Fig 1: Clic1 mediates E2-induced rapid excitation in ERα neurons in the brain.(A, D, G, J, and M) Left: IAA-94–sensitive current measured in ERαARH neuron (A), ERαMeA neuron (D), ERαBNST neuron (G), ERαLH neuron (J), and ERαPOAH neuron (M) in the absence or presence of E2 treatment (100 nM). Right: Peak inward and outward IAA-94–sensitive currents measured in the absence or presence of E2 treatment (100 nM). Data are presented as means ± SEM with individual data points. N = 5 neurons from three different mice per group. **P < 0.01, ***P < 0.001, and ****P < 0.0001 in two-sided unpaired t tests. (B, E, H, K, and N) Resting membrane potential (left) and firing frequency (right) of ERαARH neurons (B), ERαMeA neurons (E), ERαBNST neurons (H), ERαLH neurons (K), and ERαPOAH neurons (N) at the baseline and after E2 treatment (100 nM) in the presence or absence of IAA-94 (100 μM). Data are presented as individual data points. ****P < 0.0001 in two-way ANOVA analysis followed by Šídák comparisons. (C, F, I, L, and O) Resting membrane potential of ERαARH neurons (C), ERαMeA neurons (F), ERαBNST neurons (I), ERαLH neurons (L), and ERαPOAH neurons (O) at the baseline and after E2 treatment (100 nM) in the presence or absence of IAA-94 (100 μM), with TTX (1 μM), CNQX (30 μM), D-AP5 (30 μM), and bicuculline (50 μM). Data are presented as means ± SEM with individual data points. ****P < 0.0001 in two-way ANOVA analysis followed by Šídák comparisons.
Fig 2: ERα interacts with Clic1.(A) BioID to screen for ERα-interacting proteins. (B) ERα-interacting membrane proteins, with ERα (encoded by the ESR1 gene) and CLIC1 highlighted by the red circles. ERα interactors are classified on the basis of their subcellular localization. The same BioID-MS were performed in two independent biological experiments. (C) Co-immunoprecipitation showing the interaction of recombinant human ERα and Clic1 proteins in the test tube. The same results were repeated in three independent biological experiments. (D) Co-immunoprecipitation showing the interaction of overexpressed human ERα-GFP and Clic1-Flag proteins in HEK293T cells. Cells coexpressing Clic1-Flag and ERα-EGFP were treated with or without E2 for 30 min. The Clic1-Flag protein complex was then pulled down using Flag-M2 magnetic beads. Clic1 and ERα were identified by Western blotting. The same results were repeated in three independent biological experiments. (E) Co-immunoprecipitation showing the interaction of ERα-WT or ERα-C451A and Clic1 proteins in HEK293 cells. Cells coexpressing Clic1-Flag and ERα-WT-EGFP (or ERα-C451A-EGFP) were subjected to immunoprecipitation with Flag-M2 beads. Identification of Clic1 and ERα in the immunoprecipitation samples was performed by Western blotting. Data are quantified from four independent biological experiments. Data are presented as means ± SEM. ****P < 0.0001 in two-sided unpaired t test. IP, immunoprecipitation. IgG, immunoglobulin G; WT, wild type; LC-MS, liquid chromatography tandem mass spectrometry.
Fig 3: Clic1 in ERαvlVMH and ERαARH neurons mediates effects of E2 fluctuations on energy balance in female mice.(A) A schematic illustration of Clic1 genetic disruption in ERαvlVMH and ERαARH neurons in ERα-Cre mice and using WT littermates as controls. (B and C) Temporal changes in body weight in HFD-fed WT (B) and ERα-Cre (C) female mice in response to OVX + V versus OVX + E2 treatment. Data are presented as means ± SEM. N = 6, 7, or 8 mice per group. ****P < 0.0001 in two-way ANOVA analysis. (D) Body weight changes on day 48 in mice described in (B and C). (E) Averaged daily food intake in mice described in (B and C). (F and G) Fat (F) and lean (G) mass of mice described in (B and C). Data are presented as means ± SEM. N = 6, 7, or 8 mice per group. **P < 0.01 or ***P < 0.001 between OVX + V and OVX + E2 groups; #P < 0.05 or ####P < 0.0001 between WT and ERα-Cre mice in two-way ANOVA analysis followed by Šídák comparisons.
Fig 4: Clic1 mediates E2-induced rapid excitation in ERαvlVMH neurons.(A) Voltage-dependent currents in an ERαvlVMH neuron in the absence (black) and presence (blue) of IAA-94, and the subtraction (red) as the IAA-94–sensitive current. (B) Peak inward and outward IAA-94-senstive currents in ERαvlVMH neurons. Left: Sham versus OVX control female mice; right: vehicle versus E2-treated (100 nM) brain slices from gonad-intact control female mice. N = 5 or 7 neurons from three mice per group. *P < 0.05, **P < 0.01, and ***P < 0.001 in two-sided unpaired t tests. (C) Traces of the current clamp recording from ERαvlVMH neurons in response to E2 treatment (100 nM) in the presence or absence of IAA-94. (D) Resting membrane potential (left) and firing frequency (right) of ERαvlVMH neurons (N = 14) at the baseline and after E2 treatment (100 nM) in the presence or absence of IAA-94. ****P < 0.0001 in two-way analysis of variance (ANOVA) analysis followed by Šídák comparisons. (E) Traces of the current clamp recording from ERαvlVMH neurons in response to E2 treatment (100 nM) in the presence or absence of IAA-94, with TTX, CNQX, D-AP5, and bicuculline in the perfusion buffer. (F) Resting membrane potential of ERαvlVMH neurons (N = 10) at the baseline and after E2 treatment (100 nM) in the presence or absence of IAA-94, with TTX, CNQX, D-AP5, and bicuculline in the perfusion buffer. ****P < 0.0001 in two-way ANOVA analysis followed by Šídák comparisons. Data are presented as means ± SEM with individual data points. n.s., not significant.
Fig 5: Deletion of Clic1 blocks E2-induced rapid excitation in ERαvlVMH neurons.(A) An illustration of Clic1 genetic disruption in ERα neurons from one side of the vlVMH and using ERα neurons from the other side of vlVMH as controls. (B) Peak inward and outward IAA-94–senstive currents in ERαvlVMH neuron from Clic1 disrupted side (Cas9) versus the control side (GFP). N = 5 neurons from three mice per group. *P < 0.05 and ***P < 0.001 in two-sided unpaired t tests. (C) Resting membrane potential (left) and firing frequency (right) of ERαvlVMH neurons from Clic1 disrupted side (Cas9; N = 14) versus the control side (GFP, N = 21) at the baseline and after E2 treatment (100 nM). ****P < 0.0001 in two-way ANOVA analysis followed by Šídák comparisons. Note that some neurons did not have spontaneous action potential firing and therefore were not included in the firing frequency analysis. Data are presented as means ± SEM with individual data points.
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