Calmodulin-dependent KCNE4 dimerization controls membrane targeting

Abstract

The voltage-dependent potassium channel Kv1.3 participates in the immune response. Kv1.3 is essential in different cellular functions, such as proliferation, activation and apoptosis. Because aberrant expression of Kv1.3 is linked to autoimmune diseases, fine-tuning its function is crucial for leukocyte physiology. Regulatory KCNE subunits are expressed in the immune system, and KCNE4 specifically tightly regulates Kv1.3. KCNE4 modulates Kv1.3 currents slowing activation, accelerating inactivation and retaining the channel at the endoplasmic reticulum (ER), thereby altering its membrane localization. In addition, KCNE4 genomic variants are associated with immune pathologies. Therefore, an in-depth knowledge of KCNE4 function is extremely relevant for understanding immune system physiology. We demonstrate that KCNE4 dimerizes, which is unique among KCNE regulatory peptide family members. Furthermore, the juxtamembrane tetraleucine carboxyl-terminal domain of KCNE4 is a structural platform in which Kv1.3, Ca²⁺/calmodulin (CaM) and dimerizing KCNE4 compete for multiple interaction partners. CaM-dependent KCNE4 dimerization controls KCNE4 membrane targeting and modulates its interaction with Kv1.3. KCNE4, which is highly retained at the ER, contains an important ER retention motif near the tetraleucine motif. Upon escaping the ER in a CaM-dependent pattern, KCNE4 follows a COP-II-dependent forward trafficking mechanism. Therefore, CaM, an essential signaling molecule that controls the dimerization and membrane targeting of KCNE4, modulates the KCNE4-dependent regulation of Kv1.3, which in turn fine-tunes leukocyte physiology.

Figure 1

KCNE4, which specifically regulates Kv1.3, is differentially expressed in tissues and leukocytes. (A) Protein expression of KCNE4 in different rat tissues and cell lines. HEK-293 cells were used as negative control, and HEK cells transfected with KCNE4-YFP (E4YFP) and KCNE4-HA (E4HA) were used as positive controls. Different western blots with the same KCNE4-YFP and KCNE4-HA controls from different origins were merged for better visualization. Fifty micrograms of protein was loaded in each lane, and filters were immunoblotted with anti-KCNE4 antibody. Differential β-actin expression among tissues and cell lines (not shown) invalidated this protein as a control. Therefore, a KCNE4 abundance comparison among samples should not be considered. The results must be considered as qualitative data. Representative cropped blots, clearly separated by vertical black lines, are shown only for qualitative purposes. (B, C) Representative patch-clamp recordings of HEK-293 cells transfected with Kv1.3 and Kv1.5 in the absence or presence (+ KCNE4) of KCNE4. Cells were hold at -60 mV and voltage-dependent K⁺ currents were elicited by applying 200 ms depolarizing pulses to + 60 mV. (B) Kv1.3 + /- KCNE4. (C) Kv1.5 + /- KCNE4. Gray lines, presence of KCNE4 (+ KCNE4); black lines, absence of KCNE4. Data analysis was performed using FitMaster (HEKA) and SigmaPlot 10.0 software (Systat Software).

Figure 2

KCNE1 and KCNE4 exhibit notable endoplasmic reticulum retention. Representative confocal images of HEK-293 cells transfected with KCNE1 and KCNE4. (A) KCNE-transfected cells were cotransfected with a plasma membrane marker (MB). (B) KCNE-transfected HEK-293 cells were cotransfected with an endoplasmic reticulum marker (ER). Right panels, KCNE1; center panels, KCNE4; left panels, histograms showing the colocalization between KCNEs (E1 and E4) and markers (MB and ER) based on Mander’s coefficient analysis. The values represent the mean of > 40 cells. Green, KCNEs; red, cell markers; yellow, merged image. Bars represent 10 μm. (C) Sequence alignment of murine KCNE peptides. KCNE1 (UniProtKB: P23299); KCNE2 (UniProtKB: Q9D808); KCNE3 (UniProtKB: Q9WTW2); KCNE4 (UniProtKB: Q9WTW3); KCNE5 (UniProtKB: Q9QZ26). Mouse sequences are shown because murine isoforms were used throughout the study, and the results obtained were similar to the results with human isoforms. Transmembrane segments are highlighted in red. The ER-retention motifs are boxed in yellow. The specific tetraleucine motif identified in KCNE4 is colored gray.

Figure 3

KCNE4 homo-oligomerization. HEK-293 cells were transfected with different KCNE4-tagged peptides, and their homo-oligomeric formation ability was studied. (A) Molecular simulation of KCNE4 using the KCNE1 template (PDB code 2K21) as described in the Materials and Methods. The inset highlights the tetraleucine signature (L69-72A) where CaM and Kv1.3 associate. (B) Oligomerization of KCNE4. Cell lysates were incubated in the absence ( −) or presence ( +) of DMP (dimethyl pimelimidate). Filters were immunobloted against CFP (Kv1.3-CFP, KCNE4-CFP). Lower Kv1.3 arrow, Kv1.3 monomers. Upper Kv1.3 arrow, Kv1.3 tetramers. Lower KCNE4 arrow, KCNE4 monomers. Upper KCNE4 arrow, KCNE4 oligomers. Mock- and CFP-transfected cells were used as negative controls. (C) HEK-293 cells were cotransfected with KCNE4-YFP and KCNE4-HA. Immunoprecipitation was performed for KCNE4-YFP (IP: GFP). Top panel: Immunoblot (IB) against GFP. Bottom panel: immunoblot (IB) against HA. SM: starting material. SN + , supernatant in the presence of antibody. SN-, supernatant in the absence of antibody. IP + , Immunoprecipitation in the presence of the anti-GFP antibody. IP- Immunoprecipitation in the absence of the anti-GFP antibody. (D, E) HEK-293 cells were transfected with Kv1.3-, KCNE1- and KCNE4-tagged (CFP/YFP) proteins. Homo-oligomerization of Kv1.3 and KCNE4, but not KCNE1, as analyzed by the FRET acceptor-photobleaching technique. (D) Green, representative image of a KCNE4-CFP donor. Red, representative image of a KCNE4-YFP acceptor. FRET image, white square highlights a ROI showing FRET image obtained after photobleaching. Bars represent 10 μm. (E) Quantification of the FRET efficiency (%). CFP/YFP, negative control; Kv1.3CFP/Kv1.3YFP, positive control. ***, p < 0.001 vs CFP/YFP (Student’s t-test). The values represent the mean ± SEM of 22 (CFP/YFP), 33 (Kv1.3CFP/Kv1.3YFP), 28 (KCNE4CFP/KCNE4YFP) and 25 (KCNE1CFP/KCNE1YFP) cells.

Figure 4

GFP bleaching steps from HEK-293 cells transfected with Kv1.3, KCNE4 and KCNE1. (A–C) Cells were transfected with Kv1.3 loopBADGFP. (A) Representative snapshot from the TIRFM video (488 nm laser). Colored ROIs (6 × 6 pixels) indicate representative analyzed unmoving spots. Scale bar represents 16 μm. (B) Representative graph of bleaching steps from different spots that were analyzed. Red arrows point to 4 bleaching steps. (C) Relative frequency of 1–4 bleaching events counted per spot. Gray bars correspond to the experimental frequency observed. Red dashed lines correspond to the theoretical distribution of the bleaching steps with p = 0.67. (D–F) GFP bleaching steps with HEK-293 cells transfected with KCNE4 loopBADGFP. (D, E) Representative graphs of the bleaching steps from different KCNE4 spots that were analyzed. (D) Red arrows point to one bleaching step, indicating a KCNE4 monomer. (E) Two bleaching steps demonstrating KCNE4 dimers. (F) Relative frequency of 1–4 bleaching events counted per spot. Gray bars correspond to the experimental frequency observed. Red dashed lines correspond to the theoretical distribution of bleaching steps showing a dimer. (G, H) GFP bleaching steps from HEK-293 cells transfected with KCNE1 loopBADGFP. (G) Representative graph showing one bleaching step suggesting KCNE1 monomers. (H) Relative frequency of 1–4 bleaching events counted per spot. Gray bars correspond to the experimental frequency observed, demonstrating a KCNE1 monomeric structure.

Figure 5

The juxtamembrane tetraleucine motif is critical for KCNE4 dimerization. (A) Representative cartoon showing the sequence alignment of the juxtamembrane segment of KCNE4 and KCNE2 and the comparison with the introduced mutations. The transmembrane domain (TM) is shown in a box in red. The KCNE ERRMs are in yellow. The tetraleucine signature is colored gray. Amino acid substitutions are highlighted in red. For KCNE4 (ERRM), the ERRM of KCNE4 was disrupted with alanine residues (in red). For KCNE4 (L69-72A), the tetraleucine motif (gray) was also mutated to alanine (in red). For KCNE4(RM&L), both the ERRM and the tetraleucine L69-72A motifs were disrupted by alanine substitutions. The tetraleucine motif of KCNE4 was introduced in the same location in KCNE2 (KCNE2(L83-86)). (B-D) HEK-293 cells were transfected with KCNE4CFP, KCNE2-HA, Kv1.3-(CFP/YFP) and different KCNE mutants. (B) Coimmunoprecipitation assay against KCNE4-CFP and KCNE4(L69-72A)CFP in the presence of KCNE4-HA (IP:GFP). Top panel: Immunoblot against CFP (IB:GFP). Bottom panel: immunoblot against HA (IB:HA). SM: starting material. IP + : Immunoprecipitation in the presence of the anti-GFP antibody. IP-: Immunoprecipitation in the absence of the anti-GFP antibody. KCNE4-HA coimmunoprecipitates with KCNE4CFP (KCNE4WT) but not with KCNE4(L69-72A)CFP. Representative cropped blots are clearly separated by vertical white lines. (C) Non-denaturing SDS-PAGE. HEK 293 cells were transfected with KCNE4WT-CFP and KCNE4(L69-72A)CFP and cell lysates immunoblotted against GFP. Note that while KCNE4WT-CFP exhibited large molecular mass forms (red arrow), KCNE4(L69-72A)CFP only appeared as monomers (yellow arrow). (D) Quantification of the FRET efficiency of KCNE4 dimerization. Cells were cotransfected with CFP/YFP (negative controls), Kv1.3CFP/Kv1.3-YFP (positive control), KCNE4WT (CFP/YFP) and KCNE4WT-YFP and KCNE4(L69-72A)CFP. The values represent the mean ± SE of n > 25 cells. ***p < 0.001 Student’s t-test. (E) HEK-293 cells were cotransfected with KCNE4WT-HA/ KCNE4(L69-72A)CFP and KCNE4WT-HA and either KCNE2WT-CFP or KCNE2(L83-86)-CFP. Top panels: Immunoblot against CFP (IB: GFP). Bottom panel: immunoblot against HA (IB: HA). SM: starting material. IP: GFP: Immunoprecipitation in the presence of the anti-GFP antibody. KCNE4-HA coimmunoprecipitates with KCNE2(L83-86)-CFP but not with KCNE2WT-CFP.

Figure 6

The presence of CaM and Kv1.3 impairs the dimerization of KCNE4. Coimmunoprecipitation of KCNE4-CFP and KCNE4-HA in the presence ( +) or absence (-) of Kv1.3 (A) and CaM (B). The experiment in the presence of CaM (+ CaM) was performed in the absence (+ 2 mM EDTA/2 mM EGTA) or the presence (+ Ca⁺) of 2 mM Ca⁺. KCNE4 was immunoprecipitated (IP) with select anti-tag antibodies (anti-GFP and anti-HA), and membranes were blotted (IB) against Kv1.3, KCNE4 and CaM with the indicated primary antibodies (anti-Kv1.3, anti-KCNE4, anti-GFP or anti-HA). Representative cropped blots are clearly separated by vertical white lines. (C) Relative KCNE4/KCNE4 coimmunoprecipitation in the presence of Kv1.3 and CaM. Control represents KCNE4/KCNE4 with no additions. The values represent the mean of 4 independent experiments. *p < 0.05 vs control (KCNE4-CFP/KCNE4-HA in the absence of further additions, Student’s t-test). (D–F) Representative FRET experiments between KCNE4YFP and KCNE4CFP in the absence (D, control) or presence of + Kv1.3 (E) or + CaM (F). Green panels, KCNE4-CFP (donor); red panels, KCNE4-YFP (acceptor); merged postbleach panels; yellow indicates colocalization; white square highlights the acceptor photobleached ROI analyzed. Bars represent 10 μm. (G) FRET efficiency quantification of KCNE4-CFP/KCNE4-YFP in the presence of CaM and Kv1.3. The values represent the mean of 20–30 cells. *p < 0.05; **p < 0.01; ***p < 0.001 (Student’s t-test). Control, no additions; + CaM, presence of CaM; + Kv1.3, presence of Kv1.3. CFP/YFP, negative controls; Kv1.3YFP/Kv1.3CFP, positive controls. (H) Schematic showing the interfering associations impairing KCNE4 dimer formation due to multiple tetraleucine motif interactions. The presence of either CaM or Kv1.3 would disrupt KCNE4 association, inhibiting dimer formation.

Figure 7

Endoplasmic reticulum colocalization of KCNE4 in the presence of CaM. HEK-293 cells were cotransfected with KCNE4-CFP, CaM-YFP and pDsRed-ER. (A–C) Colocalization of KCNE4 to the ER in the absence of CaM. (D–K) Colocalization of KCNE4 to the ER in the presence of CaM. (D–G) Representative images of a cell expressing low levels of CaM. (H–K) Representative images of a cell expressing high levels of CaM. (A, D, H) KCNE4-CFP, red; (E, I) CaM-YFP in green; (B, F, J) ER, blue; (C, G, K) merged panels. Purple indicates colocalization of KCNE4 and ER markers. Yellow, colocalization of KCNE4 and CaM. White and light pink indicate triple colocalization. Scale bars represented 10 µm. (L) ER colocalization of KCNE4 plotted against the CaM/KCNE4 ratio. All images were captured with the same laser intensity and photomultiplication parameters. A pixel-by-pixel analysis was performed to determine the relative KCNE4/ER and the CaM/KCNE4 ratios. The regression curve and a high R value are indicated. Values were from 20–30 cells. Note: High levels of CaM correspond to low ER colocalization of KCNE4.

Figure 8

KCNE4 traffics to the plasma membrane via a COPII-dependent mechanism. HEK-293 cells were cotransfected with KCNE4WT and mutants (KCNE4(L69-72A), KCNE4(ERRM) and KCNE4(RM&L)) and the Akt-PH-pDsRed membrane marker in the absence (-Sar1, A-L) or the presence (+ Sar1, M-X) of HA-Sar1H79G. (A-C and M–O) KCNE4WT colocalization with the membrane marker. (D-F and P-R) KCNE4(L69-72A) colocalization with the membrane marker. (G-I and S-U) KCNE4(ERRM) colocalization with the membrane marker. (J-L and V-X) KCNE4(RM&L) membrane colocalization. Red panels, KCNE4. Blue panels, stained membrane. In the merged panels, purple indicates colocalization. Scale bars represent 10 µm. (Y) Quantification of membrane (MB) colocalization of KCNE4 in the absence ( −) or presence ( +) of HA-Sar1H79G (Sar1) using Mander’s coefficient. White columns, KCNE4 WT; light gray, KCNE4(L69-72A); dark gray, KCNE4(ERRM); black columns, KCNE4(RM&L). The values represent the mean of 20–30 cells. **p < 0.01; ***p < 0.001 One-way ANOVA and post hoc Tukey’s test.