小菜蛾对阿维菌素产生了10倍的抗药性[25]。电生理试验同样证明了小菜蛾GluCl的A309V和G315E(分别对应于TuGluCl1 G323D和TuGluCl3 G326E)突变可导致体外表达的受体对阿维菌素的敏感性分别下降4.8倍和493倍[53]。近期的一项研究发现,GluCls中的A251V、S46P和H272R突变在对伊维菌素具有抗药性的头虱Pediculus humanus capitis中具有较高的突变频率,可能是导致头虱对伊维菌素产生抗药性的部分原因[54]。
  越来越多的研究证明,杀虫剂靶标的表达量变化影响昆虫对杀虫剂的敏感性。乙酰胆碱酯酶(AChEs)编码基因ace在对有机磷杀虫剂抗性麦二叉蚜Schizaphis graminum中的表达量是敏感性试虫中的1.5倍,而在对氧化乐果有抗药性的棉蚜Aphis gossypii中却下调表达[5556]。鱼尼丁受体(RyRs)编码基因在对氟虫双酰胺抗性小菜蛾中的表达量是敏感品系中的2.93倍[57]。对新烟碱类杀虫剂产生抗性的褐飞虱Nilaparvata lugens和家蝇中,烟碱型乙酰胆碱受体(nAChRs)的亚基表达量显著下降[5859]。对阿维菌素抗性西花蓟马研究发现,虽然解毒代谢是导致抗药性的主要因素,但杀虫剂作用靶标的氨基酸变化和表达量改变也可能是造成抗药性的原因[6,60]。通过对阿维菌素作用靶标的分析,没有发现相关的抗性突变,但GluCl在抗性西花蓟马中的表达量是敏感性西花蓟马中的2.63倍[6]。在小菜蛾中同样发现,GluCl在阿维菌素抗性品系中的表达量显著高于敏感品系[6162]。当GluCl被干扰后,二化螟幼虫对阿维菌素的敏感性显著上升[21]。但在对烟粉虱Bemisia tabaci、小菜蛾、黏虫Mythimna separata和朱砂叶螨Tetranychus cinnabarinus的研究中发现,GluCl被干扰后试虫对阿维菌素的敏感性降低[6366]。GluCls在昆虫中具有多种生理功能,当被干扰后会对昆虫的生理状态产生多种负面作用,进而影响昆虫对杀虫剂的敏感性,这可能是导致GluCls在不同昆虫中研究结果有差异的原因。
  6 结语
  至今GluCls只在无脊椎动物中被发现,是一个理想的杀虫剂作用靶标。虽然对昆虫GluCls已有了较深入的研究,但仍有很多问题尚不清楚。除了离子通道功能,GluCls在昆虫中还有哪些具体的生理功能以及如何在这些生理功能中发挥作用?昆虫GluCls只有一个α亚基,却可以通过选择性剪接等生成多个剪接变体,不同剪接变体各有哪些生理功能?昆虫内源性GluCls是由单个剪接变体组成的同型五聚体还是由不同剪接变体组成的异型五聚体?这些问题仍需要我们去探索解决。
  害虫防治中作用于GluCls的药剂主要有伊维菌素、阿维菌素和氟虫腈等,由于农药的不合理使用,多种害虫已对这些杀虫剂产生抗药性,急需新型替代药物。关于昆虫GluCls的研究,不但可以为田间害虫抗性监测提供帮助,同时也为新型高效高选择性杀虫剂的研发提供理论基础。   参考文献
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  (责任编辑:田 喆)
  收稿日期: 20190306   修订日期: 20190404
  基金项目:国家自然科学基金青年基金(31701807);江苏省自然科学基金青年基金(BK20170491)
  致  谢: 参加本试验部分工作的还有江代礼、谭翰杰、张能和纪烨斌等同学,特此一并致谢。
  通信作者 E-mail:wangjj@ yzu.edu.cn
  #为并列第一作者
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