The macrolide ivermectin (IVM) is an important antiparasitic agent in human and veterinary medicine. While the action of IVM on L-glutamic acid (Glu)-gated chloride channels (GluCls) is well documented, various actions on other channels remain undefined. The isoxazoline ectoparasiticide fluralaner (Flu) is a noncompetitive antagonist that acts at both γ-aminobutyric acid (GABA)-gated chloride channels (GABACls) and GluCls. In this action, Flu is more potent in GABACls than in GluCls. In this study, we examined the mechanisms underlying the selective actions of IVM and Flu on housefly GluCls versus GABACls or vice versa.<br/>Two-electrode voltage clamp analyses using <i>Xenopus</i> oocytes indicated that IVM exerts three different actions on both GluCls and GABACls: (1) IVM activated currents by itself, (2) potentiated currents induced by low concentrations of agonists, and (3) inhibited currents induced by high concentrations of agonists. Although IVM exerted similar effects on both channels, GluCls were more susceptible to these IVM actions than GABACls. In particular, IVM was approximately 180-fold more potent in GluCls than in GABACls in terms of activation. Substitution of a conserved glycine in the third transmembrane region (TM3) of subunits with methionine or alanine resulted in the reduction or loss of sensitivity of the channels to the three actions, indicating that these actions result from the interaction of IVM with amino acid residues in the transmembrane subunit interface (TSI).<br/>We next examined what is responsible for the low sensitivity of GluCls to Flu. Amino acid residues in the TSI of GluCls were substituted with GABACl equivalents. Of the substitutions, the substitution of a conserved leucine in the TM3 of the GluCl subunit with phenylalanine resulted in a dramatic enhancement of the antagonist potency of Flu in the mutant. The same was true for substitutions with other aromatic amino acids but not an aliphatic one. In marked contrast to the potency enhancement in Flu, substitutions with aromatic but not aliphatic amino acids eliminated the IVM activation of GluCls. The TM3 leucine of housefly GluCls and the amino acids of other GluCls/GABACls at the equivalent position might play key roles in determining the selectivity or potency of isoxazoline antagonists and macrolide activators in these channels.

Selective actions of isoxazoline antagonists and macrolide activators on ligand-gated chloride channels

254th National Meeting (2017)

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