昆虫烟碱型乙酰胆碱受体的组成及其药理学特性研究
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摘要
烟碱型乙酰胆碱受体(nAChRs)是脊椎动物和无脊椎动物神经系统中快速介导胆碱能突触传递的配基门控离子通道。在脊椎动物中,nAChRs在神经肌肉接头以及中枢和周缘神经系统中均有表达。在昆虫中,nAChRs在神经肌肉接头处没有分布,却是中枢神经系统中最丰富的神经递质受体,是重要的杀虫剂作用靶标。目前通过分子克隆和基因组测序技术在多种昆虫中发现了大量的nAChRs亚基,为昆虫nAChRs的研究提供了坚实的基础。在昆虫nAChRs亚基功能研究中,试图体外组建昆虫功能性nAChRs一直是国际研究热点,但近20年来始终没有突破,主要有两个问题没有解决：第一,昆虫神经系统中nAChRs蛋白复合体的组成仍然未知；第二,在异源表达系统中无法获得有功能的昆虫nAChRs同型五聚体。
本文利用非洲爪蟾卵母细胞表达系统,对褐飞虱的四个nAChRs a亚基与大鼠的nAChRs β2亚基进行了体外重组,通过双电极电压钳技术及放射性配基结合实验研究了重组型受体的药理学特性,并利用免疫共沉淀技术对昆虫内源性nAChRs a亚基的组成进行了探索。通过搜索褐飞虱EST数据库及基因克隆获得了两个Ly-6/neurotoxin超家族基因,分别将其命名为Nl-lynx1和Nl-lynx2.在异源表达系统中检测了这两个lynx蛋白对重组型受体NIαl/rβ2的调节作用,并对其调节机理进行了研究。
一、昆虫烟碱型乙酰胆碱受体a1、a2亚基的异源表达与功能研究
吡虫啉等新烟碱类杀虫剂作为昆虫烟碱型乙酰胆碱受体(nAChRs)的选择性激动剂,在农业害虫和卫生害虫的防治中应用广泛。褐飞虱(Nilaparvata lugens)是亚洲许多地区的主要水稻害虫。本文在异源表达系统中对褐飞虱nAChRs两个a亚基Nlal和Nla2进行了体外重组,并对重组型受体的功能特性进行了研究。在非洲爪蟾卵母细胞中,Nlal和Nla2分别与哺乳动物的β2亚基共表达,均能够形成有功能的受体,这与以往对于昆虫a亚基的研究结果一致。两种重组受体Nlαl/rβ2和Nlα2/rβ2对乙酰胆碱的激动剂剂量反应相差不足两倍,但是,Nlα1/rβ2受体对吡虫啉的亲和力显著高于Nlα2/rβ2受体,其EC5o分别为61±7μM(n=9)和870±76μM(n=13)。
在卵母细胞中同时表达Nlα1, Nla2和哺乳动物的β2亚基,所形成的受体对激动剂的剂量反应曲线介于Nlα1/rβ2受体和Nlα2/rβ2受体对激动剂的剂量反应曲线之间,但又与计算机模拟的两种受体同时存在时的剂量反应曲线存在显著差异,由此推测,Nlα1, Nlα2和哺乳动物的β2亚基可能组成一个三亚基五聚体Nlα1/Nlα2/rβ2。改变各亚基的注射比例,激动剂的EC50没有显著变化,进一步证明三个亚基共表达时形成的受体不是亚基组成不同的nAChRs的混合物,而是单一的重组型nAChRs。为证明在褐飞虱体内Nlal和Nla2两个亚基是否存在于同一个受体中,进一步研究昆虫内源性nAChRs的组成,本文分别以Nlal和N1a2亚基胞内环的氨基酸序列为抗原制备特异性的多克隆抗体,进行了免疫共沉淀,研究结果证明,在褐飞虱体内N1a1和N1a2亚基的确存在于同一个受体中。
二、昆虫烟碱型乙酰胆碱受体a3、a8亚基的异源表达与功能研究
昆虫烟碱型乙酰胆碱受体(nAChRs)是以吡虫啉为代表的新烟碱类杀虫剂的作用靶标。目前,通过基因组序列分析和基因克隆的方法在包括黑腹果蝇(Drosophila melanogaster)在内的多种昆虫中鉴定出了大量的nAChRs亚基。系统发生关系的分析结果表明,果蝇Dβ2亚基与许多昆虫的a8亚基属于同一个亚类群中。以往的研究中并没有关于在异源表达系统中成功表达昆虫a8/Dβ2亚类群nAChRs亚基的报道,但是,有证据表明DD2亚基可以与其它两个亚基共聚,形成三亚基五聚体。
本文通过RT-PCR和RACE技术克隆了褐飞虱Nla8亚基(GenBank登录号：FJ481979),并首次报道了包含昆虫nAChRs a8亚基的三亚基五聚体Nla8/Nla3/rβ2。Nla8亚基基因全长2036bp,开放阅读框为1617bp,编码538个氨基酸,与意大利蜜蜂(Apis mellifera)、赤拟谷盗(Tribolium castaneum)、冈比亚按蚊(Anopheles gambiae)的nAChRs a8亚基具有较高的相似性,且具有nAChRs a亚基基因家族的特征结构。在非洲爪蟾卵母细胞表达系统中,褐飞虱Nla8亚基可以与哺乳动物p亚基rβ2共聚,形成重组型受体Nlα8/rβ2;也可以与Nla3亚基及rβ2共聚,形成三亚基五聚体Nlα8/Nlα3/rβ2,且该受体对激动剂的剂量反应显著高于Nlα8/rβ2nAChRs. Nlα/Nlα/rp2对吡虫啉的最大反应电流Imax是Nlα8/rβ2的29.69倍。剂量依赖反应的研究结果表明,Nlα8/rβ2对乙酰胆碱的ECs0是Nlα8/Nlα3/rβ的5.58倍,而Nlα8/rβ2对吡虫啉的EC50约是Nlα8/Nlα/rβ2的40倍。免疫共沉淀的研究结果进一步证明,Nla3与Nla8亚基存在于同一个内源性1nAChRs中。
三、昆虫烟碱型乙酰胆碱受体附属蛋白的克隆及其对受体的调节作用
在昆虫体内或离体条件下,除了nAChRs亚基组成外,一些附属蛋白,包括分子伴侣,调控因子或调节蛋白,在昆虫nAChRs的蛋白折叠与成熟、复杂结构组成、蛋白功能等方面起到十分重要的作用。本文通过搜索褐飞虱EST数据库(http://bphest.dna.affrc.go.jp/:Noda et al.,2008)及基因克隆获得了两个Ly-6/neurotoxin超家族基因,分别将其命名为Nl-lynx1和Nl-lynx2。Nl-lynx1和Nl-lynx2与萤火虫Pr-lynx1基因的相似性分别为53%和50%,与小鼠lynx1基因的相似性分别为39%和41%,与小鼠lynx2基因的相似性分别为42%和44%。尽管褐飞虱Nl-lynx1和Nl-lynx2基因与萤火虫的Pr-lynx1基因及脊椎动物的Ly-6/neurotoxin超家族的基因不具有较高的相似性,但是具有Ly-6/neurotoxin超家族基因的特征结构。在非洲爪蟾卵母细胞表达系统中,Nl-Lynx1和Nl-Lynx2能够上调重组型受体Nlα1/rβ2对激动剂反应的最大电流,但是对受体对激动剂的敏感性没有影响。Nl-Lynx1与重组型受体Nlα1/rβ2共表达时,受体对乙酰胆碱和吡虫啉的Imax分别是单独表达Nlα1/rp2时的3.56倍和1.72倍;Nl-Lynx2与重组型受体Nlal/rβ2共表达时,受体对乙酰胆碱和吡虫啉的Imax分别是单独表达Nla1/rβ2时的3.25倍和1.51倍。放射性配基结合实验的结果表明,Nl-lynx1或Nl-lynx2与受体Nla1/rβ2共表达只能增加配基的最大结合量(Bmax)不改变受体与配基结合的平衡常数(Kd),即不改变受体对配基的亲和力。进一步的研究结果表明,Nl-lynx1或Nl-lynx2对受体的脱敏特性及nAChRs亚基表达水平没有影响,且在一定范围内Nla1/rβ2对激动剂的反应电流随Nl-lynx:主射量的增加而增大,由此推测,Nl-lynx对受体的调节作用可能是通过提高蛋白质折叠和亚基组装的效率实现的。
本文的研究结果表明,两种不同的昆虫nAChRs α亚基共表达可以提高受体对激动剂的反应,但仍需哺乳动物β亚基的参与才能够在异源表达系统中构建功能受体；附属蛋白与受体亚基共表达仍然不能形成有功能的受体,由此推测昆虫nAChRs的蛋白复合体中尚有未知的蛋白需要鉴定,试图体外组建昆虫功能性nAChRs可能需要更多的未知附属蛋白。因此,本研究通过免疫共沉淀分离纯化包含某一亚基的昆虫内源性nAChRs蛋白复合体,经SDS-PAGE电泳进行分离,LC-MS/MS质谱鉴定的方法,对nAChRs蛋白复合体中的未知蛋白鉴定进行了尝试。虽然,由于抗体特异性等原因导致鉴定出的蛋白数量过多,难以筛选出目的蛋白并对其功能进行验证,但是,应用免疫共沉淀,亲和层析等方法对昆虫1nAChRs蛋白复合体进行分离纯化,结合基因组或转录组数据库,通过质谱对其组分进行鉴定的方法仍然具有可行性。应用蛋白质组学和基因组学的技术手段,鉴定昆虫nAChRs蛋白复合体的组成,体外构建功能性昆虫nAChRs,仍然是未来昆虫nAChRs结构与功能研究的热点之一
Nicotinic acetylcholine receptors (nAChRs) are neurotransmitter-gated ion channels, which mediate fast cholinergic synaptic transmission in insect and vertebrate nervous systems. In mammals and other vertebrates, nAChRs are expressed at both the neuromuscular junction and within the CNS (Central Nervous System) and PNS (Peripheral Nervous System). In insects, though not expressed at the neuromuscular junction, nAChRs play an important role in the insect CNS, where ACh is the major excitatory neurotransmitter. Many insect nAChR subunits have been identified and characterized by molecular cloning and genome sequencing. In the last two decades, studies have focused on the heterologous expression of functional insect nAChRs. However, in the case of insects such as Drosophila with all subunits identified, considerable problems remain in identification of native subunit composition and even in generating functional insect nAChRs in heterologous expression systems. Some proteins other than nAChRs themselves might play important roles in insect nAChRs formation in vivo and in vitro, such as the chaperones, regulators and modulators.
In the present study, we co-expressed four nAChRs a subunits of brown planthopper, Nilaparvata lugens, with rat β2subunit in Xenopus oocytes and characterized the hybrid nAChRs using electrophysiological recordings with a two-electrode voltage clamp and radioligand binding assay. Co-immunoprecipitation was used to examine the native nAChRs subunit composition. We also reported the identification of two members of the Ly-6/neurotoxin superfamily in the brown planthopper and their action as the modulators on insect nAChRs were evaluated in the Xenopus oocytes heterologous expression system. The mechanism of these two modulators effecting on the agonist potency was also discussed.
1. Heterologous co-expression and pharmacological characterization of two insect nAChRs a subunits:Nlal and Nla2
Neonicotinoid insecticides, such as imidacloprid, are selective agonists of insect nAChRs and are used extensively in areas of crop protection and animal health to control a variety of insect pest species. Here, we describe studies performed with nAChR subunits Nlal and Nla2cloned from the N. lugens, a major insect pest of rice crops in many parts of Asia. When Nlal or Nla2was co-expressed with the rat nAChR β2subunit in Xenopus oocytes, functional hybrid nAChRs could be generated, which is consistent with previous studies. The difference in EC50values for ACh from oocytes expressing Nlal/rP2and Nla2/rβ2nAChRs was less than two fold (EC50=37±6μM and EC5o=62±10μM, respectively; n=9). Whereas EC50values for imidacloprid were61±7μM (n=9) and870±76μM (n=13) for Nlal/rβ2and Nlα2/rβ2, respectively.
In oocytes co-injected with the Nlal, Nla2, and rP2subunit cRNAs, the experimental dose-response curve differed from theoretical dose-response curves predicted for mixed populations of Nla1/rβ2and Nla2/rβ2nAChRs, and no plateau was detected in the experimental dose-response data, which provided evidence for a single population of 'triplet' Nla1/Nla2/rβ2nAChR. Increasing the ratio of the P2subunit resulted in reduced maximal currents, but no significant difference in EC50values was detected, which also proved that one 'triplet' nAChR was generated. In addition, evidence has also been obtained from immunoprecipitation study to indicate that Nlal and Nla2subunit co-assembled in N. lugens native nAChR.
2. Heterologous co-expression and pharmacological characterization of two insect nAChRs a subunits:Nla3and Nla8
Genome sequence analysis has provided information concerning subunit diversity of nAChRs from several insect species. Phylogenetic analysis suggests that the 'DP2group' contains 'a' type subunit in most insect species including a8subunit. There have been no reports of the successful functional heterologous expression of any nAChR subunit from the insect a8/DP2subunit group previously. But there is the evidence to suggest that the Dβ2subunit might be able to co-assemble with two other subunits into a triplet nAChR. Here we cloned the nAChRs a8subunit of N. lugens (GenBank accession number FJ481979) and demonstrated for the first time the incorporation of an insect nAChR subunit from the a8/Dβ2group into a functional recombinant nAChR.
The full length of N. lugens nAChRs a8subunit is2036bp, including1617bβ ORF which code538amino acid, and share close sequence similarity with nAChR a8subunits from honey bee Apis mellifera, the malaria mosquito Anopheles gambiae, and the red flour beetle Tribolium castaneum. Heterologous expression demonstrated that when Nla8was co-expressed with rat β2subunit or co-expressed together with Nla3and rβ2, functional nAChRs were detected. However, in oocytes co-injected with the Nlα3, Nlα8, and rβ2subunit cRNAs, much larger responses were detected than those in oocytes expressing Nla8/rβ2nAChRs. The maximal agonist response (Imax) detected with imidacloprid in oocytes expressing Nla8/rβ2nAChRs was29.69fold higher than Imax in oocytes expressing Nla3/Nla8/rβ2nAChRs. EC50values for acetylcholine were95.8±11.7and17.6±3.7μM for Nla8/rβ2and Nla3/Nla8/rβ2nAChRs, respectively, and for imidacloprid values were127.9±19.3and3.2±0.5μM, respectively. These data indicated Nla3, Nla8, and rβ2subunit generated a'triple'nAChRs. Evidence has also been obtained from immuno-precipitation study to indicate that Nla3and Nla8subunit co-assemble in native nAChR.
3. Identification of two Lynx proteins and the modulation on insect nAChRs
Some proteins other than nAChRs themselves might play important roles in insect nAChRs formation in vivo and in vitro, such as the chaperone, regulator and modulator. In present study, two members of the Ly-6/neurotoxin superfamily have been identified by blasting in EST database of N. lugens with Pr-lynxl (Pyrocoelia rufa) and named as Nl-lynxl and Nl-lynx2. Although Nl-lynxl and Nl-lynx2did not show very high similarities to Pr-lynx1(53%and50%) and vertebrate Ly-6/neurotoxin superfamily, such as Mus musculus lynxl (39%and41%) and lynx2(42%and44%), both Nl-lynx1and Nl-lynx2contained the signature cysteine-rich motif of the Ly-6/neurotoxin superfamily. Similar to Pr-lynxl, both Nl-lynxl and Nl-lynx2only increased agonist-evoked currents, but did not change the agonist sensitivity on nAChRs expressed in Xenopus oocytes. In Xenopus oocytes expressing Nlal/rβ2, the addition of Nl-lynx1or Nl-lynx2increased Imax of ACh to3.56-fold and3.25-fold respectively, and increased Imax of imidacloprid to1.72-fold and1.51-fold respectively. Radioligand binding assay determined that co-expression of Nl-lynx1or Nl-lynx2with Nlal/rβ2only increased the maximum binding (Bmax), but did not change the binding affinity (Kd). Further studies showed that the co-expression of Nl-lynxl or Nl-lynx2did not change the desensitization properties of Nlal/rβ2or the expression levels of Nlalor rβ2subunit on oocyte surface. So, the higher efficiency of protein folding and subunit assembly might be the important reasons for the significantly higher level of binding detected with co-expression of Nlal/rβand Nl-lynxl or N1-lynx2.
The present study shows that co-expression of two insect nAChRs a subunits is able to increase agonist response of hybrid receptors, but functional nAChRs could not be detected in Xenopus oocytes without mammalian β subunit, even with nAChRs modulator Nl-lynx1or Nl-lynx2, which suggests additional accessory proteins need to be identified for heterologous expression of functional insect nAChRs. So, we have tried to identify more proteins of native nAChRs complex by co-immunoprecipitation, SDS-PAGE electro-phoresis and LC-MS/MS. Although the number of proteins identified is too large to find useful information, the method we used was proved to be feasible.
本文利用非洲爪蟾卵母细胞表达系统,对褐飞虱的四个nAChRs a亚基与大鼠的nAChRs β2亚基进行了体外重组,通过双电极电压钳技术及放射性配基结合实验研究了重组型受体的药理学特性,并利用免疫共沉淀技术对昆虫内源性nAChRs a亚基的组成进行了探索。通过搜索褐飞虱EST数据库及基因克隆获得了两个Ly-6/neurotoxin超家族基因,分别将其命名为Nl-lynx1和Nl-lynx2.在异源表达系统中检测了这两个lynx蛋白对重组型受体NIαl/rβ2的调节作用,并对其调节机理进行了研究。
一、昆虫烟碱型乙酰胆碱受体a1、a2亚基的异源表达与功能研究
吡虫啉等新烟碱类杀虫剂作为昆虫烟碱型乙酰胆碱受体(nAChRs)的选择性激动剂,在农业害虫和卫生害虫的防治中应用广泛。褐飞虱(Nilaparvata lugens)是亚洲许多地区的主要水稻害虫。本文在异源表达系统中对褐飞虱nAChRs两个a亚基Nlal和Nla2进行了体外重组,并对重组型受体的功能特性进行了研究。在非洲爪蟾卵母细胞中,Nlal和Nla2分别与哺乳动物的β2亚基共表达,均能够形成有功能的受体,这与以往对于昆虫a亚基的研究结果一致。两种重组受体Nlαl/rβ2和Nlα2/rβ2对乙酰胆碱的激动剂剂量反应相差不足两倍,但是,Nlα1/rβ2受体对吡虫啉的亲和力显著高于Nlα2/rβ2受体,其EC5o分别为61±7μM(n=9)和870±76μM(n=13)。
在卵母细胞中同时表达Nlα1, Nla2和哺乳动物的β2亚基,所形成的受体对激动剂的剂量反应曲线介于Nlα1/rβ2受体和Nlα2/rβ2受体对激动剂的剂量反应曲线之间,但又与计算机模拟的两种受体同时存在时的剂量反应曲线存在显著差异,由此推测,Nlα1, Nlα2和哺乳动物的β2亚基可能组成一个三亚基五聚体Nlα1/Nlα2/rβ2。改变各亚基的注射比例,激动剂的EC50没有显著变化,进一步证明三个亚基共表达时形成的受体不是亚基组成不同的nAChRs的混合物,而是单一的重组型nAChRs。为证明在褐飞虱体内Nlal和Nla2两个亚基是否存在于同一个受体中,进一步研究昆虫内源性nAChRs的组成,本文分别以Nlal和N1a2亚基胞内环的氨基酸序列为抗原制备特异性的多克隆抗体,进行了免疫共沉淀,研究结果证明,在褐飞虱体内N1a1和N1a2亚基的确存在于同一个受体中。
二、昆虫烟碱型乙酰胆碱受体a3、a8亚基的异源表达与功能研究
昆虫烟碱型乙酰胆碱受体(nAChRs)是以吡虫啉为代表的新烟碱类杀虫剂的作用靶标。目前,通过基因组序列分析和基因克隆的方法在包括黑腹果蝇(Drosophila melanogaster)在内的多种昆虫中鉴定出了大量的nAChRs亚基。系统发生关系的分析结果表明,果蝇Dβ2亚基与许多昆虫的a8亚基属于同一个亚类群中。以往的研究中并没有关于在异源表达系统中成功表达昆虫a8/Dβ2亚类群nAChRs亚基的报道,但是,有证据表明DD2亚基可以与其它两个亚基共聚,形成三亚基五聚体。
本文通过RT-PCR和RACE技术克隆了褐飞虱Nla8亚基(GenBank登录号：FJ481979),并首次报道了包含昆虫nAChRs a8亚基的三亚基五聚体Nla8/Nla3/rβ2。Nla8亚基基因全长2036bp,开放阅读框为1617bp,编码538个氨基酸,与意大利蜜蜂(Apis mellifera)、赤拟谷盗(Tribolium castaneum)、冈比亚按蚊(Anopheles gambiae)的nAChRs a8亚基具有较高的相似性,且具有nAChRs a亚基基因家族的特征结构。在非洲爪蟾卵母细胞表达系统中,褐飞虱Nla8亚基可以与哺乳动物p亚基rβ2共聚,形成重组型受体Nlα8/rβ2;也可以与Nla3亚基及rβ2共聚,形成三亚基五聚体Nlα8/Nlα3/rβ2,且该受体对激动剂的剂量反应显著高于Nlα8/rβ2nAChRs. Nlα/Nlα/rp2对吡虫啉的最大反应电流Imax是Nlα8/rβ2的29.69倍。剂量依赖反应的研究结果表明,Nlα8/rβ2对乙酰胆碱的ECs0是Nlα8/Nlα3/rβ的5.58倍,而Nlα8/rβ2对吡虫啉的EC50约是Nlα8/Nlα/rβ2的40倍。免疫共沉淀的研究结果进一步证明,Nla3与Nla8亚基存在于同一个内源性1nAChRs中。
三、昆虫烟碱型乙酰胆碱受体附属蛋白的克隆及其对受体的调节作用
在昆虫体内或离体条件下,除了nAChRs亚基组成外,一些附属蛋白,包括分子伴侣,调控因子或调节蛋白,在昆虫nAChRs的蛋白折叠与成熟、复杂结构组成、蛋白功能等方面起到十分重要的作用。本文通过搜索褐飞虱EST数据库(http://bphest.dna.affrc.go.jp/:Noda et al.,2008)及基因克隆获得了两个Ly-6/neurotoxin超家族基因,分别将其命名为Nl-lynx1和Nl-lynx2。Nl-lynx1和Nl-lynx2与萤火虫Pr-lynx1基因的相似性分别为53%和50%,与小鼠lynx1基因的相似性分别为39%和41%,与小鼠lynx2基因的相似性分别为42%和44%。尽管褐飞虱Nl-lynx1和Nl-lynx2基因与萤火虫的Pr-lynx1基因及脊椎动物的Ly-6/neurotoxin超家族的基因不具有较高的相似性,但是具有Ly-6/neurotoxin超家族基因的特征结构。在非洲爪蟾卵母细胞表达系统中,Nl-Lynx1和Nl-Lynx2能够上调重组型受体Nlα1/rβ2对激动剂反应的最大电流,但是对受体对激动剂的敏感性没有影响。Nl-Lynx1与重组型受体Nlα1/rβ2共表达时,受体对乙酰胆碱和吡虫啉的Imax分别是单独表达Nlα1/rp2时的3.56倍和1.72倍;Nl-Lynx2与重组型受体Nlal/rβ2共表达时,受体对乙酰胆碱和吡虫啉的Imax分别是单独表达Nla1/rβ2时的3.25倍和1.51倍。放射性配基结合实验的结果表明,Nl-lynx1或Nl-lynx2与受体Nla1/rβ2共表达只能增加配基的最大结合量(Bmax)不改变受体与配基结合的平衡常数(Kd),即不改变受体对配基的亲和力。进一步的研究结果表明,Nl-lynx1或Nl-lynx2对受体的脱敏特性及nAChRs亚基表达水平没有影响,且在一定范围内Nla1/rβ2对激动剂的反应电流随Nl-lynx:主射量的增加而增大,由此推测,Nl-lynx对受体的调节作用可能是通过提高蛋白质折叠和亚基组装的效率实现的。
本文的研究结果表明,两种不同的昆虫nAChRs α亚基共表达可以提高受体对激动剂的反应,但仍需哺乳动物β亚基的参与才能够在异源表达系统中构建功能受体；附属蛋白与受体亚基共表达仍然不能形成有功能的受体,由此推测昆虫nAChRs的蛋白复合体中尚有未知的蛋白需要鉴定,试图体外组建昆虫功能性nAChRs可能需要更多的未知附属蛋白。因此,本研究通过免疫共沉淀分离纯化包含某一亚基的昆虫内源性nAChRs蛋白复合体,经SDS-PAGE电泳进行分离,LC-MS/MS质谱鉴定的方法,对nAChRs蛋白复合体中的未知蛋白鉴定进行了尝试。虽然,由于抗体特异性等原因导致鉴定出的蛋白数量过多,难以筛选出目的蛋白并对其功能进行验证,但是,应用免疫共沉淀,亲和层析等方法对昆虫1nAChRs蛋白复合体进行分离纯化,结合基因组或转录组数据库,通过质谱对其组分进行鉴定的方法仍然具有可行性。应用蛋白质组学和基因组学的技术手段,鉴定昆虫nAChRs蛋白复合体的组成,体外构建功能性昆虫nAChRs,仍然是未来昆虫nAChRs结构与功能研究的热点之一
Nicotinic acetylcholine receptors (nAChRs) are neurotransmitter-gated ion channels, which mediate fast cholinergic synaptic transmission in insect and vertebrate nervous systems. In mammals and other vertebrates, nAChRs are expressed at both the neuromuscular junction and within the CNS (Central Nervous System) and PNS (Peripheral Nervous System). In insects, though not expressed at the neuromuscular junction, nAChRs play an important role in the insect CNS, where ACh is the major excitatory neurotransmitter. Many insect nAChR subunits have been identified and characterized by molecular cloning and genome sequencing. In the last two decades, studies have focused on the heterologous expression of functional insect nAChRs. However, in the case of insects such as Drosophila with all subunits identified, considerable problems remain in identification of native subunit composition and even in generating functional insect nAChRs in heterologous expression systems. Some proteins other than nAChRs themselves might play important roles in insect nAChRs formation in vivo and in vitro, such as the chaperones, regulators and modulators.
In the present study, we co-expressed four nAChRs a subunits of brown planthopper, Nilaparvata lugens, with rat β2subunit in Xenopus oocytes and characterized the hybrid nAChRs using electrophysiological recordings with a two-electrode voltage clamp and radioligand binding assay. Co-immunoprecipitation was used to examine the native nAChRs subunit composition. We also reported the identification of two members of the Ly-6/neurotoxin superfamily in the brown planthopper and their action as the modulators on insect nAChRs were evaluated in the Xenopus oocytes heterologous expression system. The mechanism of these two modulators effecting on the agonist potency was also discussed.
1. Heterologous co-expression and pharmacological characterization of two insect nAChRs a subunits:Nlal and Nla2
Neonicotinoid insecticides, such as imidacloprid, are selective agonists of insect nAChRs and are used extensively in areas of crop protection and animal health to control a variety of insect pest species. Here, we describe studies performed with nAChR subunits Nlal and Nla2cloned from the N. lugens, a major insect pest of rice crops in many parts of Asia. When Nlal or Nla2was co-expressed with the rat nAChR β2subunit in Xenopus oocytes, functional hybrid nAChRs could be generated, which is consistent with previous studies. The difference in EC50values for ACh from oocytes expressing Nlal/rP2and Nla2/rβ2nAChRs was less than two fold (EC50=37±6μM and EC5o=62±10μM, respectively; n=9). Whereas EC50values for imidacloprid were61±7μM (n=9) and870±76μM (n=13) for Nlal/rβ2and Nlα2/rβ2, respectively.
In oocytes co-injected with the Nlal, Nla2, and rP2subunit cRNAs, the experimental dose-response curve differed from theoretical dose-response curves predicted for mixed populations of Nla1/rβ2and Nla2/rβ2nAChRs, and no plateau was detected in the experimental dose-response data, which provided evidence for a single population of 'triplet' Nla1/Nla2/rβ2nAChR. Increasing the ratio of the P2subunit resulted in reduced maximal currents, but no significant difference in EC50values was detected, which also proved that one 'triplet' nAChR was generated. In addition, evidence has also been obtained from immunoprecipitation study to indicate that Nlal and Nla2subunit co-assembled in N. lugens native nAChR.
2. Heterologous co-expression and pharmacological characterization of two insect nAChRs a subunits:Nla3and Nla8
Genome sequence analysis has provided information concerning subunit diversity of nAChRs from several insect species. Phylogenetic analysis suggests that the 'DP2group' contains 'a' type subunit in most insect species including a8subunit. There have been no reports of the successful functional heterologous expression of any nAChR subunit from the insect a8/DP2subunit group previously. But there is the evidence to suggest that the Dβ2subunit might be able to co-assemble with two other subunits into a triplet nAChR. Here we cloned the nAChRs a8subunit of N. lugens (GenBank accession number FJ481979) and demonstrated for the first time the incorporation of an insect nAChR subunit from the a8/Dβ2group into a functional recombinant nAChR.
The full length of N. lugens nAChRs a8subunit is2036bp, including1617bβ ORF which code538amino acid, and share close sequence similarity with nAChR a8subunits from honey bee Apis mellifera, the malaria mosquito Anopheles gambiae, and the red flour beetle Tribolium castaneum. Heterologous expression demonstrated that when Nla8was co-expressed with rat β2subunit or co-expressed together with Nla3and rβ2, functional nAChRs were detected. However, in oocytes co-injected with the Nlα3, Nlα8, and rβ2subunit cRNAs, much larger responses were detected than those in oocytes expressing Nla8/rβ2nAChRs. The maximal agonist response (Imax) detected with imidacloprid in oocytes expressing Nla8/rβ2nAChRs was29.69fold higher than Imax in oocytes expressing Nla3/Nla8/rβ2nAChRs. EC50values for acetylcholine were95.8±11.7and17.6±3.7μM for Nla8/rβ2and Nla3/Nla8/rβ2nAChRs, respectively, and for imidacloprid values were127.9±19.3and3.2±0.5μM, respectively. These data indicated Nla3, Nla8, and rβ2subunit generated a'triple'nAChRs. Evidence has also been obtained from immuno-precipitation study to indicate that Nla3and Nla8subunit co-assemble in native nAChR.
3. Identification of two Lynx proteins and the modulation on insect nAChRs
Some proteins other than nAChRs themselves might play important roles in insect nAChRs formation in vivo and in vitro, such as the chaperone, regulator and modulator. In present study, two members of the Ly-6/neurotoxin superfamily have been identified by blasting in EST database of N. lugens with Pr-lynxl (Pyrocoelia rufa) and named as Nl-lynxl and Nl-lynx2. Although Nl-lynxl and Nl-lynx2did not show very high similarities to Pr-lynx1(53%and50%) and vertebrate Ly-6/neurotoxin superfamily, such as Mus musculus lynxl (39%and41%) and lynx2(42%and44%), both Nl-lynx1and Nl-lynx2contained the signature cysteine-rich motif of the Ly-6/neurotoxin superfamily. Similar to Pr-lynxl, both Nl-lynxl and Nl-lynx2only increased agonist-evoked currents, but did not change the agonist sensitivity on nAChRs expressed in Xenopus oocytes. In Xenopus oocytes expressing Nlal/rβ2, the addition of Nl-lynx1or Nl-lynx2increased Imax of ACh to3.56-fold and3.25-fold respectively, and increased Imax of imidacloprid to1.72-fold and1.51-fold respectively. Radioligand binding assay determined that co-expression of Nl-lynx1or Nl-lynx2with Nlal/rβ2only increased the maximum binding (Bmax), but did not change the binding affinity (Kd). Further studies showed that the co-expression of Nl-lynxl or Nl-lynx2did not change the desensitization properties of Nlal/rβ2or the expression levels of Nlalor rβ2subunit on oocyte surface. So, the higher efficiency of protein folding and subunit assembly might be the important reasons for the significantly higher level of binding detected with co-expression of Nlal/rβand Nl-lynxl or N1-lynx2.
The present study shows that co-expression of two insect nAChRs a subunits is able to increase agonist response of hybrid receptors, but functional nAChRs could not be detected in Xenopus oocytes without mammalian β subunit, even with nAChRs modulator Nl-lynx1or Nl-lynx2, which suggests additional accessory proteins need to be identified for heterologous expression of functional insect nAChRs. So, we have tried to identify more proteins of native nAChRs complex by co-immunoprecipitation, SDS-PAGE electro-phoresis and LC-MS/MS. Although the number of proteins identified is too large to find useful information, the method we used was proved to be feasible.
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