Fig 2: NHERF‐1 primarily contributes to TWIK‐1/TREK‐1‐mediated K+ currents in cultured astrocytes. (A) Normalized traces of various K+ channel inhibitors‐sensitive currents of cultured astrocytes. (B) The bar graph shows normalized various K+ channel inhibitors‐sensitive currents densities at +50 mV and −150 mV. (C) Current densities showing before and after application of spadin to GFP control (GFP) or GFP‐NHERF‐1 transfected astrocytes. (D) Summary bar graph of C at +50 mV and −150 mV. (E) Spadin‐sensitive currents were calculated by difference between currents before and after 10 μM spadin application in C. (F) Summary bar graph of GFP control (GFP/spadin sensitive) or GFP‐NHERF‐1 (GFP‐NHERF‐1/spadin sensitive) spadin‐subtracted currents plotted at +50 mV and −150 mV. The number on each bar indicates n for each condition. All values are mean ± SEM. p‐values were obtained using one‐way ANOVA followed by Bonferroni‘s post hoc test or Student's t‐test. ns: not significant, *p < 0.05, **p < 0.01, and ***p < 0.001.

Fig 3: NHERF‐1 regulates the surface expression of TREK‐1 in astrocytes. (A) Co‐IP data shows interactions between TREK‐1 and NHERF‐1 in astrocytes. Whole‐cell lysates were immunoprecipitated using anti‐NHERF‐1 antibody and then blotted using anti‐TREK‐1 antibody. (B) Left; representative images of Duolink PLA assays in astrocytes. Scale bar, 20 μm. Right; PLA signals were counted using ImageJ software and average numbers of spots per cell are presented in the graph. (C) Cell surface biotinylation results from astrocytes transfected with scrambled shRNA (Sc shRNA) or Nherf‐1 shRNA. (D) The summary bar graph shows the summary of C. (E) Cell surface biotinylation experiments with astrocytes transfected with GFP control (GFP) or GFP‐NHERF‐1. (F) Data from three independent experiments are presented in the normalized bar graph as in (E) data obtained from three independent experiments. The number on each bar indicates n for each condition. All values are mean ± SEM. p‐values were obtained using Student's t‐test. *p < 0.05 and ***p < 0.001.

Fig 4: NHERF‐1 overexpression‐mediated astrocytes dysfunction and seizure behavior were regained by TREK‐1 N4. (A) Diagram showing injection locations of AAV‐GFAPp‐NHERF‐1‐2A‐mCherry and/or AAV‐GFAPp‐TREK‐1 N4‐2A‐GFP in hippocampal CA1 SR. (B) Left; representative fluorescence images of dual immunostaining of AAV‐GFAPp‐TREK‐1 N4‐2A‐GFP (green), AAV‐GFAPp‐NHERF‐1‐2A‐mCh (red) and GFAP (purple) with DAPI staining of nuclei (blue) in mouse hippocampus. Scale bar, 50 μm. Right; enlarged images of B. Scale bar, 20 μm. (C) Representative traces of passive conductance induced by voltage stepping from −160 mV to +40 mV in AAV‐GFAPp‐NHERF‐1‐2A‐mCherry and/or AAV‐GFAPp‐TREK‐1 N4‐2A‐GFP expressing astrocytes of SR with or without 10 μM spadin (SP) respectively. (D) I‐V curves of passive conductance in C. (E) Bar graphs showing averaged currents at −160 mV and +40 mV. (F) SP‐sensitive currents were calculated by difference between currents before and after spadin application in D. (G) Summary bar graph of SP‐subtracted currents plotted at −160 mV and +40 mV (N = 2 or 3, respectively). (H) Schematic representation of the experimental timeline. Each virus was injected into mice, and 2 weeks later, an additional injection of 35 mg/kg KA was administered intraperitoneally (ip) into the abdominal cavity. (I) Time course of mean behavioral seizure score following KA injection. (J) Cumulative seizure score over 20 min for each group. (K) Latency to Score 3 seizure for each group. The number on each bar indicates n for each condition. All values are mean ± SEM. p‐values were obtained with one‐way ANOVA followed by Bonferroni's post hoc test. ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Fig 5: Heterozygous NHERF‐1 KO mice showed an enhanced TREK‐1‐mediated astrocytic passive conductance. (A) Representative traces of passive conductance induced by voltage stepping from −160 mV to +40 mV in hippocampal astrocytes from NHERF‐1 WT or Het mice treated with or without 10 μM spadin (SP). (B) I‐V curves of passive conductance in A. (C) Bar graphs showing averaged currents at −160 mV and +40 mV. (D) SP‐sensitive currents were calculated by difference between currents before and after SP application in B. (E) Summary bar graph of SP‐subtracted currents plotted at −160 mV and +40 mV (N = 4, respectively). All values are mean ± SEM. p‐values were obtained with one‐way ANOVA followed by Bonferroni's post hoc test or Student's t‐test. ns: not significant, *p < 0.05, **p < 0.01, and ***p < 0.001.