Defolliculated Xenopus laevis oocytes were purchased from Ecocyte Bioscience and maintained in incubation solution (100 mM NaCl, 2 mM KCl, 1.8 mM CaCl2.2H2O, 1 mM MgCl2.6H2O, 5 mM HEPES, 2.5 mM C3H3NaO3, pH 7.5 supplemented with penicillin 100 U/mL and streptomycin 100 μg/mL) at 19°C. Each oocyte was microinjected with 57 ng of cRNA encoding Phh-GluCl or Phh-RDL using a Drummond nanoject II microinjector. Three to five days after cRNA injection, two-electrode voltage-clamp recordings were performed with an oocyte clamp OC-725C amplifier (Warner instrument) at a holding potential of -80mV or -60 mV to assess the expression of the GluCl or RDL channels. Currents were recorded and analyzed using the pCLAMP 10.4 package (Molecular Devices). Concentration-response relationships for agonists were carried out by challenging oocytes with 10 s applications of increasing concentrations of compounds. The peak current values were normalized to the response to 1000 μM glutamate or 100 μM GABA giving the maximum current amplitude for GluCl and RDL channels, respectively. For Phh-GluCl, the dose-response relationship for ivermectin was performed by applying each concentration of ivermectin for 20–30 s. Each oocyte received only one ivermectin concentration (≤ 30 nM) followed with a high ivermectin concentration (≥ 100 nM). Data were normalized to a first 100 μM glutamate apllication. For RDL, the effect of antagonists was evaluated, by a first preincubation of each antagonist alone for 90 s, followed by the co-application with 100 μM GABA for 10 s. Oocytes were then washed with recording buffer and sequentially preincubated with an increasing antagonist concentration followed by coapplication with 100 μM GABA. The observed responses were normalized to the response induced by 100 μM GABA alone performed prior to challenging with the antagonist. The concentration of agonist required to mediate 50% of the maximum response (EC50), the concentration of antagonist required to inhibit 50% of the agonist response (IC50) and the Hill coefficient (nH) were determined using non-linear regression on normalized data with GraphPad Prism software. Concentration-response curves for agonists were fitted with the following equation:

Where Y is the normalized response, H is the Hill slope and X is the logarithm of concentration. The same equation was used for the concentration-response curves for antagonists by replacing logEC50 by logIC50. Results are expressed as mean +/- SEM and statistical analysis were performed using One-Way ANOVA with Tukey’s Multiple Comparisons Test. The competitive experiments on Phh-RDL were performed as previously described [42] with a concentration-response relationship in the presence of GABA alone or with 1 nM or 10 nM lotilaner. A pre-incubation of 1 nM or 10 nM lotilaner for 90 s was followed by a coappllication with increasing concentrations of GABA (1–1000 μM) for 10 s with a 30 s wash between each concentration. Current amplitudes were normalized with a first 100 μM GABA application. The corresponding concentration response curves were fitted using the following four parameter Hill equation:

Where Bottom is the response at the minimal concentration and Top is the maximal response. Statistical comparison were performed using unpaired Student’s t test. Chloride and sodium permeabilities were conducted as described previously [53]. In brief, the reversal potential of the GABA-induced currents was measured with a 1500-ms-long ramp of voltage from -60 to +60 mV in standard saline recording solution (100 mM NaCl, 2.5 mM KCl, 1 mM CaCl2, 5 mM HEPES, pH 7.3) as well as in recording solutions containing a concentration range of either chloride (NaCl replaced by sodium acetate) or sodium (NaCl replaced by tetraethylammonium chloride).

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