Fig 2: MOR and TREK-1 co-localizes in astrocytic soma and process. (A) Subcellular distribution (soma, process and microdomain) of MOR in astrocyte (left). MOR is stained with immunogold with silver enhancement (dark specks, arrowheads), and GFP, representing astrocyte, is stained with immunoperoxidase (dark amorphous deposits, arrows). Cellular distribution of double labeling of TREK-1/TWIK-1 (middle) and MOR/TWIK-1 (right) in astrocyte. TREK-1 or MOR is stained with immunogold with silver enhancement (dark specks, arrowheads), and TWIK-1 is stained with immunoperoxidase (dark amorphous deposits, arrows). The soma, process, and microdomain of the astrocyte was colored blue. Presynaptic axon terminal (pre) and postsynaptic dendrite (post) were colored red and green, respectively. N is nucleus. Scale bar indicates 500 nm. (B) Summary bar graph for the frequency of MOR detection in each subcellular structure of astrocytes. Numbers in the bar graph are the number of MOR-positive soma, processes or microdomains out of total number of profiles observed. The data was collected from three animals. One-way analysis of variance (ANOVA) with Tukey’s multiple comparison test (***P < 0.001). (C,D) Summary bar graph for the gold particle density of TREK-1 (C) or MOR (D) in each subcellular structure of TWIK-1-positive astrocytes. Numbers in the bar graph indicate the number of images analyzed from three different animals. One-way ANOVA with Tukey’s multiple comparison test (***P < 0.001).

Fig 3: Knockdown of ß-COP downregulates TWIK1/TREK1-mediated currents in astrocytes. (A) Average current-voltage (I-V) relationship of whole-cell K+ currents in cultured astrocyte transfected with Sc shRNA, TREK1 shRNA, ß-COP shRNA, or TREK1 shRNA + ß-COP shRNA. (B) Summary bar graph showing averaged currents at +50 mV and -150 mV in (A). All values are mean ± SEM. SEM, standard error of the mean. p-values were obtained with one-way ANOVA followed by Tukey’s post hoc test. N.S: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001. (C) TREK1-sensitive currents were calculated by the difference between transfection of TREK1 shRNA without or with ß-COP shRNA. (D) I-V relationship of whole-cell K+ currents in cultured astrocyte transfected with Sc shRNA, TWIK1 shRNA, ß-COP shRNA, or TWIK1 shRNA + ß-COP shRNA. (E). Summary bar graph showing averaged currents +50 mV and -150 mV in (D). All values are mean ± SEM. SEM, standard error of the mean. p-values were obtained with one-way ANOVA followed by Tukey’s post hoc test. N.S: not significant, ** p < 0.01, *** p < 0.001. (F) TWIK1-sensitive currents were calculated by the difference between transfection of TWIK1 shRNA without or with ß-COP shRNA.

Fig 4: Spadin inhibits TWIK-1 mediated K+ currents in a TREK-1-dependent manner in cultured astrocytes. (A) Schematic representation of the genomic structure of the TWIK-1 gene. Target sequences of gRNA are located within exon2 of mouse TWIK-1 gene. Examples of indel mutations are displayed. (B) Immunocytochemical images of control gRNA (Con gRNA) or TWIK-1 gRNA transfected cultured astrocytes stained with anti-TWIK-1 antibody. (C) Current densities before and after application of 10 µM spadin (SP) to control gRNA or TWIK-1 gRNA transfected astrocytes. (D) Summary bar graph of control gRNA or TWIK-1 gRNA currents with or without spadin (SP) plotted at +50 mV and -150 mV. The number on each bar indicates n for each condition. All values are mean ± s.e.m. p-values were obtained with one-way ANOVA followed by Turkey’s post hoc test. n.s: not significant, * p < 0.05 and *** p < 0.001. (E) Subtract spadin (SP-sub) treatment currents from before application of spadin currents in control gRNA or TWIK-1 gRNA transfected astrocytes. (F) Summary bar graph of con gRNA (Con gRNA-SP sub) or TWIK-1 gRNA (TWIK-1 gRNA-SP sub) spadin-subtracted currents plotted at +50 mV and -150 mV. Th number on each bar indicates n for each condition. All values are mean ± s.e.m. p-values were obtained with Student’s t-test. * p < 0.05 and *** p < 0.001.

Fig 5: Sensitivity of cooling-evoked responses to arachidonic acid, riluzole, chloroform and nifedipine supports the involvement of TREK channels.