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：
10.1101/2021.08.13.456240
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发表时间：
2021-08
期刊：
影响因子：
7.7
通讯作者：
D. Korona;Benedict Dirnberger;Carlo N G Giachello;R. Queiroz;David P. Minde;M. Deery;G. Johnson;Karin H. Müller;L. Firth;F. Earley;S. Russell;K. Lilley
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作者：
D. Korona;Benedict Dirnberger;Carlo N G Giachello;R. Queiroz;David P. Minde;M. Deery;G. Johnson;Karin H. Müller;L. Firth;F. Earley;S. Russell;K. Lilley
果蝇烟碱乙酰胆碱受体（nAChRs）是一种配体门控离子通道，是杀虫剂的靶标。众所周知，肽神经毒素可以通过与其靶亚基结合来阻断nachr，然而，为了有效设计杀虫剂，需要更好地了解受体亚基的组成。为了便于分析nAChR，我们使用CRISPR/Cas9策略在共同遗传背景下为所有10个nAChR亚基基因生成空等位基因。我们通过注射幼虫和苯乙烯马来酸脂质颗粒（SMALPs）下拉实验研究了nAChR亚基与肽神经毒素的相互作用。对于无效等位基因，我们确定了α-Bungarotoxin （α-Btx）和ω-Hexatoxin-Hv1a （Hv1a）给药的影响，确定了与这些毒素结合有关的潜在受体亚基。我们采用下拉法确定α-Btx与d - α5、d - α6、d - α7亚基的相互作用。最后，我们报道了荧光标记的内源性Dα6在神经系统发育过程中的定位。综上所述，本研究阐明了原生果蝇nAChR亚基与杀虫肽毒素的相互作用，并为昆虫nAChR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 receptor subunit composition is needed for effective design of insecticides. 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 Dα5, Dα6, Dα7 subunits. Finally, we report the localization of fluorescent tagged endogenous Dα6 during nervous system 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.