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Research ArticleArticle

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

Yunosuke Nakata, Toshinori Fuse, Kohei Yamato, Miho Asahi, Kunimitsu Nakahira, Fumiyo Ozoe and Yoshihisa Ozoe
Molecular Pharmacology November 2017, 92 (5) 546-555; DOI: https://doi.org/10.1124/mol.117.109413

Abstract

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.

Introduction

Ligand-gated ion channels (LGICs) play vital roles in regulating neuronal excitation and inhibition in animals. These channels are either: 1) cation-selective channels, the activation of which depolarizes the postsynaptic membrane toward firing an action potential; or 2) anion-selective channels, the activation of which hyperpolarizes the membrane or suppresses the depolarization generated by cation channels (Smart and Paoletti, 2012). Nicotinic acetylcholine receptors and GABA receptors, which are members of the Cys-loop family of LGICs, are examples of such cation and anion channels, respectively (Miller and Smart, 2010). Inhibitory glutamate receptors, which are found only in invertebrates, are also Cys-loop LGIC family members. The Cys-loop LGICs are pentamers, the subunits of which are assembled to form a central ion-permeable channel. Each subunit consists of a large N-terminal extracellular domain, four hydrophobic α-helical transmembrane segments (TMs), an intracellular loop between TM3 and TM4, and a short extracellular C terminus. The orthosteric agonist-binding site is located at the subunit interface of the extracellular domain. These channels are important targets for drugs and insecticides (Ozoe, 2013; Alexander et al., 2015).

Glutamate-gated chloride channels (GluCls) are the main targets for the insecticidal, acaricidal, and nematicidal macrolides avermectins (AVMs), which allosterically activate and modulate various ion channels, including GABA-gated chloride channels (GABACls), glycine-gated chloride channels, pH-gated chloride channels, α7 acetylcholine–gated cation channels, ATP-gated P2X receptor cation channels, and G protein–gated inwardly rectifying potassium channels (Cully et al., 1994, 1996; Krause et al., 1998; Dawson et al., 2000; Shan et al., 2001; Silberberg et al., 2007; Fuse et al., 2016; Nakatani et al., 2016; Chen et al., 2017). GluCls are activated by nanomolar AVMs, whereas other channels require concentrations in the micromolar range. AVM B1 (abamectin) is used to control phytophagous mites and insect pests on agricultural and horticultural crops (Lasota and Dybas, 1991). Ivermectin (IVM) (Fig. 1), 22,23-dihydroAVM B1, is widely used to control endoparasites and ectoparasites in animals and to treat onchocerciasis and lymphatic filariasis caused by parasitic worms in humans (Ōmura and Crump, 2014; Wolstenholme et al., 2016; Laing et al., 2017). GABACls are targets for chlorinated hydrocarbon and phenylpyrazole insecticides, which stabilize the closed conformation of GABACls by interacting with TM2 amino acid residues on the cytoplasmic side within the channel pore (Ozoe, 2013). Isoxazoline and m-benzamidobenzamide insecticides are new-generation antagonists that inhibit insect GABACls (Ozoe et al., 2010, 2013; Nakao et al., 2013; Gassel et al., 2014; Shoop et al., 2014; Asahi et al., 2015; McTier et al., 2016; Nakao and Banba, 2016). These insecticides inhibited GABACls with mutations conferring insensitivity to the conventional antagonists (Nakao et al., 2013; Asahi et al., 2015). The substitution of a conserved TM3 Gly located in the transmembrane subunit interface (TSI) of Drosophila GABACls diminished or eliminated the inhibitory effects of a m-benzamidobenzamide (meta-diamide) insecticide on GABA responses, whereas the conventional antagonists remained effective (Nakao et al., 2013). These findings indicate that these insecticides have modes of action distinct from that of the conventional channel-blocking antagonists.

Fig. 1.
Fig. 1.

Chemical structures of fluralaner, its analogs, and IVM B1a.

Fluralaner (Bravecto) (Fig. 1) is a recently marketed isoxazoline ectoparasiticide that is used for flea and tick protection in pets (Taenzler et al., 2014; Wengenmayer et al., 2014). This compound inhibits agonist responses in both GABACls and GluCls, with selectivity for the former over the latter (Ozoe et al., 2010), whereas IVM is a selective activator or modulator for GluCls (Fuse et al., 2016). In the present study, we sought to identify the mechanism underlying the difference in fluralaner and IVM sensitivity between GluCls and GABACls. We report that the substitution of a single amino acid in the TSI of GluCls with a positionally equivalent amino acid from GABACls results in a drastic increase in the antagonist potency of fluralaner and a dramatic elimination of the IVM activation of currents and the IVM potentiation of the glutamate responses.

Materials and Methods

Chemicals.

Fluralaner (99%), A1209 (99%), and A341 (99%) were synthesized according to a previously reported method (Mita et al., 2005, 2009, 2010). GABA and IVM (B1a ≥ 90%, B1b ≤ 5%) were purchased from Sigma-Aldrich (St. Louis, MO). Other general chemicals including sodium hydrogen l-glutamate were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), unless otherwise noted.

Wild-Type and Mutant GluCl cDNAs.

Two LGIC splice variants that show robust agonist responses were used in this study. cDNAs encoding GluCl (variant A) (accession number AB177546) and GABACl (RDL variant ac) (accession numbers AB177547, AB824728, AB824729) subunits from the housefly (Musca domestica; susceptible reference strain from the World Health Organization) were subcloned into the plasmid vectors pcDNA3 and pBluescript KS(−), respectively (Eguchi et al., 2006; Ozoe et al., 2013). The introduction of mutations into the cDNAs was performed using a QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA) and verified by DNA sequencing.

Two-Electrode Voltage-Clamp Electrophysiology.

The lobes of ovaries from female African clawed frogs (Xenopus laevis) anesthetized by immersion in a 0.1% (w/v) Tricaine solution were surgically removed. Follicle cells were treated with collagenase (2 mg/ml; Sigma-Aldrich) in a calcium-free standard oocyte solution (Ca2+-free SOS) (100 mM NaCl, 2 mM KCl, 1 mM MgCl2, 5 mM HEPES, pH 7.6) for 1–2 hours at 20°C. After the treatment, the oocytes were washed with Ca2+-free SOS supplemented with 2.5 mM sodium pyruvate, gentamycin (50 μg/ml; Thermo Fisher Scientific, Waltham, MA), penicillin (100 U/ml; Thermo Fisher Scientific), and streptomycin (100 μg/ml; Thermo Fisher Scientific) and were incubated for 1–2 days at 16°C.

The Musca GluCl and Rdl cDNAs containing a T7 promoter site upstream of the coding region were amplified by polymerase chain reaction (PCR). The PCR products were purified using an illustra GFX PCR DNA and Gel Band Preparation Kit (GE Healthcare Bio-Sciences, Pittsburgh, PA). After sequence verification, the amplified cDNA templates (100 ng) were in vitro transcribed into capped poly(A) complementary RNAs (cRNAs) using an mMESSAGE mMACHINE T7 Ultra Kit (Thermo Fisher Scientific). The quality and quantity of the prepared cRNAs were evaluated by agarose gel electrophoresis and absorption spectroscopy, respectively. Purified cRNA (5 ng) was injected into each oocyte using a Nanoliter 2000 injector (World Precision Instruments, Sarasota, FL). The injected oocytes were incubated for 2–4 days at 16°C.

The oocytes expressing Musca GluCls or GABACls were placed in a chamber perfused with SOS (100 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM HEPES, pH 7.6). Glass microelectrodes were filled with 2 M KCl to yield a resistance of 0.5–1.6 MΩ. Electrophysiological recordings were performed using an Oocyte Clamp OC-726C amplifier (Warner Instruments, Hamden, CT) at a holding potential of −80 mV at 20°C. The data were digitized using a Laboratory-Trax-4/16 converter (World Precision Instruments) and analyzed using the Data-Trax2 software (World Precision Instruments). IVM, fluralaner, and fluralaner analogs dissolved in dimethyl sulfoxide were diluted with SOS to produce solutions containing the indicated concentrations for each compound and <0.01% dimethyl sulfoxide. To analyze the antagonism of the GluCls or GABACls by fluralaner and its analogs, glutamate or GABA dissolved at the EC50 of each channel (Table 1) in SOS was applied to the oocytes for 3 seconds at 30- to 60-second intervals with the perfusion of fluralaner until maximum inhibition was achieved. Oocytes were perfused with the solution for 3 minutes to analyze channel activation by IVM. The ability of IVM to potentiate and antagonize glutamate responses was analyzed in a manner similar to the evaluation of fluralaner antagonism but using glutamate at its EC5 and EC90, respectively. The EC5 and EC90 are the concentrations at which the potentiation and antagonism of glutamate responses are observed, respectively (Fuse et al., 2016). All experiments were replicated using at least six oocytes from at least two frogs. The data are presented as the mean ± S.E.M. The EC50 and IC50 values were obtained from dose-response relationships by the four-parameter logistic regression using OriginPro 8J SR4 (version 8.0951; LightStone, Tokyo, Japan). Unpaired t tests were performed to evaluate statistical significance; P values for L315 mutants are reported with a Bonferroni correction for multiple tests.

View this table:
TABLE 1

Potencies of glutamate, fluralaner, fluralaner analogs, and IVM in wild-type and mutant forms of Musca GluCls and GABACls expressed in the Xenopus oocytes

Homology Modeling and Docking Simulation.

The amino acid sequences of the Musca GluCl-A subunit and the Caenorhabditis elegans GluCl-α subunit were aligned using ClustalW2. A Musca GluCl homology model was constructed using MOE software (version 2014.04; Chemical Computing Group, Montreal, QC, Canada). The X-ray crystal structure of the C. elegans GluCl-α channel (Protein Data Bank code, 3RHW) was used as a template.

Results

Responses of Wild-Type and Mutant GluCls to Glutamate.

The amino acid at position 36′ (index number starting from a conserved TM2 cationic residue numbered 0′) in TM3 is particularly important in determining the sensitivity of IVM in LGICs (Lynagh and Lynch, 2010). To examine the effects of intersubunit amino acids on the potencies of fluralaner and IVM against GluCls, we first substituted four GluCl amino acids (Ile253, Met257, Leu315, and Thr316) near Gly at position 36′ (G36′) (Gly312 in the Musca GluCl subunit) with the positionally equivalent amino acids of the Musca GABACl (RDL) subunit (Fig. 2), generating four single mutants (I253A, M257L, L315F, and T316V) and one double mutant (M257L/T316V). Because the amino acid at position 253 of the Musca GluCl subunit was identical to the amino acid of the Musca GABACl RDL subunit at the equivalent position, Ile253 was substituted with Ala. All wild-type and mutant GluCls expressed in Xenopus oocytes responded to glutamate to elicit currents (Fig. 3A). The L315F mutant was ∼36-fold less sensitive to glutamate compared with the wild-type channel (Table 1). Although this mutation may allosterically affect glutamate binding to the orthosteric site, the dose-response curve indicated that the channel functioned normally to induce currents in response to glutamate.

Fig. 2.
Fig. 2.

Location of the amino acid substitution in Musca GluCls. (A) Amino acid alignment of the TM1 and TM3 of Musca GluCl and GABACl (RDL) subunits. Substituted amino acids are highlighted in red. (B) Top view of the channel domain with the side chains of substituted amino acids indicated by highlighting. (C) Close-up of the side chains of substituted amino acids in the TSI.

Fig. 3.
Fig. 3.

Inhibition of glutamate-induced currents by fluralaner in wild-type and mutant Musca GluCls. (A) Dose-response curves of glutamate-induced currents. Data points indicate the mean ± S.E.M. values (n = 6) normalized relative to maximal current amplitudes. (B) Current trace of glutamate (EC50)-induced currents during fluralaner perfusion in the wild-type channel. Note that the slight current recovery in the last glutamate application is within the range encompassed by variation. (C) Current trace of glutamate (EC50)-induced currents during fluralaner perfusion in the L315F mutant. (D) Dose-response curves of fluralaner inhibition of agonist-induced current in wild-type and mutant GluCls compared with that in the wild-type GABACl. Normalized relative to responses induced by the EC50 values of agonists. Data points indicate mean ± S.E.M. values (n = 6).

Fluralaner Inhibition of Glutamate-Induced Currents in Mutant GluCls.

Fluralaner inhibited glutamate-induced currents in wild-type Musca GluCls expressed in Xenopus oocytes (Fig. 3, B and D), with an IC50 of 146 nM (Table 1). However, wild-type Musca GluCls were ∼24-fold less sensitive to fluralaner than were wild-type Musca GABACls, as previously reported (Ozoe et al., 2010). Fluralaner was ∼2-fold less potent toward the I253A mutant than in the wild-type GluCl, whereas it was ∼4-fold and ∼2-fold more potent in the M257L and M257L/T316V mutants, respectively, than in the wild-type GluCl (Fig. 3D; Table 1). The potency of fluralaner in the T316V mutant did not differ significantly from that in the wild type. Notably, the L315F mutant was strongly inhibited by fluralaner, with an IC50 of 1.06 nM, indicating that this mutant is ∼138-fold more sensitive to fluralaner inhibition than the wild type (Fig. 3, C and D; Table 1). The IC50 of fluralaner in the L315F mutant was even ∼6-fold smaller than its IC50 in the inhibition of GABA-induced currents in Musca GABACls (Fig. 3D; Table 1).

IVM Actions on Mutant GluCls.

IVM alone activated slow, irreversible currents in wild-type Musca GluCls expressed in Xenopus oocytes (Fig. 4A), with an EC50 of 18.8 nM (Table 1). The wild-type Musca GluCls were ∼66-fold to ∼184-fold more sensitive to IVM than were wild-type Musca GABACls (Table 1). The potency of IVM was ∼5-fold higher in the I253A mutant than in the wild type, whereas the potencies of the M257L and T316V mutants were not significantly different from that of the wild-type GluCl (Fig. 4C; Table 1). The difference in the maximal current amplitude between the wild type and mutants could be ascribed to differences in the expression levels of channels in oocytes (Fig. 4C). Surprisingly, the L315F mutant, which showed an enhanced sensitivity to fluralaner, lacked sensitivity to IVM in terms of activation (Fig. 4, B and C). Next, we examined IVM potentiation and antagonism of glutamate-induced currents in the L315F mutant because IVM exerts a triple action on GluCls and GABACls depending on the conditions (Fuse et al., 2016). In L315F GluCls, IVM did not potentiate the currents induced by a low concentration of glutamate (EC5, 50 μM) (Fig. 5, A and B) but inhibited those induced by a high concentration of glutamate (EC90, 3 mM), with an IC50 (±S.E.M.) of 5.48 ± 1.20 nM (n = 6), which is not significantly different from the IC50 (4.92 ± 2.23 nM) for the wild type (Fig. 5, C and D).

Fig. 4.
Fig. 4.

Responses of wild-type and mutant Musca GluCls to IVM. (A) Current trace showing the IVM activation of wild-type GluCls. (B) Current trace when IVM was applied to the L315F mutant. (C) Dose-response curves of IVM-induced currents in GluCls normalized relative to responses induced by the EC50 values of glutamate. Data points indicate mean ± S.E.M. values (n = 6).

Fig. 5.
Fig. 5.

Responses of wild-type and mutant Musca GluCls to IVM. (A) Current trace showing the absence of the IVM potentiation of glutamate (EC5) responses in L315F GluCls. (B) Dose-response curves for evaluating the IVM potentiation of glutamate responses in wild-type and L315F GluCls compared with responses induced by glutamate (EC5) alone. Data points indicate mean ± S.E.M. values (n = 6). (C) Current trace showing the IVM inhibition of glutamate (EC90)-induced currents in the L315F mutant. (D) Dose-response curves of the IVM antagonism of wild-type and L315F GluCls normalized relative to responses induced by the EC90 values of glutamate. Data points indicate mean ± S.E.M. values (n = 6).

Effects of Fluralaner Analogs on L315F Mutant GluCls.

As fluralaner showed marked antagonism of the L315F GluCl, we examined whether a similar potency enhancement could be observed for fluralaner analogs (Figs. 6 and 7). The isoxazolines A1209 and A341 (Fig. 1) are fluralaner analogs that show >500-fold and >100-fold higher antagonism of Musca GABACls, respectively, compared with Musca GluCls (Fig. 6, A, B, and D; Fig. 7, A, B, and D; Table 1). The L315F mutation resulted in >6000-fold and >100-fold enhancement of the potency of A1209 and A341 in GluCls (Fig. 6, C and D; Fig. 7, C and D; Table 1), which were, respectively, much greater than and comparable to the enhancement observed for fluralaner. The L315F GluCl was outstandingly sensitive to A1209 (Table 1). Although the isoxazoline A341 exhibited little antagonism in wild-type GluCls, the antagonist potency of this compound in L315F GluCls was comparable to that in wild-type GABACls (Fig. 7).

Fig. 6.
Fig. 6.

Inhibition of glutamate-induced currents by a fluralaner analog, A1209, in wild-type and mutant Musca GluCls. (A) Current trace showing the inhibition of GABA (EC50)-induced currents in wild-type GABACls. (B) Current trace showing the inhibition of glutamate (EC50)-induced currents in wild-type GluCls. (C) Current trace showing the inhibition of glutamate (EC50)-induced currents in L315F GluCls. (D) Dose-response curves of the A1209 inhibition of glutamate- and GABA-induced currents normalized relative to responses induced by the EC50 values of agonists. Data points indicate mean ± S.E.M. values (n = 6).

Fig. 7.
Fig. 7.

Inhibition of glutamate-induced currents by a fluralaner analog, A341, in wild-type and mutant Musca GluCls. (A) Current trace showing the inhibition of GABA (EC50)-induced currents in wild-type GABACls. (B) Current trace showing the inhibition of glutamate (EC50)-induced currents in wild-type GluCls. (C) Current trace showing the inhibition of glutamate (EC50)-induced currents in L315F GluCls. (D) Dose-response curves of the A341 inhibition of glutamate- and GABA-induced currents normalized relative to responses induced by the EC50 values of agonists. Data points represent mean ± S.E.M. values (n = 6).

Effects of Aromatic Amino Acid Substitution at Position 315 on the Potency of Fluralaner.

As the L315F substitution enhanced the potency of fluralaner against Musca GluCls, we examined the effects of other amino acid substitutions on the potency of fluralaner (Fig. 8). We injected cRNAs encoding five mutants (L315Y, L315W, L315H, L315A, and L315M) into oocytes. The aromatic amino acids included Tyr, Trp, and His, which has the aromatic heterocycle imidazole in the side chain. Ala and Met were chosen as hydrophobic aliphatic amino acids. Although the oocytes injected with the cRNAs for L315W and L315A failed to respond to glutamate, the other oocytes did respond to glutamate (Fig. 8A). Fluralaner inhibition of glutamate-induced currents was ∼24-fold and ∼8-fold more potent in the L315Y and L315H mutants, respectively, than in the wild type (Fig. 8, B, C, and E; Table 1). In contrast, the potency of fluralaner did not differ significantly between the L315M mutant and the wild-type channel (Fig. 8, D, E; Table 1). These findings indicate that aromatic amino acids at position 315 are effective in enhancing the antagonist potency of fluralaner in Musca GluCls but that an aliphatic amino acid is not.

Fig. 8.
Fig. 8.

Inhibition of glutamate-induced currents by fluralaner in Musca L315 mutant GluCls. (A) Dose-response curves of glutamate-induced currents in wild-type and mutant GluCls normalized relative to maximal current amplitudes. Data points indicate mean ± S.E.M. values (n = 6). (B) Current trace of glutamate (EC50)-induced currents during fluralaner perfusion in the L315Y mutant. (C) Current trace of glutamate (EC50)-induced currents during fluralaner perfusion in the L315H mutant. (D) Current trace of glutamate (EC50)-induced current during fluralaner perfusion in the L315M mutant. (E) Dose-response curves of fluralaner inhibition of glutamate-induced currents in wild-type and mutant GluCls normalized relative to responses induced by the EC50 values of glutamate. Data points indicate mean ± S.E.M. values (n = 6).

Effects of Aromatic Amino Acid Substitution at Position 315 on the Potency of IVM.

We examined the effects of the L315Y, L315H, and L315M substitutions on IVM-induced currents in Musca GluCls (Fig. 9). The former two substitutions abolished the IVM-induced activation of Musca GluCls (Fig. 9, A, B, and D). In contrast, IVM activated currents in the L315M mutant, although the potency was ∼2-fold lower than in the wild-type channel (Fig. 7, C and D; Table 1). These findings indicate that aromatic amino acids, but not an aliphatic amino acid, at position 315 eliminate the IVM-induced activation of GluCls.

Fig. 9.
Fig. 9.

Responses of Musca L315 mutant GluCls to IVM. (A) Current trace showing the absence of the IVM activation in L315Y GluCls. (B) Current trace when IVM was applied to L315H GluCls. (C) Current trace showing the IVM activation of L315M GluCls. (D) Dose-response curves showing the presence and absence of IVM-induced currents in mutants normalized relative to responses induced by the EC50 values of glutamate. Data points indicate mean ± S.E.M. values (n = 6).

Discussion

The TSI of pentameric LGICs plays critical roles in the actions of insecticides and other drugs (Nakao et al., 2013; Forman and Miller, 2016). The TSI in mammalian GABACls has been extensively studied as a binding site for general anesthetics such as propofol, etomidate, and barbiturates (Forman and Miller, 2016). Interestingly, both convulsive and anesthetic barbiturates modulate GABACls by binding to this region (Jayakar et al., 2015). IVM activates currents and potentiates and antagonizes the agonist-induced currents of LGICs including GABACls and GluCls, with the latter being more sensitive than the former, by possibly binding in the TSI (Hibbs and Gouaux, 2011; Estrada-Mondragon and Lynch, 2015; Fuse et al., 2016). A single TM3 amino acid in the TSI, G36′ (Fig. 2), which is conserved in IVM-sensitive LGICs, has been shown to be critical for these actions of IVM (Lynagh and Lynch, 2010; Fuse et al., 2016). The substitution of G36′ with bulkier amino acids results in a reduction or the loss of sensitivity to IVM. The importance of G36′ in insecticide actions was indicated by a report that a G36′D mutation was identified in the abamectin-resistant strain of two-spotted spider mites (Tetranychus urticae) (Kwon et al., 2010). A G36′E substitution disrupted T. urticae GluCl activation by abamectin and milbemycin (Mermans et al., 2017). Furthermore, G36′ mutations were reported to diminish or eliminate the ability of a meta-diamide insecticide to inhibit GABA-induced currents in the Drosophila GABACls (Nakao et al., 2013). Interestingly, the analogous mutation of a positionally equivalent Gly was identified in the nicotinic acetylcholine receptor α7 subunit of spinosad-resistant western flower thrips (Frankliniella occidentalis) and the nicotinic acetylcholine receptor α6 subunit of spinosad-resistant tomato leafminers (Tuta absoluta) (Puinean et al., 2013; Silva et al., 2016). Together, these reports indicate that a conserved Gly in TM3 plays a key role and that the TSI seems to form the sites of action for a variety of ligands.

The ectoparasiticide fluralaner was shown to exhibit selective antagonism of GABACls over GluCls (Ozoe et al., 2010). To examine whether any amino acid substitution enhances the low potency of fluralaner in GluCls, we focused on replacing amino acids around G36′ in the TSI of Musca GluCls with the positionally equivalent amino acids of Musca GABACls, which were sensitive to fluralaner (Table 1). We have shown that the substitution of an amino acid, Leu315, located one α-helical turn below G36′, with aromatic amino acids (but not with an aliphatic amino acid) dramatically enhances the potency of fluralaner (Figs. 3 and 8). These findings may explain the high potency of fluralaner in GABACls, which possess Phe at the equivalent position. Our docking simulation of the S enantiomer of fluralaner, which is the active component (Ozoe et al., 2010), to a Musca GluCl homology model revealed that the aromatic ring of fluralaner lies near Leu315 (Fig. 10). It remains to be clarified whether the enhanced potency depends on a π-π stacking interaction (Zhao et al., 2015) between the aromatic ring of substituted amino acids at position 315 and the phenyl group of fluralaner.

Fig. 10.
Fig. 10.

Docking of the S enantiomer of fluralaner into the TSI of a wild-type Musca GluCl homology model. The α-helical transmembrane segments (TM1–TM4) of two adjacent subunits are shown in different colors. The docked fluralaner molecule lies near Leu315, suggesting a π-π stacking interaction between the phenyl group of fluralaner and the aromatic ring of an amino acid substituted at position 315 of Musca GluCls. The CPK coloring is used for the fluralaner stick model.

In contrast to the enhancement of the potency of fluralaner, we found that the same aromatic substitution abolished the direct IVM activation of GluCls and the IVM potentiation of glutamate-induced currents, while the antagonism remained unchanged (Figs. 4, 5, and 9). The L315M mutant, which has a nonaromatic amino acid at position 315, retained the ability to be activated by IVM. This finding is consistent with the fact that the homomeric AVM-sensitive C. elegans GluCl-α channel has Met at an equivalent position, whereas the AVM-insensitive β channel has Gln at this position (Cully et al., 1994) (Supplemental Fig. 1). It will be interesting to investigate whether this Gln is responsible for the insensitivity to AVMs. The amino acid Thr316 of the Musca GluCl subunit (Fig. 2), adjacent to Leu315, is positionally equivalent to the amino acid that was reported to form a hydrogen bond with IVM in an X-ray crystal study of the C. elegans GluCl-α channel (Hibbs and Gouaux, 2011). The T316V substitution did not change the potency of IVM in the present study (Fig. 3D; Table 1), suggesting that the hydrogen bonding does not contribute substantially to IVM binding in Musca GluCls. Instead, our data suggest that Leu315 is located adjacent to bound IVM. How the substitution of the amino acid Leu315 impairs the IVM-induced activation of GluCls and the IVM-induced potentiation of glutamate responses remains to be examined.

Finally, we generated the Musca RDL subunit with an inverse mutation (the substitution of Phe with Leu at an equivalent position) (Fig. 2A) to evaluate whether this mutation results in low sensitivity of GABACls to fluralaner. However, because this mutation led to a spontaneously open channel, we were unable to determine the potency of fluralaner in this mutant.

In conclusion, we have shown that Leu315 located in the TSI of Musca GluCls and the positionally equivalent amino acid of GABACls play key roles in determining the selectivity of fluralaner and IVM toward these channels. This finding may be applicable to other GluCls and GABACls, given that the TM3 sequence is highly conserved among insect and mite species (Supplemental Fig. 1). As predicted, a very recent publication has indicated that activation by IVM was strongly reduced and that activation by okaramine B, an insecticidal indole alkaloid, was completely abolished in the silkworm (Bombyx mori) GluCl containing an L319F mutation, which is equivalent to the L315F mutation in the Musca GluCl (Furutani et al., 2017). More importantly, we have shown in the present study that the L315F mutation has opposite impacts on the selectivity of fluralaner and IVM for GluCls. This implies that even if IVM-insensitive arthropod pests with an equivalent mutation emerge, these arthropod pests would be sensitive to fluralaner. These findings should prove useful for understanding the modes of action of these parasiticides and further development of pest control agents.

Acknowledgments

The authors thank T. Kita, K. Nomura, and M. Takashima for technical assistance and advice.

Authorship Contributions

Participated in research design: Nakata, Asahi, Nakahira, F. Ozoe, and Y. Ozoe.

Conducted experiments: Nakata, Fuse, Yamato, and F. Ozoe.

Performed data analysis: Nakata, Fuse, and Yamato.

Wrote or contributed to the writing of the manuscript: Nakata, Fuse, and Y. Ozoe.

Footnotes

Abbreviations

AVM
avermectin
cRNA
complementary RNA
G36′
Gly at position 36′
GABACl
GABA-gated chloride channel
GluCl
glutamate-gated chloride channel
IVM
ivermectin
LGIC
ligand-gated ion channel
PCR
polymerase chain reaction
SOS
standard oocyte solution
TM
transmembrane segment
TSI
transmembrane subunit interface

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Actions of Isoxazoline Ectoparasiticide on Chloride Channels

Yunosuke Nakata, Toshinori Fuse, Kohei Yamato, Miho Asahi, Kunimitsu Nakahira, Fumiyo Ozoe and Yoshihisa Ozoe
Molecular Pharmacology November 1, 2017, 92 (5) 546-555; DOI: https://doi.org/10.1124/mol.117.109413
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