Abstract
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.
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