Fig 1: Run-up of the TREK-1c current in a TR-1 cell line and effects of NEM and pitstop2 on the run-up. (A) Run-up of the TREK-1c current. Immediately after whole-cell access, the TREK-1c current was evoked by step-pulses of 400 ms from −100 to 40 mV from a holding potential of −70 mV (0 min). The TREK-1c current gradually increased and reached a plateau at 5 min. The application of 3 mM bupivacaine nearly completely inhibited the current. (B) The current-voltage relationship of the TREK-1c current showed outward rectification. (C and D) Inhibition of the run-up by NEM and brefeldin-A. Immediately after the first recording (gray bar), 1 mM NEM (C) or 5 µg/ml brefeldin-A (D) was added to Tyrode solution. Whole-cell conductance was normalized to that of 0 min. (* p < 0.05, the Student's t-test, n = 7). (E) Facilitation of the run-up by pitstop2. Pitstop2 (30 µM) was added to Tyrode solution immediately after whole-cell access (gray bar; * p < 0.05, n = 6).
Fig 2: Involvement of ezrin in the surface localization of TREK-1c channels. (A) Inhibition of the run-up by an ezrin inhibitor. TREK-1c conductance gradually increased in control TR-1 cells, but the addition of the ezrin inhibitor, NSC668394 (100 µM), to Tyrode solution significantly inhibited the run-up (*p < 0.05, the Student's t-test, n = 8). (B) The percentage of cells in which mCherry-TREK-1c proteins were localized at the plasma membrane was significantly decreased by the NSC668394 treatment (p < 0.05, the χ2-test, 100 cells from 3 independent experiments). (C) The co-localization of ezrin with mCherry-TREK-1c proteins at the plasma membrane and dissociation by NSC668394. MT-1 cells were immunostained with an anti-ezrin antibody. Anti-ezrin immunoreactivity was co-localized with mCherry-TREK at the plasma membrane in control cells. In contrast, in NSC668394-treated cells, mCherry-TREK fluorescence was dissociated from the anti-ezrin antibody and plasma membrane. (D) Involvement of ezrin in the association between TREK and actin. MT-1 cells were incubated with 100 µM NSC668394 or 0.1% DMSO for 5 min. Solubilized lysates, which were immunoprecipitated with an anti-mCherry antibody, were then immunoblotted with an anti-actin antibody. The addition of NSC668394 decreased actin precipitation, suggesting the involvement of ezrin in the association of TREK-1c with actin fibers. Arrow and arrowheads indicate the position of actin and IgG, respectively.
Fig 3: Inhibition of the run-up by a C-terminus peptide that interacts with Mtap2. (A) Whole cell voltage-clamp recordings were made from TR-1 cells with an internal solution containing 10 μM TREK298–311 or TREK335–360 peptide, which corresponds to the site for the interaction with AKAP150 or Mtap2, respectively. No significant differences were observed in initial conductance among the 3 groups. Whereas TREK-298–311 slightly decreased the run-up, TREK-335–360 significantly inhibited it: conductance was significantly lower than that in control cells 5 and 7 min after whole-cell access (* p < 0.05, ANOVA followed by the Student's t-test, n = 6). (B) Co-immunoprecipitation of Mtap2 with mCherry-TREK. Lysate of MT-1 cells was immnunoprecipitated with anti-mCherry antibody and precipitate was analyzed with Mtap2 antibody. The arrowhead indicates the position of Mtap2. (C) Inhibition by TREK335–360 peptide. Citrate-treated and neutralized lysate was allowed to reassemble and immunoprecipitated with anti-mCherry antibody in the presence or absence of TREK335–360 peptide. Precipitate was analyzed with immunoblotting with anti-Mtap2 antibody. Addition of the peptide inhibited the co-immunoprecipitation.
Fig 4: Additive effects of ezrin and Mtap2 on the run-up of the TREK-1c current. (A) A whole-cell patch-clamp was made from TR-1 cells with internal solution containing 10 µM TREK-298–311, which corresponds to the binding site with AKAP150. NSC668394 (100 µM) was applied immediately after the first recording. The effects of the co-application of NSC668394 and TREK298–311 were similar to those of NSC668394 alone (compare with Fig. 4A). (B) The co-application of NSC668394 with TREK-335–360, which corresponds to the binding site with Mtap2, diminished the TREK-1c current. Conductance was significantly lower than that of TREK-335–360 alone (* p < 0.05, the Student's t-test, n = 7). This result suggests the additive involvement of Mtap2 and ezrin in the run-up.
Fig 5: Effects of CMS and fluoxetine treatment on TREK-1 protein expression in frontal cortex and hippocampus of rat brains. Expression of TREK-1 protein was obviously enhanced in frontal cortex (A) of CMS rats and could be reversed by fluoxetine. While no obvious changes could be observed in hippocampus (B). ##P<0.01 vs. control group, **P<0.05 vs. CMS+saline group, n=4.
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