Received for publication May 18, 2006.
Revised July 3, 2006.
Accepted for publication July 25, 2006.

Role in the selectivity of neonicotinoids of insect-specific basic residues in loop D of the nicotinic acetylcholine receptor agonist binding site

Abstract

The insecticide imidacloprid and structurally-related neonicotinoids act selectively on insect nicotinic acetylcholine receptors (insect 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 42 nAChR and Drosophila melanogaster D2/chicken 2 hybrid nAChR expressed in Xenopus laevis oocytes. Although mutations of T77 in loop D of the 2 subunit resulted in a barely detectable effect on the imidacloprid concentration-response curve for the 42 nAChR, T77R;E79V double mutations shifted the curve dramatically to higher affinity binding of imidacloprid. Similarly, T77K;E79R and T77N;E79R double mutations in the D22 nAChR also resulted in a shift to a higher affinity for imidacloprid, which exceeded that observed for a single mutation of T77 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 42 and D22 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 offering an explanation for the observed functional changes resulting from site-directed mutagenesis.

Key words: Nicotinic cholinergic, Func. analysis receptor/ion channel mutants

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