Department of Genetics, University of Cambridge, Downing Street, Cambridge, United Kingdom.
Cambridge Centre for Proteomics, Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom.
Elife. 2022 May 16;11:e74322. doi: 10.7554/eLife.74322.
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 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 α5 (D5), Dα6, D7 subunits. Finally, we report the localisation of fluorescent tagged endogenous Dα6 during CNS development. Taken together, this study elucidates native nAChR subunit interactions with insecticidal peptide toxins and provides a resource for the in vivo analysis of insect nAChRs.
烟碱型乙酰胆碱受体（nAChRs）是配体门控离子通道，是杀虫剂的作用靶点。肽神经毒素通过与靶亚基结合来阻断 nAChRs，然而，为了进行有效的杀虫剂设计，需要更好地了解这种机制。为了便于分析 nAChRs，我们使用了 CRISPR/Cas9 策略，在常见的遗传背景下生成了十个亚基基因的缺失等位基因。我们通过幼虫注射和苯乙烯马来酸脂粒（SMALPs）下拉测定研究了 nAChR 亚基与肽神经毒素的相互作用。对于缺失等位基因，我们确定了α-银环蛇毒素（α-Btx）和ω-六桶毒素-Hv1a（Hv1a）给药的影响，确定了这些毒素结合中涉及的潜在受体亚基。我们采用下拉测定法证实了 α-Btx 与α5（D5）、Dα6、D7 亚基的相互作用。最后，我们报告了荧光标记的内源性 Dα6 在 CNS 发育过程中的定位。总之，本研究阐明了天然 nAChR 亚基与杀虫肽毒素的相互作用，并为体内分析昆虫 nAChRs 提供了资源。
