Soualah Z , Taly A , Crespin L , Saulais O , Henrion D , Legendre C , Tricoire-Leignel H , Legros C , Mattei C .

             synaptic signaling
             extrasynaptic signaling via GABA

	FIGURE 1. The 2D structure of fipronil (Pubchem CID: 3352).
	FIGURE 2. GABA sensitivity of ternary and binary α6-containing GABAARs. (A) Stoichiometric organization of α6-containing GABAARs used in this work (see Baumann et al., 2001; Baur et al., 2010; Sigel and Steinmann, 2012). (B) Concentration-response curves for α6β3γ2S, α6β3δ, and α6β3 GABAARs. Data were best fitted by non-linear regression to the Hill equation with variable slope α6β3γ2S: EC50 = 3.85 ± 0.28 μM and Hill-coefficient = 1.24 ± 0.08, R2 = 0.98; α6β3δ: EC50 = 0.75 ± 0.09 μM and Hill-coefficient = 0.99 ± 0.09, R2 = 0.95; α6β3: EC50 = 0.43 ± 0.01 μM and Hill-coefficient = 1.83 ± 0.09, R2 = 0.99). Data are mean ± SEM (n = 4–8) of at least two independent experiments.
	FIGURE 3. Inhibitory effects of FPN on GABA-induced currents in membrane-transplanted oocytes. (A) Representative traces of currents evoked by increasing concentrations of GABA in an oocyte transplanted with rat cerebellum membranes. (B) Effects of FPN (1, 10, 100 μM) on GABA-evoked currents. Control experiments denote the addition of 10–4 M of GABA (left), before simultaneous addition of GABA and FPN (right). (C) Histograms showing the concentration-dependent inhibitory effects of FPN on GABA-evoked currents elicited by oocyte transplantation with rat cerebellum membranes. The number of recorded oocytes is indicated inside the bars. Data are mean ± SEM. Multiple comparisons were performed using one-way ANOVA tests followed by Tukey’s post hoc correction (*p < 0.05, ns, not significant).
	FIGURE 4. FPN effects on GABA-induced currents elicited by α6β3γ2S and α6β3δ GABAARs. (A,C) Concentration-response curves for α6β3γ2S and α6β3δ GABAARs. Data were best fitted by non-linear regression to the Hill equation with variable slope. (B,D) Comparison of EC50 and GABA-currents densities obtained with α6β3γ2S (B) and α6β3δ (D) GABAARs, in control (CTRL) and in the presence of FPN (10 μM). For α6β3γ2S, pEC50 was 5.83 ± 0.21 without FPN and 5.77 ± 0.28 with FPN; Emax was 11.6 ± 0.6 pA/pF without FPN and 3.4 ± 0.2 with FPN. For α6β3δ, pEC50 was 6.24 ± 0.12 without FPN and 6.33 ± 0.10 with FPN; Emax was 1.8 ± 0.3 pA/pF without FPN and 0.8 ± 0.2 with FPN. The number of recorded oocytes is indicated inside the bars. For both receptors, the normality of maximal current density distribution was validated using Shapiro-Wilk test and data were analyzed with unpaired t-test. EC50 values did not pass Shapiro-Wilk test and they were analyzed with non-parametric Mann and Whitney test (ns: non-significant, *p < 0.5, ****p < 0.0001. Data are mean ± SEM of at least two independent experiments (n = 5–6 cells) (see also Table 1).
	FIGURE 5. Comparison of inhibitory effects of FPN on α6β3γ2S, α6β3δ, and α6β3 GABAARs. (A) Left: Concentration-inhibition curves of FPN on α6β3γ2S, α6β3δ, and α6β3 GABAARs stimulated with GABA (EC50). Data were best fitted by non-linear regression to the Hill equation with variable slope. For α6β3γ2S, pIC50 was 4.86 ± 0.10 and Hill coefficient = 0.80 ± 0.05, R2 = 0.96; for α6β3δ, pIC50 was 4.78 ± 0.05 and Hill coefficient = 0.90 ± 0.12, R2 = 0.93; for α6β3, pIC50 was 5.93 ± 0.20 and Hill coefficient = 1.08 ± 0.29, R2 = 0.99. Right: Representative responses to concentration-inhibition of FPN on α6β3γ2S, α6β3δ, and α6β3 GABAARs stimulated with GABA (EC50). (B,C) Analysis of the inhibitory effects of FPN on α6β3γ2S, α6β3δ and α6β3 GABAARs. Potency (pIC50, B) and efficacy (maximum inhibition obtained with 300 μM FPN, C) of FPN on the three α6-containing GABAARs were compared. Normality of data distribution was validated using Shapiro-Wilk test and data were analyzed with one-way ANOVA followed by multiple comparison test with Tukey test (ns: non-significant, ****p < 0.0001). Data are mean ± SEM of at least two independent experiments (n = 4–9 cells) (see also Table 2).
	FIGURE 6. Effect of FPN on leak currents elicited by expression of β3-subunits in Xenopus oocytes. (A) Representative current traces obtained by increasing concentrations of FPN (1, 10, and 10 μM). (B) Analysis of FPN-induced currents. The current density increases as a function of FPN concentrations: it was 0.0058 ± 0.0021 nA/pF, 0.0177 ± 0.0018 nA/pF, 0.0661 ± 0.0105 nA/pF for 1, 10, and 100 μM FPN, respectively. The number of recorded oocytes is indicated inside the bars. Data are mean ± SEM of at least two independent experiments. Multiple comparisons were performed using one-way ANOVA tests followed by Tukey’s post hoc correction (***p < 0.001, ****p < 0.0001, ns, not significant).
	FIGURE 7. Binding modes of FPN obtained by docking on the mouse α6-containing GABAAR. (A) Model of the receptor viewed from the membrane plane. The protein is shown in cartoon representation with a different color code for each polypeptide. The position of the membrane is represented by a sphere positioned at the level of lipid head groups as determined by the Orientations of Proteins in Membranes database. (B) Model of the receptor viewed from above the membrane (rotation of 90° from A). (C,D) Binding modes of FPN obtained by docking on the mouse α6β3γ2S (C) and α6β3 (D) GABAARs. Close-up showing the FPN-binding pocket (FPN appears in sticks). FPN interacts with an upper site (Ser272)—nearby the extracellular part of the membrane—and the second site (Val257)—located near the intracellular part of the membrane.
	Figure S1

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