KCNE4-dependent functional consequences of Kv1.3-related leukocyte physiology

Figure 1. Kv1.3 and KCNE4 are differentially expressed in leukocytes. The presence of Kv1.3 and KCNE4 expression was analyzed in human Jurkat T lymphocytes and mouse CY15 dendritic cells. (A) Kv1.3 and KCNE4 protein expression in leukocytes. HEK 293 cells were used as a negative control. Although Jurkat and CY15 dendritic cells shared Kv1.3 and KCNE4 expression, the abundance of KCNE4 in T cells was much lower and minimally detected by western blot. In addition, Kv1.5 was abundantly expressed in CY15 cells. Representative cropped blots, clearly separated by vertical white lines, are shown only for qualitative purposes. Voltage-dependent K+ currents were elicited in Jurkat (B) and CY15 cells (C). Cells were held at -60 mV, and 250 ms pulse potentials were applied as indicated. (D) Representative confocal images of Kv1.3 (Da and Dd, in green) and KCNE4 (Db and De, in red) in Jurkat T lymphocytes (Da–Dc) and CY15 dendritic cells (Dd–Df). Scale bars: 10 µm. Given the limited expression of KCNE4 in T-cells, IPI was performed in Jurkat cells. (E) KCNE4 coimmunoprecipitated with Kv1.3 in dendritic cells. Lysates were immunoprecipitated against Kv1.3 (IP: Kv1.3) and immunoblotted (IB) against…

Figure 2. KCNE4 expression impairs Kv1.3 surface expression and inhibits Kv currents in Jurkat T cells. Confocal imaging of Jurkat T cells transfected with YFP, Kv1.3YFP, KCNE4CFP and KCNE4CFP with YFP-Kv1.3. Nuclei were stained with DAPI (blue). (Aa–Ad) Jurkat nontransfected cells (control). (Ba–Bd) YFP-transfected cells (YFP). (Ca–Cd) Kv1.3YFP transfected cells. (Da–Dd) KCNE4CFP transfected cells. (Ea–Ed) Kv1.3YFP and KCNE4CFP cotransfected cells. (Aa, Ba, Ca, Da, Ea) Kv1.3 in green. (Ab, Bb, Cb, Db, Eb) KCNE4 in red. (Ac, Bc, Cc, Dc, Ec) DAPI in blue. Merged yellow indicates colocalization between green and red (Ad, Bd, Cd, Dd, Ed). Scale bar: 5 µm. (F) FRET analysis of the Kv1.3-KCNE4 protein interaction by flow cytometry in Jurkat T lymphocytes. Values are mean ± SE, n = 5–7, *p < 0.05 (Student’s t-test). Kv1.3CFP-Kv1.3YFP was used as a positive control, whereas Kv1.3CFP-KCNE2YFP served as the negative control. (G, H) Representative current traces obtained from control Jurkat T cells (G) and cells positively transfected with KCNE4CFP (H). Cells were clamped at -60 mV, and K+ currents elicited by 250 ms voltage steps from -80 mV to +80 mV in 10-mV increments. (I) I/Imax plotted against…

Figure 3. KCNE4 overexpression modulates Kv1.3-related physiological events in Jurkat T lymphocytes. Jurkat T cells were electroporated with KCNE4CFP, and positively transfected cells were selected for specific assays. (A) Kv1.3 and KCNE4 expression in Jurkat cells. (B) Percentage of Jurkat T cell proliferation. Cells were serum starved overnight and cultured for an additional 24 h in the presence of FBS. The alamarBlue dye was used. *p < 0.05, vs. control; n = 5–7 independent experiments (Student’s t-test). (C) Cell cycle analysis with Hoechst 33342 Jurkat T cells. Cell cycle distribution (% of cells in each phase) after 24 h in the presence of FBS. G0/G1-phase, dark gray bars; S-phase, white bars; G2/M-phase of the cell cycle, light gray bars. Mean ± SE (n = 3 independent experiments). (D) Relative cell size of Jurkat cells measured in a Countess™ automated cell counter. The size of control cells corresponded to a diameter of 10.9 ± 0.6 µm. Values are the mean ± SE from n = 11 independent experiments (*p < 0.05, + KCNE4 vs. control; Student’s t-test). (E) KCNE4CFP overexpression hampered IL-2 production in activated Jurkat T lymphocytes. Cells were cultured in the presence or absence of…

Figure 4. KCNE4 impaired Kv1.3 accumulation in the IS but did not disrupt IS formation. Human Jurkat T lymphocytes and human Raji B lymphocytes were used to generate cell conjugates. (A–C) Activated B-cells (10 µg/mL SEE toxin) were cocultured in the absence (Ba–Bd) or presence (Ca–Cd) of Jurkat cells, and confocal images were obtained. (Aa–Ad) Jurkat T-cells in the absence of B cells. Endogenous Kv1.3 (green), CD3 (marker of T-cells, red), and CD19 (marker of B-cells, blue) were detected. (Ad, Bd, Cd) merge panels. Note that triple colocalization (white) in Cd localizes Kv1.3 in the IS, as identified by CD3 staining (Cb). Bars are 20 µm. (D) Accumulation ratio of mGFP-Kv1.3 at the IS vs. KCNE4-mCherry total intensity (n = 40). (E) CD3 recruitment into the IS vs. KCNE4 intensity. The horizontal red line represents the threshold level (1.5) for Kv1.3 and CD3 accumulation in the IS. Values greater or less than 1.5 indicated positive or negative accumulation of proteins at the IS, respectively.

Figure 5. KCNE4 depletion alters CY15 dendritic cell size and proliferation. CY15 cells were treated with shRNA lentiviral particles against KCNE4, and FBS-dependent proliferation was analyzed. (A) Representative western blot of KCNE4 protein expression in control CY15 dendritic cells treated with LvKCNE4 and LvScramble particles. HEK 293 cells were used as a negative control. β-actin was used as a loading control. Results from two independent clones (a and b) are shown. (B) Relative cell size of control CY15 dendritic cells (white bars), LvScramble (black bars) and LvKCNE4 (gray bars) cells. Control size was 10.4 ± 0.3 µm diameter. *p < 0.05 vs control; n = 5–8 independent experiments (Student’s t-test). (C) Percentage of FBS-dependent proliferation during 24 h of control CY15 (white bar) and LvKCNE4 (gray bar) dendritic cells in the absence of puromycin (*p < 0.05, vs control; n = 5 independent experiments; Student’s t-test). (D) FBS-dependent proliferation of LvScramble (white circle) and LvKCNE4 (gray circle) dendritic cells over 24 h in the presence of increasing puromycin concentrations. (p < 0.01 one-way ANOVA, n = 5–8). Values are the mean ± SE.

Figure 6. LPS-dependent activation increases the Kv1.3/KCNE4 ratio in CY15 dendritic cells. Cells were treated for 24 h with LPS (100 ng/ml), and the protein expression of selected K+ channel proteins was studied at 0, 6 and 24 h. (A) Confocal images of CY15 cells stained with Kv1.3 upon LPS treatment. Scale bars: 20 µm. (B) Representative voltage-dependent K+ currents elicited in CY15 cells treated with (LPS 24 h) or without (LPS 0 h) LPS during 24 h. Cells were held at -60 mV and 250 ms pulses to +60 mV were applied. (C) Peak current densitiy (pA/pF) of K+ currents from CY15 cells in the absence (0 h) or the presence (24 h) of LPS. White bars, LPS 0 h (control); black bars, LPS 24 h. Values are the mean ± SE of 4–6 cells. *p < 0.05 vs 0 h, Student’s t test. (D) Representative western blot of Kv1.5, Kv1.3 and KCNE4. The expression of iNOS monitored cellular activation in the presence of LPS. β-actin was used as a loading control. Representative cropped blots, clearly delineated by vertical white lines, are shown. (E) Relative expression of protein abundance. Kv1.3, black circles; Kv1.5, gray circles; KCNE4, white circles. (F) Coimmunoprecipitation of Kv1.3 and KCNE4 in CY15 cells in the…

Figure 7. LPS-dependent activation of CY15 dendritic cells increases the abundance of Kv1.3 at the cell surface. CY15 cells were incubated in the presence (LPS) or the absence (control) of LPS for 24 h. Cells were first stained with WGA (membrane marker) and then immunolabeled against Kv1.3. (A–D) Control cells in the absence of LPS. (E–H) Cells treated with LPS. Green, Kv1.3; red, WGA; merged panels show colocalization between green and red. (D, H) Histogram of the pixel by pixel analysis of the section indicated by the arrow in (C, G), respectively. Bars represent 10 μm. (I) Mander's overlap coefficient (MOC) quantifying the degree of colocalization between Kv1.3 and membrane surface (WGA) staining. White bar, control; black bar, LPS. Values are mean ± SE of n > 30 cells. ***p < 0.01 vs control (Student’s t-test).