Effects of intersubunit amino acid substitutions on GABA receptor sensitivity to the ectoparasiticide fluralaner.

The isoxazoline ectoparasiticide fluralaner exerts antiparasitic effects by inhibiting the function of γ-aminobutyric acid (GABA) receptors (GABARs). The present study was conducted to identify the amino acid residues that contribute to the high sensitivity of insect GABARs to fluralaner. We generated housefly (Musca domestica) GABARs with amino acid substitutions in the first through third α-helical transmembrane segments (TM1-TM3) of the RDL subunit using site-directed mutagenesis and examined the effects of the substitutions on the sensitivity of GABARs expressed in Xenopus oocytes to fluralaner using two-electrode voltage clamp electrophysiology. The Q271L substitution in TM1 caused a significant reduction in the sensitivity to fluralaner. Although the I274A and I274F substitutions in TM1 did not affect fluralaner sensitivity, the I274C substitution significantly enhanced the sensitivity to fluralaner. In contrast, the L278C substitution in TM1 reduced fluralaner sensitivity. Substitutions of Gly333 in TM3 led to substantial reductions in the sensitivity to fluralaner. These findings indicate that Gln271, Ile274, Leu278, and Gly333, which are situated in the outer half of the transmembrane subunit interface, are closely related to the antagonism of GABARs by fluralaner.

A Single Amino Acid Substitution in the Third Transmembrane Region Has Opposite Impacts on the Selectivity of the Parasiticides Fluralaner and Ivermectin for Ligand-Gated Chloride Channels.

Fluralaner (Bravecto) is a recently marketed isoxazoline ectoparasiticide. This compound potently inhibits GABA-gated chloride channels (GABACls) and less potently glutamate-gated chloride channels (GluCls) in insects. The mechanism underlying this selectivity is unknown. Therefore, we sought to identify the amino acid residues causing the low potency of fluralaner toward GluCls. We examined the fluralaner sensitivity of mutant housefly (Musca domestica) GluCls in which amino acid residues in the transmembrane subunit interface were replaced with the positionally equivalent amino acids of Musca GABACls. Of these amino acids, substitution of an amino acid (Leu315) in the third transmembrane region (TM3) with an aromatic amino acid dramatically enhanced the potency of fluralaner in the GluCls. In stark contrast to the enhancement of fluralaner potency, this mutation eliminated the activation of currents and the potentiation but not the antagonism of glutamate responses that are otherwise all elicited by the macrolide parasiticide ivermectin (IVM). Our findings indicate that the amino acid Leu315 in Musca GluCls plays significant roles in determining the selectivity of fluralaner and IVM for these channels. Given the high sequence similarity of TM3, this may hold true more widely for the GluCls and GABACls of other insect species.

Electrophysiological characterization of ivermectin triple actions on Musca chloride channels gated by l-glutamic acid and γ-aminobutyric acid.

Ivermectin (IVM) is a macrocyclic lactone that exerts antifilarial, antiparasitic, and insecticidal effects on nematodes and insects by acting on l-glutamic acid-gated chloride channels (GluCls). IVM also acts as an allosteric modulator of various other ion channels. Although the IVM binding site in the Caenorhabditis elegans GluCl was identified by X-ray crystallographic analysis, the mechanism of action of IVM in insects is not well defined. We therefore examined the action of IVM on the housefly (Musca domestica) GluCl and γ-aminobutyric acid (GABA)-gated ion channel (GABACl). For both channels, IVM induced currents by itself, potentiated currents induced by low concentrations of agonists, and inhibited currents induced by high concentrations of agonists. Despite exerting common actions on both types of channels, GluCls were more susceptible to IVM actions than GABACls, indicating that GluCls are the primary target of IVM. Substitution of an amino acid residue in the third transmembrane segment (G312M in GluCls, and G333A and G333M in GABACls) resulted in significantly reduced levels or loss of activation, potentiation, and antagonism of the channels, indicating that these three actions result from the interaction of IVM with amino acid residues in the transmembrane intersubunit crevice.