RESUMO
BACKGROUND AND PURPOSE: Neonicotinoid insecticides interact with the orthosteric site formed at subunit interfaces of insect nicotinic ACh (nACh) receptors. However, their interactions with the orthosteric sites at α-non α and α-α subunit interfaces remain poorly understood. The aim of this study was to elucidate the mechanism of neonicotinoid actions using the Drosophila Dα1-chicken ß2 hybrid nACh receptor. EXPERIMENTAL APPROACH: Computer models of the (Dα1)3 (ß2)2 nACh receptor in complex with imidacloprid and thiacloprid were generated. Amino acids in the Dα1 subunit were mutated to corresponding amino acids in the human α4 subunit to examine their effects on the agonist actions of neonicotinoids on (Dα1)3 (ß2)2 and (Dα1)2 (ß2)3 nACh receptors expressed in Xenopus laevis oocytes using voltage-clamp electrophysiology. KEY RESULTS: The (Dα1)3 (ß2)2 nACh receptor models indicated that amino acids in loops D, E and G probably determine the effects of neonicotinoids. The amino acid mutations tested had minimal effects on the EC50 for ACh. However, the R57S mutation in loop G, although having minimal effect on imidacloprid's actions, reduced the affinity of thiacloprid for the (Dα1)3 (ß2)2 nACh receptor, while scarcely affecting thiacloprid's action on the (Dα1)2 (ß2)3 nACh receptor. Both the K140T and the combined R57S;K140T mutations reduced neonicotinoid efficacy but only for the (Dα1)3 (ß2)2 nACh receptor. Combining the E78K mutation with the R57S;K140T mutations resulted in a selective reduction of thiacloprid's affinity for the (Dα1)3 (ß2)2 nACh receptor. CONCLUSIONS AND IMPLICATIONS: These findings suggest that a triangle of residues from loops D, E and G contribute to the selective actions of neonicotinoids on insect-vertebrate hybrid nACh receptors. LINKED ARTICLES: This article is part of a themed section on Nicotinic Acetylcholine Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.11/issuetoc.
