Drosophila nicotinic acetylcholine receptor subunits and their native interactions with insecticidal peptide toxins.
Drosophila nicotinic acetylcholine receptor subunits and their native interactions with insecticidal peptide toxins.
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DOI：
10.7554/elife.74322
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发表时间：
2022-05-16
期刊：
影响因子：
7.7
通讯作者：
Pless, Stephan A.
中科院分区：
文献类型：
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作者：
Korona, Dagmara;Dirnberger, Benedict;Giachello, Carlo N. G.;Queiroz, Rayner M. L.;Popovic, Rebeka;Mueller, Karin H.;Minde, David-Paul;Deery, Michael J.;Johnson, Glynnis;Firth, Lucy C.;Earley, Fergus G.;Russell, Steven;Lilley, Kathryn S.;Pless, Stephan A.
果蝇烟碱型乙酰胆碱受体(NAChRs)是一种配体门控离子通道，是杀虫剂的靶标。已知的多肽神经毒素通过与其靶亚基结合来阻断nAChRs，然而，为了有效地设计杀虫剂，需要更好地了解这一机制。为了便于nAChRs的分析，我们使用CRISPR/Cas9策略在相同的遗传背景下生成所有10个nAChR亚单位基因的空等位基因。我们通过幼虫注射和苯乙烯马来酸脂质颗粒(SMALPs)下拉试验研究了nAChR亚基与多肽神经毒素的相互作用。对于无效等位基因，我们确定了α-银环蛇毒素(α-Btx)和ω-Hexatoxin-Hv1a(Hv1a)的作用，确定了与这些毒素结合有关的潜在受体亚基。我们用下拉实验证实了α-Btx与果蝇α5(Dα5)、Dα6、Dα7亚基的相互作用。最后，我们报道了荧光标记的内源性Dα6在果蝇中枢神经系统发育过程中的定位。综上所述，本研究阐明了果蝇nAChR亚基与杀虫多肽毒素的相互作用，为昆虫nAChRs的体内分析提供了资源。
Drosophila nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that represent a target for insecticides. Peptide neurotoxins are known to block nAChRs by binding to their target subunits, however, a better understanding of this mechanism is needed for effective insecticide design. To facilitate the analysis of nAChRs we used a CRISPR/Cas9 strategy to generate null alleles for all ten nAChR subunit genes in a common genetic background. We studied interactions of nAChR subunits with peptide neurotoxins by larval injections and styrene maleic acid lipid particles (SMALPs) pull-down assays. For the null alleles, we determined the effects of α-Bungarotoxin (α-Btx) and ω-Hexatoxin-Hv1a (Hv1a) administration, identifying potential receptor subunits implicated in the binding of these toxins. We employed pull-down assays to confirm α-Btx interactions with the Drosophila α5 (Dα5), Dα6, Dα7 subunits. Finally, we report the localisation of fluorescent tagged endogenous Dα6 during Drosophila CNS development. Taken together, this study elucidates native Drosophila nAChR subunit interactions with insecticidal peptide toxins and provides a resource for the in vivo analysis of insect nAChRs.