Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels mediating fast cholinergic synaptic transmission in nervous system. In insects, nAChRs are the target sites for several naturally occurring and synthetic compounds, including the neonicotinoid insecticides. So far, one of the major strategies to explore the interaction of nAChR and ligands is based on the heterologous expression of nAChR, which is tough, and needs to be explored. In this study, we expressed and purified extracellular domain of rat a7 subunit (Rα7-ECD), the binding site of the ligands in E. coli and determined the interactions and kinetic constants of neonicotinoid insecticides with Rα7-ECD. The recombinant Rα7-ECD is water-soluble and appears to be correctly folded. The interactions of three neonicotinoid pesticides with Rα7-ECD were assessed by surface plasmon resonance (SPR) biosensor. The results revealed a fast association and fast disassociation binding mode of Rα7-ECD/pesticides complexes with the KD value of clothianidin (6.414E-9 M) > imidacloprid (9.030E-9 M) > acetamiprid (2.874E-6 M), respectively. This study demonstrated that the nAChR expressed from E. coli was functional, and SPR biosensor technology would be a good alternative for characterizing members of nAChR receptor family.

中文翻译：

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可溶性功能性α7-烟碱乙酰胆碱受体在大肠杆菌中的表达及其与新烟碱类农药的高亲和力结合

烟碱乙酰胆碱受体 (nAChRs) 是配体门控离子通道，介导神经系统中的快速胆碱能突触传递。在昆虫中，nAChRs 是几种天然和合成化合物的靶点，包括新烟碱类杀虫剂。迄今为止，探索nAChR与配体相互作用的主要策略之一是基于nAChR的异源表达，这很困难，需要探索。在这项研究中，我们表达并纯化了大鼠 a7 亚基 (Rα7-ECD) 的胞外结构域，即大肠杆菌中配体的结合位点，并确定了新烟碱类杀虫剂与 Rα7-ECD 的相互作用和动力学常数。重组 Rα7-ECD 是水溶性的，并且似乎正确折叠。通过表面等离子体共振 (SPR) 生物传感器评估了三种新烟碱类农药与 Rα7-ECD 的相互作用。结果表明，Rα7-ECD/农药复合物与噻虫胺（6.414E-9 M）>吡虫啉（9.030E-9 M）>啶虫脒（2.874E-6 M）的KD值具有快速结合和快速解离结合模式，分别。这项研究表明，从大肠杆菌中表达的 nAChR 是有功能的，SPR 生物传感器技术将是表征 nAChR 受体家族成员的一个很好的选择。