Arthropod anion-permeable channel receptors, γ-aminobutyric acid (GABA) receptors (GABARs) and glutamic acid receptors (GluClRs), are important targets for pest control chemicals. Fluralaner, an isoxazoline ectoparasiticide, antagonizes the agonist action at GABARs more potently than at GluClRs, with a novel mode of action. Although fluralaner has been shown to act at a site that differs from that for conventional GABAR antagonist insecticides, its site of action remains to be defined. We previously reported that the replacement of L315 of the third transmembrane α-helix (TM3) of the housefly GluClR subunit with an amino acid at the corresponding position (i.e., F at 336) of the housefly GABAR subunit drastically enhanced the sensitivity of GluClRs to fluralaner. To provide further insight into the site of action of fluralaner, we substituted amino acid residues at the transmembrane subunit interface of the housefly GABAR with various amino acids and examined changes in the sensitivity of mutant GABARs to GABA and fluralaner using the <i>Xenopus</i> oocyte expression system and two-electrode voltage clamp electrophysiology. The substitution of G333 in TM3 led to a substantial reduction in sensitivity to GABA and fluralaner. As the substitutions of D329 and F336 resulted in no or unstable GABA responses, the roles of these amino acids could not be investigated. Q271L substitution in the first transmembrane α-helix (TM1) caused a significant decrease in sensitivity to GABA and fluralaner. I274A and I274F substitutions in TM1 affected neither GABA nor fluralaner sensitivity, although I274C substitution significantly enhanced sensitivity to GABA and fluralaner. L278C substitution in TM1 resulted in slightly enhanced GABA sensitivity and reduced fluralaner sensitivity. These results indicate that Q271 and L278 in TM1 and G333 in TM3 are deeply involved in the antagonism of GABARs by fluralaner.

Effects of amino acid substitutions at the intersubunit cavity on the sensitivity of the GABA receptor to fluralaner

2019 Fall National Meeting

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