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Effects of intersubunit amino acid substitutions on GABA receptor sensitivity to the ectoparasiticide fluralaner
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Pesticide Biochemistry and Physiology journal homepage: www.elsevier.com/locate/pest
Effects of intersubunit amino acid substitutions on GABA receptor sensitivity to the ectoparasiticide fluralaner Kohei Yamatoa, Yunosuke Nakataa, Madoka Takashimaa, Fumiyo Ozoea, Miho Asahib, ⁎ Masaki Kobayashib, Yoshihisa Ozoea, a b
Faculty of Life and Environmental Science, Shimane University, Matsue, Shimane 690-8504, Japan Biological Research Laboratories, Nissan Chemical Corporation, Shiraoka, Saitama 349-0294, Japan
A R T I C LE I N FO
A B S T R A C T
Keywords: GABA receptor Transmembrane subunit interface Isoxazoline 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.
1. Introduction γ-Aminobutyric acid (GABA) is a free amino acid that plays vital roles as a ubiquitous inhibitory neurotransmitter in the nervous system. GABA released from presynaptic neurons binds to the GABA receptor (GABAR), which is a member of the pentameric ligand-gated chloride channel (pLGICs) family, and triggers rapid chloride flow through the channel into postsynaptic neurons or muscle cells, thereby offsetting the effect of excitatory postsynaptic cation influx (Mody and Pearce, 2004). The binding of GABA to the extracellular domain of GABAR induces conformational changes in the channel domain, each subunit of which is composed of four consecutive α-helical transmembrane segments (TM1–TM4), to open the pore (Miller and Smart, 2010; Masiulis et al., 2019). In the case of arthropods, GABAergic function is mediated by homopentameric GABARs formed by RDL subunits. Arthropod GABARs serve as important targets for pest control chemicals (Ozoe, 2013). Fluralaner (Fig. 1), an isoxazoline ectoparasiticide, potently antagonizes agonist activity at GABARs, and it also acts, albeit less potently, on glutamate-gated chloride channels (GluClRs) (Ozoe et al., 2010).
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Fluralaner has been shown to act at a site that is different from that targeted by conventional GABAR antagonist insecticides (Asahi et al., 2015), and its site of action remains to be defined. We previously reported that the replacement of Leu315 in the third transmembrane segment (TM3) of the housefly (Musca domestica) GluClR subunit with the amino acid found at the corresponding position in the housefly GABAR RDL subunit (i.e., Phe at the 336 position) dramatically enhanced the sensitivity of GluClRs to fluralaner (Nakata et al., 2017). In the present study, to determine the site of action of fluralaner, we substituted amino acid residues in the transmembrane subunit interface (TSI) of the housefly GABAR with various amino acids (Fig. 2) and examined the changes in the sensitivity of mutant GABARs expressed in Xenopus oocytes to fluralaner using two-electrode voltage clamp (TEVC) electrophysiology. Here, we report that four amino acids in the TSI are associated with the high sensitivity of GABARs to fluralaner.
Corresponding author. E-mail address: [email protected] (Y. Ozoe).
https://doi.org/10.1016/j.pestbp.2019.11.001 Received 9 September 2019; Received in revised form 17 October 2019; Accepted 3 November 2019 0048-3575/ © 2019 Elsevier Inc. All rights reserved.
Please cite this article as: Kohei Yamato, et al., Pesticide Biochemistry and Physiology, https://doi.org/10.1016/j.pestbp.2019.11.001
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microelectrodes were filled with 2 M KCl to yield a resistance of 0.5–1.6 MΩ. The data were digitized using a Lab-Trax-4/16 converter (World Precision Instruments) and analyzed using Data-Trax2 software (World Precision Instruments). The same oocyte was exposed to increasing concentrations of GABA dissolved in SOS for 3 s. The oocyte was perfused with SOS between each application for approximately 1 min to ensure full recovery from desensitization. Fluralaner was dissolved in dimethyl sulfoxide (DMSO) and diluted with SOS to produce the desired concentration. The DMSO concentrations were < 0.01%, which had no adverse effects on GABA responses. To analyze the antagonism of GABARs by fluralaner, GABA dissolved in SOS at a concentration equivalent to the EC50 of each GABAR (Table 1) was repeatedly applied to the oocytes for 3 s at 30–60s intervals, while the oocytes were perfused with a fluralaner solution until steady-state inhibition was reached after stable GABA responses. The steady-state inhibition percentage obtained for each concentration was used to draw a dose-response curve. All experiments were replicated using 4–10 oocytes from at least two frogs. The data are presented as the mean ± SEM. The EC50 and IC50 values were determined according to the dose-response relationships by four-parameter logistic regression using OriginPro 8 J SR4 (ver. 8.0951) (LightStone, Tokyo, Japan). Unpaired t-tests were performed to evaluate the statistical significance.
Fig. 1. Chemical structure of fluralaner.
2. Materials and methods 2.1. Chemicals Fluralaner (99%) was synthesized as previously reported (Mita et al., 2005, 2009, 2010). GABA, tricaine (ethyl 3-aminobenzoate methanesulfonate), and collagenase were purchased from Sigma-Aldrich (St. Louis, MO, USA). Gentamycin, penicillin, and streptomycin were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Other chemicals were purchased from FujiFilm Wako Pure Chemical Corporation (Osaka, Japan), unless otherwise noted. 2.2. Wild-type and mutant Rdl cDNAs The cDNA encoding the wild-type GABAR RDL subunit (variant ac; RDLac) (accession no. AB177547, AB824728, and AB824729) from the housefly (Musca domestica) was cloned into the plasmid vector pBluescript KS(−) in our previous studies (Eguchi et al., 2006; Ozoe et al., 2013). The introduction of mutations into the cDNA sequence was performed using a QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA).
2.6. Homology modeling and docking simulations Prior to the construction of the homology model, the amino acid sequences of the Musca RDLac subunit and the Caenorhabditis elegans GluCl-α subunit were aligned using ClustalW2. A Musca GABAR homology model was constructed using MOE software (version 2018.01; Chemical Computing Group, Montreal, Canada). The X-ray crystal structure of the C. elegans GluCl-α channel (PDB code: 3RHW) was used as a template. The obtained model was optimized geometrically using the AMBER10:EHT force field. Potential docking sites were searched using SiteFinder of MOE. A fluralaner molecule created using MOE Builder was docked into the TSI of the generated model using the ASEDock program (2016.5 version, Chemical Computing Group) with default parameters. The energies of the receptor and ligands were minimized using the AMBER10:EHT force field.
2.3. Isolation of Xenopus oocytes Female African clawed frogs (Xenopus laevis) were purchased from Shimizu Laboratory Supplies Co., Ltd. (Kyoto, Japan). A standard oocyte solution (SOS) containing 100 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, and 5 mM HEPES (pH 7.6) was used in experiments with Xenopus oocytes. The lobes of the ovary were surgically removed from female frogs anesthetized by immersion in a 0.1% (w/v) tricaine solution. Follicle cells were treated with collagenase (2 mg/ml) in Ca2+-free SOS for 1–2 h at 20 °C. After the oocytes were washed with Ca2+-free SOS, they were incubated in SOS supplemented with 2.5 mM sodium pyruvate, gentamycin (50 μg/ml), penicillin (100 U/ml), and streptomycin (100 μg/ml) for one day at 16 °C.
3. Results The ectoparasiticide fluralaner (formerly reported as A1443) has not only high antiparasitic activity against fleas and ticks but also high insecticidal activity against houseflies (Ozoe et al., 2010). Fluralaner is a selective inhibitor of insect GABARs. Fig. 3A shows typical GABAactivated current traces observed during the perfusion of wild-type Musca GABARs with 10 nM fluralaner. The currents induced by GABA (EC50) were reduced after every application in a use-dependent manner until they reached a steady state. The IC50 value of fluralaner for wildtype GABAR was determined to be 4.77 ± 1.93 nM (Table 1). Fluralaner successfully docked into the TSI of the Musca GABAR homology model, with an amide side chain directed inward (Fig. 2A and B). We set out to examine the effects of the substitution of TM1 and TM3 amino acids on the inhibitory potency of fluralaner. We generated 17 GABAR mutants in which six amino acid residues in the outer half of the TSI were substituted with various amino acids (Table 1). The selected TM1 and TM3 amino acids from adjacent subunits face the TSI in our homology model (Fig. 2A and B). Nine out of the 17 mutants failed to show a robust GABA response or seemed to undergo spontaneous opening of their channels, likely because the TSI contains functionally important, irreplaceable amino acid residues that are required for channel gating and subunit assembly. However, the other mutants, including three TM2 mutants, showed GABA responses with various sensitivities, as described in the following sections. Their sensitivities to fluralaner were compared with the sensitivity of the wild type in terms
2.4. Preparation of Musca Rdl cRNAs and their injection into oocytes The Musca Rdl cDNA, including the upstream T7 promoter site, was amplified by PCR. The PCR products were purified using an illustra GFX PCR DNA and Gel Band Preparation Kit (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). After sequence verification, the amplified cDNA templates (100 ng) were in vitro transcribed into capped poly(A) cRNAs using a 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. The purified cRNA (5 ng) was injected into each oocyte using a Nanoliter 2000 injector (World Precision Instruments, Sarasota, FL, USA). The injected oocytes were incubated for two days at 16 °C. 2.5. TEVC electrophysiology An oocyte expressing Musca GABARs was placed in a chamber perfused with SOS. Electrophysiological recordings were performed using an Oocyte Clamp OC-726C amplifier (Warner Instruments, Hamden, CT, USA) at a holding potential of −80 mV at 20 °C. Glass 2
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Fig. 2. Amino acid residues and docked fluralaner in the transmembrane subunit interface (TSI) illustrated in a Musca GABAR homology model. (A) Side view of the TSI formed by two adjacent subunits. (B) Top view of the TSI. (C) Amino acid sequences of the TM1, TM2, and TM3 of the Musca RDL subunit. Substituted amino acids are highlighted.
wild type (Fig. 3B and C), resulting in a 68-fold increase in the IC50 value of fluralaner (Table 1). This substitution also led to a 42-fold increase in the EC50 of GABA (Fig. 3D; Table 1) and a 4-fold increase in the IC50 of fipronil (Fig. 3E; Table 2), which is a noncompetitive GABAR antagonist that binds to a site within the channel of GABAR (Ozoe, 2013). We substituted Gln271 with Asn, which has the ability to form a hydrogen bond similar to Gln. The effects of the Q271N substitution on the potency of fluralaner were not determined, as this substitution led to the spontaneous opening of the channel (data not shown). The I274C substitution caused the enhanced sensitivity of GABARs to fluralaner (Fig. 3C), resulting in a 56-fold decrease in the IC50 of fluralaner (Table 1) and an approximately 4-fold decrease in the EC50 of GABA (Fig. 3D; Table 1). The inhibition of GABA (EC50)-induced currents was gradually increased during the perfusion of fluralaner and finally reached approximately 50% inhibition at 100 pM (Fig. 3F). Neither the I274A nor I274F substitutions affected the potency of either GABA or fluralaner (Fig. 3C and D; Table 1). The L278C substitution reduced the sensitivity of GABARs to fluralaner (Fig. 3C), resulting in an 11-fold increase in the IC50 of fluralaner (Table 1), whereas it increased the sensitivity to GABA by approximately 3-fold (Fig. 3D; Table 1). The effects of the L278A and L278F substitutions on the potency of fluralaner were not determined, because these mutants produced only a small current in response to GABA and a spontaneous current in the absence of GABA, respectively (data not shown).
Table 1 Potencies of GABA and fluralaner in Musca GABAR wild type (WT) and mutants expressed in Xenopus oocytes. Region
TM1
TM2
TM3
Channel type
GABA EC50 (μM)
Fluralaner IC50 (nM)
WT Q271L Q271N I274A I274F I274C L278A L278F L278C A299N A299G A299S D329G D329E G333M G333S G333A F336L F336I F336M F336W
6.45 ± 0.67 271 ± 10⁎ ND 25.4 ± 6.5 7.16 ± 1.16 1.44 ± 0.14⁎ ND ND 2.32 ± 0.44⁎ 23.1 ± 2.3⁎ 8.45 ± 1.71 9.43 ± 5.16 ND ND 85.6 ± 9.6⁎ 152 ± 13⁎ 158 ± 9⁎ ND ND ND ND
4.77 ± 1.93 323 ± 51⁎ NT 2.67 ± 1.41 5.18 ± 1.49 0.0856 ± 0.0080⁎⁎ NT NT 53.3 ± 10.4⁎ 4.22 ± 1.45 40.9 ± 19.0 22.8 ± 14.1 NT NT > 10,000 > 10,000 1380 ± 230⁎ NT NT NT NT
ND, not determined. NT, not tested. The data are mean ± S.E.M. of 4–10 experiments. ⁎ p < .01 (relative to WT). ⁎⁎ p < .05 (relative to WT).
3.2. Substitutions in TM2
of the half maximal inhibitory concentration (IC50) for currents induced by the EC50 of GABA in each mutant.
We substituted an amino acid residue in TM2 to examine the sensitivity of the three mutants to fluralaner (Fig. 4). The Ala at the 299 position lies within the channel pore of Musca GABAR (Fig. 2A). The A299N, A299G, and A299S substitutions, which are equivalent to the mutations that are responsible for target-site insensitivity to fipronil in insect pests (Nakao, 2017), did not significantly affect the potency of fluralaner (p > .05) (Fig. 4B; Table 1).
3.1. Substitutions in TM1 We substituted three amino acid residues in the TM1 of the RDL subunit to generate eight Musca GABAR mutants (Fig. 2; Table 1). These amino acids face the TSI in the GABAR homology model. The doseresponse curves indicated that the Q271L substitution resulted in a reduction in the sensitivity of GABAR to fluralaner compared with the 3
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Fig. 3. Ligand responses of wild-type and mutant Musca GABARs with amino acid substitutions in TM1. (A) Fluralaner inhibition of GABA-induced currents in the wild type. Typical current trace. (B) Fluralaner inhibition of GABA-induced currents in the Q271L mutant. Typical current trace. (C) Dose-response curves of the fluralaner inhibition of GABA (EC50)-induced currents in TM1 mutants and the wild type. Normalized to currents induced by GABA alone. (D) Concentration-response curves of GABA-induced currents in TM1 mutants and the wild type. Normalized to maximum currents induced by GABA. (E) Dose-response curves of the fipronil inhibition of GABA (EC50)-induced currents in the Q271L and G333 M mutants and the wild type. Normalized to currents induced by GABA alone. (F) Fluralaner inhibition of GABA (EC50)-induced currents in the I274C mutant. Typical current trace.
3.3. Substitutions in TM3
Table 2 Potencies of fipronil in Musca GABAR wild type (WT) and mutants expressed in Xenopus oocytes. Region
Channel type
Fipronil IC50 (nM)
TM1 TM3
WT Q271L G333 M
14.6 ± 2.7 55.5 ± 5.5⁎ 131 ± 12⁎
We substituted three TM3 amino acid residues that face the subunit interface to generate nine mutants (Fig. 2; Table 1). The effects of the D329G and D329E substitutions on GABAR sensitivity to fluralaner were not tested, as the D329G mutant failed to activate a current, and the D329E mutant induced only a small current upon the application of GABA (data not shown). The G333M and G333S substitutions substantially reduced the sensitivity of GABARs to fluralaner (Fig. 5A), resulting in IC50 values of > 10 μM (Table 1). The G333A mutant was more sensitive to
The data are mean ± S.E.M. of 4–6 experiments. ⁎ p < .01 (relative to WT).
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Fig. 4. Ligand responses of mutant Musca GABARs with amino acid substitutions in TM2. (A) Concentration-response curves of GABA-induced currents in TM2 mutants and the wild type. Normalized to maximum currents induced by GABA. (B) Dose-response curves of the fluralaner inhibition of GABA (EC50)-induced currents in TM2 mutants and the wild type. Normalized to currents induced by GABA alone.
fluralaner than the G333M and G333S mutants (Fig. 5A and B), although this mutant was still approximately 300-fold less sensitive to fluralaner than the wild type (Table 1). Mutations at the 333 position resulted in 13- to 24-fold increases in the EC50 value of GABA (Fig. 5C and D; Table 1). The G333M substitution caused GABAR to be approximately 9-fold less sensitive to fipronil than the wild type (Fig. 3E; Table 2). As the F336 L substitution resulted in the spontaneous opening of the channel (data not shown), GABAR sensitivity to fluralaner was not determined for this mutant. The substitution of Phe336 with Ile, Met,
and Trp also resulted in the spontaneous opening of the channel (data not shown). 4. Discussion In the present study, we sought to identify the TSI amino acids that determine the high sensitivity of insect GABARs to the isoxazoline ectoparasiticide fluralaner. To this end, we substituted amino acids facing the outer half of the TSI in the Musca GABAR. The TSIs of pLGICs have a crevice between the α-helical TM3 of the principal (+) subunit and the
Fig. 5. Ligand responses of mutant Musca GABARs with amino acid substitutions in TM3. (A) Dose-response curves of the fluralaner inhibition of GABA (EC50)induced currents in TM3 mutants and the wild type. Normalized to currents induced by GABA alone. (B) Fluralaner inhibition of GABA (EC50)-induced currents in the G333A mutant. Typical current trace. (C) GABA-induced currents in the G333A mutant. Typical current trace. (D) Concentration-response curves of GABA-induced currents in TM3 mutants and the wild type. Normalized to maximum currents induced by GABA. 5
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α-helical TM1 of the complementary (−) subunit (Miller and Smart, 2010) (Fig. 2). Numerous studies have indicated that the TSI is a binding site for drugs, such as anesthetics and neurosteroids (Chua and Chebib, 2017; Laverty et al., 2017; Miller et al., 2017). The TSIs of GluClRs and nicotinic acetylcholine receptors are sites of action for the anthelmintic/insecticide avermectin and the insecticide spinosyn, respectively (Hibbs and Gouaux, 2011; Puinean et al., 2013). We have previously reported that the low fluralaner sensitivity in the Musca GluClR (IC50 = 146 nM) was dramatically enhanced (IC50 = 1.06 nM) by substituting an amino acid in the TSI with the corresponding amino acid in the Musca GABAR, which is highly sensitive to fluralaner (IC50 = 6.05 nM) (Nakata et al., 2017). As a next step, this finding led us to investigate the roles of TSI amino acids in the high sensitivity of the Musca GABAR to fluralaner. We first performed ligand-docking simulation studies using a Musca GABAR homology model to determine whether fluralaner binds in the TSI cavity. Consequently, the docking model showed that fluralaner successfully docked into the TSI, and we were able to predict several potential amino acid residues that could interact with fluralaner (Fig. 2). Notably, the docking model revealed that the carbonyl oxygen atom of Gln271 in TM1 could act as a hydrogen bond acceptor for the side chain amide group of fluralaner. To test this prediction, Gln271 was substituted with Leu, which has no ability to form a hydrogen bond. As a result, this substitution was found to cause a marked reduction in the sensitivity of GABAR to fluralaner, supporting the validity of our prediction. The substitution of Ile274, which is located one α-helical turn below Gln271, with a small or a large hydrophobic side chain amino acid, i.e., Ala or Phe, did not significantly change the sensitivity of GABAR to fluralaner. A hydrophobic side chain, regardless of its size, might favor an interaction with the hydrophobic moiety around the isoxazoline ring of fluralaner. In contrast to the results for the I274A and I274F mutants, the I274C substitution resulted in further enhancement of the high fluralaner sensitivity of GABAR to the subnanomolar level (Fig. 3C; Table 1). This mutant showed a typical response to GABA but a slight decrease in the EC50 value of GABA. There is no nearby Cys capable of forming a disulfide bond with the substituted Cys274 in the Musca GABAR. It remains to be seen whether the sulfhydryl group of Cys274 has a peculiar interaction with the heteroatoms of the isoxazoline ring of fluralaner, given the moderate hydrogen-bonding ability of Cys (Zhou et al., 2009). The substitution of Leu278, located two α-helical turns below Gln271, with Cys reduced the fluralaner sensitivity of GABAR. The attempts to test mutants in which Leu278 was substituted with Ala and Phe were unsuccessful because these mutants did not activate large inward currents in response to GABA. Taken together, these findings indicate that Gln271, Ile274, and Leu278 in TM1 are associated with the high (low nanomolar) sensitivity of Musca GABAR to fluralaner. Although two (Gln and Ile) of these amino acids are conserved at the equivalent position of Musca GluClR, we previously showed that Leu315 (equivalent to Phe336 in GABAR) in TM3 determines the lower (high nanomolar) sensitivity of GluClR (Nakata et al., 2017). We next examined whether mutations of an amino acid residue in TM2 could affect the sensitivity of GABARs to fluralaner. Ala299 is situated at the so-called 2′ position of the TM2 in the RDL subunit of the Musca GABAR and most likely is located within the channel pore (Ozoe, 2013). The mutation of the equivalent Ala to Ser, Gly or Asn in insect pests is responsible for target site insensitivity to cyclodiene and phenylpyrazole insecticides (Nakao, 2017; Ozoe et al., 2015). The mutations examined in the present study did not significantly affect the sensitivity of GABAR to fluralaner, indicating that the binding site of fluralaner differs from that of cyclodienes and phenylpyrazoles. Our findings are consistent with those in previous studies (Asahi et al., 2015; Ozoe et al., 2010). A conserved Gly in TM3 has been shown to play a critical role in the sensitivity of pLGICs to ivermectin and the antagonism of insect GABARs by broflanilide (Lynagh and Lynch, 2010; Nakao et al., 2013).
Mutations of this conserved Gly to Asp in GluClR1 and its equivalent Gly residue to Glu in GluClR3 have been implicated in abamectin resistance in the two-spotted spider mite (Tetranychus urticae) (Dermauw et al., 2012; Kwon et al., 2010). The latter mutation was further confirmed to result in insensitivity to abamectin using a heterologous expression system (Mermans et al., 2017). In the present study, three Gly333 mutants were generated to examine the role of this amino acid in the action of fluralaner. The substitution of Gly333 with Met or Ser eliminated GABAR sensitivity to fluralaner, and the G333A mutant showed over 100-fold lower sensitivity than the wild type. These findings are consistent with the results for ivermectin and broflanilide, as the bulkier amino acid side chains exerted greater effects on the reduction in the sensitivity to these compounds (Lynagh and Lynch, 2010; Nakao et al., 2013). A bulky side chain may occlude the binding site or may block these compounds from accessing to the binding site. Alternatively, as Gly is an amino acid that confers flexibility to proteins, the replacement of Gly with a bulky amino acid may lead to the loss of the flexibility of TM3, which may change the channel gating kinetics (Atif et al., 2017) and thereby affect the action of fluralaner. Since our previous study using Musca GluClRs indicated that the presence of an aromatic amino acid one α-helical turn below the conserved Gly plays an important role in the high sensitivity of pLGICs to fluralaner (Nakata et al., 2017), we wanted to test the F336L mutation to see whether this inverse mutation reduced GABAR sensitivity to fluralaner. Unfortunately, we could not confirm this conclusion that was derived from the study of GluClRs because all mutants that were generated were nonfunctional. Phe336 may be an indispensable amino acid for channel assembly or gating in Musca GABARs. As the TSI amino acids most likely play important roles in GABAinduced channel gating, several mutations in the TSI induced changes in the agonist sensitivity of GABARs (i.e., EC50 values). The Q271L mutant and the three Gly333 mutants with reduced sensitivity to fluralaner showed low sensitivities to GABA, and the I274C mutant with increased sensitivity to fluralaner exhibited higher sensitivity to GABA than the wild type. However, the L278C mutant, which showed reduced sensitivity to fluralaner, showed higher sensitivity to GABA than the wild type. There seems to be no specific relationship between the IC50 values of fluralaner and the EC50 values of GABA. Therefore, the reduction in the GABA sensitivity of GABAR seems not to be linked to the mode of action of fluralaner. As GABAR is an allosteric protein, we also examined whether the Q271L and G333M substitutions affected the antagonistic effect of fipronil, which binds to a site adjacent to Ala299 within the channel pore. The Q271L mutant showed a slightly reduced sensitivity to fipronil, and the G333 M substitution caused an approximately 9-fold reduction in the sensitivity to fipronil. These results indicated that the amino acid substitutions in the TSI exerted certain effects on fipronil binding that were reduced compared to their effects on fluralaner binding. However, this does not imply that fluralaner shares a common binding site with fipronil because the substitutions of Ala299 did not change the potency of fluralaner. In conclusion, we have shown that four amino acid residues in the TSI play critical roles in the antagonistic effect of fluralaner on insect GABARs (Fig. 6). Fluralaner is a powerful inhibitor of GABA-activated currents in wild-type Musca GABARs. TEVC experiments indicated that inhibition was gradually enhanced and reached a steady state after repeated 3-s applications of GABA during the perfusion of fluralaner. Channel activation by GABA induces a conformational change in the TSI (Bali and Akabas, 2012; Bali et al., 2009) and expands the space in the TSI (Stewart et al., 2013), as revealed by a substituted cysteine accessibility method and a GABA-dependent substituted Cys crosslinking method. According to these findings, fluralaner could penetrate into the expanded TSI to induce channel blockage, thereby exerting its insecticidal effect; however, additional experiments are needed to reach a definitive conclusion.
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Fig. 6. Summary illustrating the effects of intersubunit amino acid substitutions on GABAR sensitivity to fluralaner.
References Asahi, M., Kobayashi, M., Matsui, H., Nakahira, K., 2015. Differential mechanisms of action of the novel γ-aminobutyric acid receptor antagonist ectoparasiticides fluralaner (A1443) and fipronil. Pest Manag. Sci. 71, 91–95. https://doi.org/10.1002/ps. 3768. Atif, M., Estrada-Mondragon, A., Nguyen, B., Lynch, J.W., Keramidas, A., 2017. Effects of glutamate and ivermectin on single glutamate-gated chloride channels of the parasitic nematode H. contortus. PLoS Pathog. 13, e1006663. https://doi.org/10.1371/ journal.ppat.1006663. Bali, M., Akabas, M.H., 2012. Gating-induced conformational rearrangement of the γaminobutyric acid type A receptor β-α subunit interface in the membrane-spanning domain. J. Biol. Chem. 287, 27762–27770. https://doi.org/10.1074/jbc.M112. 363341. Bali, M., Jansen, M., Akabas, M.H., 2009. GABA-induced intersubunit conformational movement in the GABAA receptor α1M1-β2M3 transmembrane subunit interface: experimental basis for homology modeling of an intravenous anesthetic binding site. J. Neurosci. 29, 3083–3092. https://doi.org/10.1523/JNEUROSCI.6090-08.2009. Chua, H.C., Chebib, M., 2017. GABAA receptors and the diversity in their structure and pharmacology. Adv. Pharmacol. 79, 1–34. https://doi.org/10.1016/bs.apha.2017. 03.003. Dermauw, W., Ilias, A., Riga, M., Tsagkarakou, A., Grbić, M., Tirry, L., Van Leeuwen, T., Vontas, J., 2012. The cys-loop ligand-gated ion channel gene family of Tetranychus urticae: implications for acaricide toxicology and a novel mutation associated with abamectin resistance. Insect Biochem. Mol. Biol. 42, 455–465. https://doi.org/10. 1016/j.ibmb.2012.03.002. Eguchi, Y., Ihara, M., Ochi, E., Shibata, Y., Matsuda, K., Fushiki, S., Sugama, H., Hamasaki, Y., Niwa, H., Wada, M., Ozoe, F., Ozoe, Y., 2006. Functional characterization of Musca glutamate- and GABA-gated chloride channels expressed independently and coexpressed in Xenopus oocytes. Insect Mol. Biol. 15, 773–783. https://doi.org/10.1111/j.1365-2583.2006.00680.x. Hibbs, R.E., Gouaux, E., 2011. Principles of activation and permeation in an anion-selective Cys-loop receptor. Nature 474, 54–60. https://doi.org/10.1038/nature10139. Kwon, D.H., Yoon, K.S., Clark, J.M., Lee, S.H., 2010. A point mutation in a glutamategated chloride channel confers abamectin resistance in the two-spotted spider mite, Tetranychus urticae Koch. Insect Mol. Biol. 19, 583–591. https://doi.org/10.1111/j. 1365-2583.2010.01017.x. Laverty, D., Thomas, P., Field, M., Andersen, O.J., Gold, M.G., Biggin, P.C., Gielen, M., Smart, T.G., 2017. Crystal structures of a GABAA receptor chimera reveal new endogenous neurosteroid-binding sites. Nature Struct. Mol. Biol. 24, 977–985. https:// doi.org/10.1038/nsmb.3477. Lynagh, T., Lynch, J.W., 2010. A glycine residue essential for high ivermectin sensitivity in Cys-loop ion channel receptors. Int. J. Parasitol. 40, 1477–1481. https://doi.org/ 10.1016/j.ijpara.2010.07.010.
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Pesticide Biochemistry and Physiology journal homepage: www.elsevier.com/locate/pest
Effects of intersubunit amino acid substitutions on GABA receptor sensitivity to the ectoparasiticide fluralaner Kohei Yamatoa, Yunosuke Nakataa, Madoka Takashimaa, Fumiyo Ozoea, Miho Asahib, ⁎ Masaki Kobayashib, Yoshihisa Ozoea, a b
Faculty of Life and Environmental Science, Shimane University, Matsue, Shimane 690-8504, Japan Biological Research Laboratories, Nissan Chemical Corporation, Shiraoka, Saitama 349-0294, Japan
A R T I C LE I N FO
A B S T R A C T
Keywords: GABA receptor Transmembrane subunit interface Isoxazoline 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.
1. Introduction γ-Aminobutyric acid (GABA) is a free amino acid that plays vital roles as a ubiquitous inhibitory neurotransmitter in the nervous system. GABA released from presynaptic neurons binds to the GABA receptor (GABAR), which is a member of the pentameric ligand-gated chloride channel (pLGICs) family, and triggers rapid chloride flow through the channel into postsynaptic neurons or muscle cells, thereby offsetting the effect of excitatory postsynaptic cation influx (Mody and Pearce, 2004). The binding of GABA to the extracellular domain of GABAR induces conformational changes in the channel domain, each subunit of which is composed of four consecutive α-helical transmembrane segments (TM1–TM4), to open the pore (Miller and Smart, 2010; Masiulis et al., 2019). In the case of arthropods, GABAergic function is mediated by homopentameric GABARs formed by RDL subunits. Arthropod GABARs serve as important targets for pest control chemicals (Ozoe, 2013). Fluralaner (Fig. 1), an isoxazoline ectoparasiticide, potently antagonizes agonist activity at GABARs, and it also acts, albeit less potently, on glutamate-gated chloride channels (GluClRs) (Ozoe et al., 2010).
⁎
Fluralaner has been shown to act at a site that is different from that targeted by conventional GABAR antagonist insecticides (Asahi et al., 2015), and its site of action remains to be defined. We previously reported that the replacement of Leu315 in the third transmembrane segment (TM3) of the housefly (Musca domestica) GluClR subunit with the amino acid found at the corresponding position in the housefly GABAR RDL subunit (i.e., Phe at the 336 position) dramatically enhanced the sensitivity of GluClRs to fluralaner (Nakata et al., 2017). In the present study, to determine the site of action of fluralaner, we substituted amino acid residues in the transmembrane subunit interface (TSI) of the housefly GABAR with various amino acids (Fig. 2) and examined the changes in the sensitivity of mutant GABARs expressed in Xenopus oocytes to fluralaner using two-electrode voltage clamp (TEVC) electrophysiology. Here, we report that four amino acids in the TSI are associated with the high sensitivity of GABARs to fluralaner.
Corresponding author. E-mail address: [email protected] (Y. Ozoe).
https://doi.org/10.1016/j.pestbp.2019.11.001 Received 9 September 2019; Received in revised form 17 October 2019; Accepted 3 November 2019 0048-3575/ © 2019 Elsevier Inc. All rights reserved.
Please cite this article as: Kohei Yamato, et al., Pesticide Biochemistry and Physiology, https://doi.org/10.1016/j.pestbp.2019.11.001
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microelectrodes were filled with 2 M KCl to yield a resistance of 0.5–1.6 MΩ. The data were digitized using a Lab-Trax-4/16 converter (World Precision Instruments) and analyzed using Data-Trax2 software (World Precision Instruments). The same oocyte was exposed to increasing concentrations of GABA dissolved in SOS for 3 s. The oocyte was perfused with SOS between each application for approximately 1 min to ensure full recovery from desensitization. Fluralaner was dissolved in dimethyl sulfoxide (DMSO) and diluted with SOS to produce the desired concentration. The DMSO concentrations were < 0.01%, which had no adverse effects on GABA responses. To analyze the antagonism of GABARs by fluralaner, GABA dissolved in SOS at a concentration equivalent to the EC50 of each GABAR (Table 1) was repeatedly applied to the oocytes for 3 s at 30–60s intervals, while the oocytes were perfused with a fluralaner solution until steady-state inhibition was reached after stable GABA responses. The steady-state inhibition percentage obtained for each concentration was used to draw a dose-response curve. All experiments were replicated using 4–10 oocytes from at least two frogs. The data are presented as the mean ± SEM. The EC50 and IC50 values were determined according to the dose-response relationships by four-parameter logistic regression using OriginPro 8 J SR4 (ver. 8.0951) (LightStone, Tokyo, Japan). Unpaired t-tests were performed to evaluate the statistical significance.
Fig. 1. Chemical structure of fluralaner.
2. Materials and methods 2.1. Chemicals Fluralaner (99%) was synthesized as previously reported (Mita et al., 2005, 2009, 2010). GABA, tricaine (ethyl 3-aminobenzoate methanesulfonate), and collagenase were purchased from Sigma-Aldrich (St. Louis, MO, USA). Gentamycin, penicillin, and streptomycin were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Other chemicals were purchased from FujiFilm Wako Pure Chemical Corporation (Osaka, Japan), unless otherwise noted. 2.2. Wild-type and mutant Rdl cDNAs The cDNA encoding the wild-type GABAR RDL subunit (variant ac; RDLac) (accession no. AB177547, AB824728, and AB824729) from the housefly (Musca domestica) was cloned into the plasmid vector pBluescript KS(−) in our previous studies (Eguchi et al., 2006; Ozoe et al., 2013). The introduction of mutations into the cDNA sequence was performed using a QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA).
2.6. Homology modeling and docking simulations Prior to the construction of the homology model, the amino acid sequences of the Musca RDLac subunit and the Caenorhabditis elegans GluCl-α subunit were aligned using ClustalW2. A Musca GABAR homology model was constructed using MOE software (version 2018.01; Chemical Computing Group, Montreal, Canada). The X-ray crystal structure of the C. elegans GluCl-α channel (PDB code: 3RHW) was used as a template. The obtained model was optimized geometrically using the AMBER10:EHT force field. Potential docking sites were searched using SiteFinder of MOE. A fluralaner molecule created using MOE Builder was docked into the TSI of the generated model using the ASEDock program (2016.5 version, Chemical Computing Group) with default parameters. The energies of the receptor and ligands were minimized using the AMBER10:EHT force field.
2.3. Isolation of Xenopus oocytes Female African clawed frogs (Xenopus laevis) were purchased from Shimizu Laboratory Supplies Co., Ltd. (Kyoto, Japan). A standard oocyte solution (SOS) containing 100 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, and 5 mM HEPES (pH 7.6) was used in experiments with Xenopus oocytes. The lobes of the ovary were surgically removed from female frogs anesthetized by immersion in a 0.1% (w/v) tricaine solution. Follicle cells were treated with collagenase (2 mg/ml) in Ca2+-free SOS for 1–2 h at 20 °C. After the oocytes were washed with Ca2+-free SOS, they were incubated in SOS supplemented with 2.5 mM sodium pyruvate, gentamycin (50 μg/ml), penicillin (100 U/ml), and streptomycin (100 μg/ml) for one day at 16 °C.
3. Results The ectoparasiticide fluralaner (formerly reported as A1443) has not only high antiparasitic activity against fleas and ticks but also high insecticidal activity against houseflies (Ozoe et al., 2010). Fluralaner is a selective inhibitor of insect GABARs. Fig. 3A shows typical GABAactivated current traces observed during the perfusion of wild-type Musca GABARs with 10 nM fluralaner. The currents induced by GABA (EC50) were reduced after every application in a use-dependent manner until they reached a steady state. The IC50 value of fluralaner for wildtype GABAR was determined to be 4.77 ± 1.93 nM (Table 1). Fluralaner successfully docked into the TSI of the Musca GABAR homology model, with an amide side chain directed inward (Fig. 2A and B). We set out to examine the effects of the substitution of TM1 and TM3 amino acids on the inhibitory potency of fluralaner. We generated 17 GABAR mutants in which six amino acid residues in the outer half of the TSI were substituted with various amino acids (Table 1). The selected TM1 and TM3 amino acids from adjacent subunits face the TSI in our homology model (Fig. 2A and B). Nine out of the 17 mutants failed to show a robust GABA response or seemed to undergo spontaneous opening of their channels, likely because the TSI contains functionally important, irreplaceable amino acid residues that are required for channel gating and subunit assembly. However, the other mutants, including three TM2 mutants, showed GABA responses with various sensitivities, as described in the following sections. Their sensitivities to fluralaner were compared with the sensitivity of the wild type in terms
2.4. Preparation of Musca Rdl cRNAs and their injection into oocytes The Musca Rdl cDNA, including the upstream T7 promoter site, was amplified by PCR. The PCR products were purified using an illustra GFX PCR DNA and Gel Band Preparation Kit (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). After sequence verification, the amplified cDNA templates (100 ng) were in vitro transcribed into capped poly(A) cRNAs using a 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. The purified cRNA (5 ng) was injected into each oocyte using a Nanoliter 2000 injector (World Precision Instruments, Sarasota, FL, USA). The injected oocytes were incubated for two days at 16 °C. 2.5. TEVC electrophysiology An oocyte expressing Musca GABARs was placed in a chamber perfused with SOS. Electrophysiological recordings were performed using an Oocyte Clamp OC-726C amplifier (Warner Instruments, Hamden, CT, USA) at a holding potential of −80 mV at 20 °C. Glass 2
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Fig. 2. Amino acid residues and docked fluralaner in the transmembrane subunit interface (TSI) illustrated in a Musca GABAR homology model. (A) Side view of the TSI formed by two adjacent subunits. (B) Top view of the TSI. (C) Amino acid sequences of the TM1, TM2, and TM3 of the Musca RDL subunit. Substituted amino acids are highlighted.
wild type (Fig. 3B and C), resulting in a 68-fold increase in the IC50 value of fluralaner (Table 1). This substitution also led to a 42-fold increase in the EC50 of GABA (Fig. 3D; Table 1) and a 4-fold increase in the IC50 of fipronil (Fig. 3E; Table 2), which is a noncompetitive GABAR antagonist that binds to a site within the channel of GABAR (Ozoe, 2013). We substituted Gln271 with Asn, which has the ability to form a hydrogen bond similar to Gln. The effects of the Q271N substitution on the potency of fluralaner were not determined, as this substitution led to the spontaneous opening of the channel (data not shown). The I274C substitution caused the enhanced sensitivity of GABARs to fluralaner (Fig. 3C), resulting in a 56-fold decrease in the IC50 of fluralaner (Table 1) and an approximately 4-fold decrease in the EC50 of GABA (Fig. 3D; Table 1). The inhibition of GABA (EC50)-induced currents was gradually increased during the perfusion of fluralaner and finally reached approximately 50% inhibition at 100 pM (Fig. 3F). Neither the I274A nor I274F substitutions affected the potency of either GABA or fluralaner (Fig. 3C and D; Table 1). The L278C substitution reduced the sensitivity of GABARs to fluralaner (Fig. 3C), resulting in an 11-fold increase in the IC50 of fluralaner (Table 1), whereas it increased the sensitivity to GABA by approximately 3-fold (Fig. 3D; Table 1). The effects of the L278A and L278F substitutions on the potency of fluralaner were not determined, because these mutants produced only a small current in response to GABA and a spontaneous current in the absence of GABA, respectively (data not shown).
Table 1 Potencies of GABA and fluralaner in Musca GABAR wild type (WT) and mutants expressed in Xenopus oocytes. Region
TM1
TM2
TM3
Channel type
GABA EC50 (μM)
Fluralaner IC50 (nM)
WT Q271L Q271N I274A I274F I274C L278A L278F L278C A299N A299G A299S D329G D329E G333M G333S G333A F336L F336I F336M F336W
6.45 ± 0.67 271 ± 10⁎ ND 25.4 ± 6.5 7.16 ± 1.16 1.44 ± 0.14⁎ ND ND 2.32 ± 0.44⁎ 23.1 ± 2.3⁎ 8.45 ± 1.71 9.43 ± 5.16 ND ND 85.6 ± 9.6⁎ 152 ± 13⁎ 158 ± 9⁎ ND ND ND ND
4.77 ± 1.93 323 ± 51⁎ NT 2.67 ± 1.41 5.18 ± 1.49 0.0856 ± 0.0080⁎⁎ NT NT 53.3 ± 10.4⁎ 4.22 ± 1.45 40.9 ± 19.0 22.8 ± 14.1 NT NT > 10,000 > 10,000 1380 ± 230⁎ NT NT NT NT
ND, not determined. NT, not tested. The data are mean ± S.E.M. of 4–10 experiments. ⁎ p < .01 (relative to WT). ⁎⁎ p < .05 (relative to WT).
3.2. Substitutions in TM2
of the half maximal inhibitory concentration (IC50) for currents induced by the EC50 of GABA in each mutant.
We substituted an amino acid residue in TM2 to examine the sensitivity of the three mutants to fluralaner (Fig. 4). The Ala at the 299 position lies within the channel pore of Musca GABAR (Fig. 2A). The A299N, A299G, and A299S substitutions, which are equivalent to the mutations that are responsible for target-site insensitivity to fipronil in insect pests (Nakao, 2017), did not significantly affect the potency of fluralaner (p > .05) (Fig. 4B; Table 1).
3.1. Substitutions in TM1 We substituted three amino acid residues in the TM1 of the RDL subunit to generate eight Musca GABAR mutants (Fig. 2; Table 1). These amino acids face the TSI in the GABAR homology model. The doseresponse curves indicated that the Q271L substitution resulted in a reduction in the sensitivity of GABAR to fluralaner compared with the 3
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Fig. 3. Ligand responses of wild-type and mutant Musca GABARs with amino acid substitutions in TM1. (A) Fluralaner inhibition of GABA-induced currents in the wild type. Typical current trace. (B) Fluralaner inhibition of GABA-induced currents in the Q271L mutant. Typical current trace. (C) Dose-response curves of the fluralaner inhibition of GABA (EC50)-induced currents in TM1 mutants and the wild type. Normalized to currents induced by GABA alone. (D) Concentration-response curves of GABA-induced currents in TM1 mutants and the wild type. Normalized to maximum currents induced by GABA. (E) Dose-response curves of the fipronil inhibition of GABA (EC50)-induced currents in the Q271L and G333 M mutants and the wild type. Normalized to currents induced by GABA alone. (F) Fluralaner inhibition of GABA (EC50)-induced currents in the I274C mutant. Typical current trace.
3.3. Substitutions in TM3
Table 2 Potencies of fipronil in Musca GABAR wild type (WT) and mutants expressed in Xenopus oocytes. Region
Channel type
Fipronil IC50 (nM)
TM1 TM3
WT Q271L G333 M
14.6 ± 2.7 55.5 ± 5.5⁎ 131 ± 12⁎
We substituted three TM3 amino acid residues that face the subunit interface to generate nine mutants (Fig. 2; Table 1). The effects of the D329G and D329E substitutions on GABAR sensitivity to fluralaner were not tested, as the D329G mutant failed to activate a current, and the D329E mutant induced only a small current upon the application of GABA (data not shown). The G333M and G333S substitutions substantially reduced the sensitivity of GABARs to fluralaner (Fig. 5A), resulting in IC50 values of > 10 μM (Table 1). The G333A mutant was more sensitive to
The data are mean ± S.E.M. of 4–6 experiments. ⁎ p < .01 (relative to WT).
4
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Fig. 4. Ligand responses of mutant Musca GABARs with amino acid substitutions in TM2. (A) Concentration-response curves of GABA-induced currents in TM2 mutants and the wild type. Normalized to maximum currents induced by GABA. (B) Dose-response curves of the fluralaner inhibition of GABA (EC50)-induced currents in TM2 mutants and the wild type. Normalized to currents induced by GABA alone.
fluralaner than the G333M and G333S mutants (Fig. 5A and B), although this mutant was still approximately 300-fold less sensitive to fluralaner than the wild type (Table 1). Mutations at the 333 position resulted in 13- to 24-fold increases in the EC50 value of GABA (Fig. 5C and D; Table 1). The G333M substitution caused GABAR to be approximately 9-fold less sensitive to fipronil than the wild type (Fig. 3E; Table 2). As the F336 L substitution resulted in the spontaneous opening of the channel (data not shown), GABAR sensitivity to fluralaner was not determined for this mutant. The substitution of Phe336 with Ile, Met,
and Trp also resulted in the spontaneous opening of the channel (data not shown). 4. Discussion In the present study, we sought to identify the TSI amino acids that determine the high sensitivity of insect GABARs to the isoxazoline ectoparasiticide fluralaner. To this end, we substituted amino acids facing the outer half of the TSI in the Musca GABAR. The TSIs of pLGICs have a crevice between the α-helical TM3 of the principal (+) subunit and the
Fig. 5. Ligand responses of mutant Musca GABARs with amino acid substitutions in TM3. (A) Dose-response curves of the fluralaner inhibition of GABA (EC50)induced currents in TM3 mutants and the wild type. Normalized to currents induced by GABA alone. (B) Fluralaner inhibition of GABA (EC50)-induced currents in the G333A mutant. Typical current trace. (C) GABA-induced currents in the G333A mutant. Typical current trace. (D) Concentration-response curves of GABA-induced currents in TM3 mutants and the wild type. Normalized to maximum currents induced by GABA. 5
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α-helical TM1 of the complementary (−) subunit (Miller and Smart, 2010) (Fig. 2). Numerous studies have indicated that the TSI is a binding site for drugs, such as anesthetics and neurosteroids (Chua and Chebib, 2017; Laverty et al., 2017; Miller et al., 2017). The TSIs of GluClRs and nicotinic acetylcholine receptors are sites of action for the anthelmintic/insecticide avermectin and the insecticide spinosyn, respectively (Hibbs and Gouaux, 2011; Puinean et al., 2013). We have previously reported that the low fluralaner sensitivity in the Musca GluClR (IC50 = 146 nM) was dramatically enhanced (IC50 = 1.06 nM) by substituting an amino acid in the TSI with the corresponding amino acid in the Musca GABAR, which is highly sensitive to fluralaner (IC50 = 6.05 nM) (Nakata et al., 2017). As a next step, this finding led us to investigate the roles of TSI amino acids in the high sensitivity of the Musca GABAR to fluralaner. We first performed ligand-docking simulation studies using a Musca GABAR homology model to determine whether fluralaner binds in the TSI cavity. Consequently, the docking model showed that fluralaner successfully docked into the TSI, and we were able to predict several potential amino acid residues that could interact with fluralaner (Fig. 2). Notably, the docking model revealed that the carbonyl oxygen atom of Gln271 in TM1 could act as a hydrogen bond acceptor for the side chain amide group of fluralaner. To test this prediction, Gln271 was substituted with Leu, which has no ability to form a hydrogen bond. As a result, this substitution was found to cause a marked reduction in the sensitivity of GABAR to fluralaner, supporting the validity of our prediction. The substitution of Ile274, which is located one α-helical turn below Gln271, with a small or a large hydrophobic side chain amino acid, i.e., Ala or Phe, did not significantly change the sensitivity of GABAR to fluralaner. A hydrophobic side chain, regardless of its size, might favor an interaction with the hydrophobic moiety around the isoxazoline ring of fluralaner. In contrast to the results for the I274A and I274F mutants, the I274C substitution resulted in further enhancement of the high fluralaner sensitivity of GABAR to the subnanomolar level (Fig. 3C; Table 1). This mutant showed a typical response to GABA but a slight decrease in the EC50 value of GABA. There is no nearby Cys capable of forming a disulfide bond with the substituted Cys274 in the Musca GABAR. It remains to be seen whether the sulfhydryl group of Cys274 has a peculiar interaction with the heteroatoms of the isoxazoline ring of fluralaner, given the moderate hydrogen-bonding ability of Cys (Zhou et al., 2009). The substitution of Leu278, located two α-helical turns below Gln271, with Cys reduced the fluralaner sensitivity of GABAR. The attempts to test mutants in which Leu278 was substituted with Ala and Phe were unsuccessful because these mutants did not activate large inward currents in response to GABA. Taken together, these findings indicate that Gln271, Ile274, and Leu278 in TM1 are associated with the high (low nanomolar) sensitivity of Musca GABAR to fluralaner. Although two (Gln and Ile) of these amino acids are conserved at the equivalent position of Musca GluClR, we previously showed that Leu315 (equivalent to Phe336 in GABAR) in TM3 determines the lower (high nanomolar) sensitivity of GluClR (Nakata et al., 2017). We next examined whether mutations of an amino acid residue in TM2 could affect the sensitivity of GABARs to fluralaner. Ala299 is situated at the so-called 2′ position of the TM2 in the RDL subunit of the Musca GABAR and most likely is located within the channel pore (Ozoe, 2013). The mutation of the equivalent Ala to Ser, Gly or Asn in insect pests is responsible for target site insensitivity to cyclodiene and phenylpyrazole insecticides (Nakao, 2017; Ozoe et al., 2015). The mutations examined in the present study did not significantly affect the sensitivity of GABAR to fluralaner, indicating that the binding site of fluralaner differs from that of cyclodienes and phenylpyrazoles. Our findings are consistent with those in previous studies (Asahi et al., 2015; Ozoe et al., 2010). A conserved Gly in TM3 has been shown to play a critical role in the sensitivity of pLGICs to ivermectin and the antagonism of insect GABARs by broflanilide (Lynagh and Lynch, 2010; Nakao et al., 2013).
Mutations of this conserved Gly to Asp in GluClR1 and its equivalent Gly residue to Glu in GluClR3 have been implicated in abamectin resistance in the two-spotted spider mite (Tetranychus urticae) (Dermauw et al., 2012; Kwon et al., 2010). The latter mutation was further confirmed to result in insensitivity to abamectin using a heterologous expression system (Mermans et al., 2017). In the present study, three Gly333 mutants were generated to examine the role of this amino acid in the action of fluralaner. The substitution of Gly333 with Met or Ser eliminated GABAR sensitivity to fluralaner, and the G333A mutant showed over 100-fold lower sensitivity than the wild type. These findings are consistent with the results for ivermectin and broflanilide, as the bulkier amino acid side chains exerted greater effects on the reduction in the sensitivity to these compounds (Lynagh and Lynch, 2010; Nakao et al., 2013). A bulky side chain may occlude the binding site or may block these compounds from accessing to the binding site. Alternatively, as Gly is an amino acid that confers flexibility to proteins, the replacement of Gly with a bulky amino acid may lead to the loss of the flexibility of TM3, which may change the channel gating kinetics (Atif et al., 2017) and thereby affect the action of fluralaner. Since our previous study using Musca GluClRs indicated that the presence of an aromatic amino acid one α-helical turn below the conserved Gly plays an important role in the high sensitivity of pLGICs to fluralaner (Nakata et al., 2017), we wanted to test the F336L mutation to see whether this inverse mutation reduced GABAR sensitivity to fluralaner. Unfortunately, we could not confirm this conclusion that was derived from the study of GluClRs because all mutants that were generated were nonfunctional. Phe336 may be an indispensable amino acid for channel assembly or gating in Musca GABARs. As the TSI amino acids most likely play important roles in GABAinduced channel gating, several mutations in the TSI induced changes in the agonist sensitivity of GABARs (i.e., EC50 values). The Q271L mutant and the three Gly333 mutants with reduced sensitivity to fluralaner showed low sensitivities to GABA, and the I274C mutant with increased sensitivity to fluralaner exhibited higher sensitivity to GABA than the wild type. However, the L278C mutant, which showed reduced sensitivity to fluralaner, showed higher sensitivity to GABA than the wild type. There seems to be no specific relationship between the IC50 values of fluralaner and the EC50 values of GABA. Therefore, the reduction in the GABA sensitivity of GABAR seems not to be linked to the mode of action of fluralaner. As GABAR is an allosteric protein, we also examined whether the Q271L and G333M substitutions affected the antagonistic effect of fipronil, which binds to a site adjacent to Ala299 within the channel pore. The Q271L mutant showed a slightly reduced sensitivity to fipronil, and the G333 M substitution caused an approximately 9-fold reduction in the sensitivity to fipronil. These results indicated that the amino acid substitutions in the TSI exerted certain effects on fipronil binding that were reduced compared to their effects on fluralaner binding. However, this does not imply that fluralaner shares a common binding site with fipronil because the substitutions of Ala299 did not change the potency of fluralaner. In conclusion, we have shown that four amino acid residues in the TSI play critical roles in the antagonistic effect of fluralaner on insect GABARs (Fig. 6). Fluralaner is a powerful inhibitor of GABA-activated currents in wild-type Musca GABARs. TEVC experiments indicated that inhibition was gradually enhanced and reached a steady state after repeated 3-s applications of GABA during the perfusion of fluralaner. Channel activation by GABA induces a conformational change in the TSI (Bali and Akabas, 2012; Bali et al., 2009) and expands the space in the TSI (Stewart et al., 2013), as revealed by a substituted cysteine accessibility method and a GABA-dependent substituted Cys crosslinking method. According to these findings, fluralaner could penetrate into the expanded TSI to induce channel blockage, thereby exerting its insecticidal effect; however, additional experiments are needed to reach a definitive conclusion.
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Fig. 6. Summary illustrating the effects of intersubunit amino acid substitutions on GABAR sensitivity to fluralaner.
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