ABSTRACT
Bifenazate is a very selective acaricide that controls the spider mite, Tetranychus urticae. Bifenazate is the first example of a carbazate acaricide. Its mode of action remains unclear. Bifenazate and its active metabolite diazene induce paralysis in spider mites, suggesting that they may act on the nervous system. Here we have employed a homologue (TuGABAR) of RDL (Resistance to dieldrin), a subunit of ionotropic γ-aminobutyric acid (GABA) receptor, from T. urticae to investigate the action of bifenazate and its active metabolite diazene on this receptor function. Although neither acaricide showed a GABA agonist action, 30 µM of bifenazate or diazene significantly enhanced the GABA-induced response of TuGABAR in a dose-dependent manner, shifting the EC(50) of GABA from 24.8 µM to 4.83 µM and 10.8 µM, respectively. This action demonstrates a positive allosteric modulator effect of bifenazate on T. urticae GABA receptors. This synergistic action is likely the result of bifenazate binding to a site distinct from that of the GABA binding site causing a conformational change that affects the magnitude of the GABA response. Precisely how the observed GABA synergist action correlates with the acaricidal activity of bifenazate, if at all, has yet to be determined.
Subject(s)
Acaricides/pharmacology , Carbamates/pharmacology , Chloride Channels/drug effects , Hydrazines/pharmacology , Imides/pharmacology , Ion Channel Gating/drug effects , Receptors, GABA/drug effects , Tetranychidae/drug effects , gamma-Aminobutyric Acid/metabolism , Acaricides/metabolism , Animals , Binding Sites , Carbamates/metabolism , Chloride Channels/chemistry , Chloride Channels/genetics , Chloride Channels/metabolism , Cloning, Molecular , Dose-Response Relationship, Drug , Hydrazines/metabolism , Imides/metabolism , Membrane Potentials , Protein Conformation , Receptors, GABA/chemistry , Receptors, GABA/genetics , Receptors, GABA/metabolism , Structure-Activity Relationship , Tetranychidae/metabolism , Xenopus laevisABSTRACT
Neonicotinoid insecticides are widely used for crop protection based on their selective actions on insect nicotinic acetylcholine receptors (insect nAChRs). Loops C and D in insect nAChRs have been shown to possess structural features favorable for neonicotinoid-nAChR interactions. However, it remains to be resolved whether such features serve either co-operatively, or independently, to enhance neonicotinoid sensitivity of nAChRs. We therefore examined using voltage-clamp electrophysiology the effects on the response to imidacloprid of combinatorial substitutions of residues in loops C and D of the chicken alpha4beta2 nAChR by those present in insect nAChRs. The E219P mutation in loop C of the alpha4 subunit resulted in enhanced responses to imidacloprid of alpha4beta2, whereas E219S and E219T mutations barely influenced its actions. On the other hand, mutations in loop D (T77R; E79V and T77N; E79R) alone shifted the imidacloprid concentration-response curve to the left (lower concentrations). Interestingly, all three mutations did, however, further enhance the agonist efficacy of imidacloprid when combined with the mutations in loop D. Such synergistic effects of the two loops on the interactions with imidaclprid were observed irrespective of subunit stoichiometry. Computational modeling of the ligand binding domain of the wild-type and mutant alpha4beta2 nAChRs using the crystal structure of the acetylcholine binding protein from Lymnaea stagnalis also indicated that interactions with loop F of loops C and D may contribute to determining the response to imidacloprid.
