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Role in the Selectivity of Neonicotinoids of Insect-Specific Basic Residues in Loop D of the Nicotinic Acetylcholine Receptor Agonist Binding Site

Department of Applied Biological Chemistry, School of Agriculture, Kinki University, Nara, Japan (M.S., M.Y., M.I., K.M.); Graduate School of Agriculture, Kyoto University, Kyoto, Japan (M.A.); MRC Functional Genetics Unit, Department of Physiology, Anatomy and Genetics, Le Gros Clark Building, University of Oxford, Oxford, United Kingdom (D.B.S.)

The insecticide imidacloprid and structurally related neonicotinoids act selectively on insect nicotinic acetylcholine receptors (nAChRs). To investigate the mechanism of neonicotinoid selectivity, we have examined the effects of mutations to basic amino acid residues in loop D of the nAChR acetylcholine (ACh) binding site on the interactions with imidacloprid. The receptors investigated are the recombinant chicken 4β2 nAChR and Drosophila melanogaster D2/chicken β2 hybrid nAChR expressed in Xenopus laevis oocytes. Although mutations of Thr77 in loop D of the β2 subunit resulted in a barely detectable effect on the imidacloprid concentration-response curve for the 4β2 nAChR, T77R;E79V double mutations shifted the curve dramatically to higher affinity binding of imidacloprid. Likewise, T77K;E79R and T77N;E79R double mutations in the D2β2 nAChR also resulted in a shift to a higher affinity for imidacloprid, which exceeded that observed for a single mutation of Thr77 to basic residues. By contrast, these double mutations scarcely influenced the ACh concentration-response curve, suggesting selective interactions with imidacloprid of the newly introduced basic residues. Computational, homology models of the agonist binding domain of the wild-type and mutant 4β2 and D2β2 nAChRs with imidacloprid bound were generated based on the crystal structures of acetylcholine binding proteins of Lymnaea stagnalis and Aplysia californica. The models indicate that the nitro group of imidacloprid interacts directly with the introduced basic residues at position 77, whereas those at position 79 either prevent or permit such interactions depending on their electrostatic properties, thereby explaining the observed functional changes resulting from site-directed mutagenesis.

Address correspondence to: Kazuhiko Matsuda PhD, Department of Applied Biological Chemistry, School of Agriculture, Kinki University, 3327-204 Nakamachi, Nara 631-8505, Japan. Email: kmatsuda{at}nara.kindai.ac.jp

This article has been cited by other articles:

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