The unconventional biogenesis of Kv7.1-KCNE1 complexes

Fig. 1. Kv7.1 and KCNE1 recapitulate cardiac IKs currents and associate in T-tubules in cardiomyocytes. (A to H) dSTORM in rat ventricular cardiomyocytes. Fluorophore-coupled antibodies were used to stain native proteins. (A) Staining for RyR in cardiac T-tubules. (B) Kv7.1 (red) and (C) merge image. (D to F) Staining for Kv7.1 and KCNE1 in cardiac T-tubules. (D) Kv7.1, red; (E) KCNE1, green; (F) colocalization of Kv7.1 and KCNE1, yellow. Scale bars, 5 μm. (G) Magnified area from the square in (F). The insets delineate the representative regions of interest analyzed in (H). (H) Kv7.1-KCNE1 colocalization at a 0-nm distance determined by dSTORM analysis. Color code in (G) and (H): gray, PM; white, T-tubules; blue, nontubule area excluding the previous structures. The black bar represents whole-cell colocalization. Statistical analysis determined a P < 0.001 by analysis of variance (ANOVA) with a Tukey post hoc test. **P < 0.01 and ***P < 0.001 (insets, n > 10 from five independent cells). (I) Kv7.1 coimmunoprecipitates with KCNE1 in rat cardiac samples. Total crude protein extracts from rat ventricular cardiomyocytes were immunoprecipitated (IP) with anti-Kv7.1 antibodies and…

Fig. 2. Kv7.1 and KCNE1 mostly interact at the PM. (A to F) Confocal images of KCNE1-YFP and Kv7.1-CFP in entire cells (A to C) and CUPs (D to F). Cells were cotransfected with KCNE1-YFP (A and D, in green) and Kv7.1-CFP (B and E, in red). (C and F) Merged image showing colocalization in yellow. (G) Colocalization analysis by Manders’ (M) coefficient in entire cells (black bar) or CUPs (white bar). The values show the means ± SEM. ***P < 0.001 CUP versus entire cell (n = 9 to 15, Student’s t test). (H to Q) Representative results of FRET acceptor photobleaching experiments on entire cells (H to L) and CUPs (M to Q). The prebleaching images (H to I and M and N) show the expression of KCNE1-YFP (H and M) and Kv7.1-CFP (I and N). After bleaching the acceptor molecule (KCNE1-YFP), postbleaching images were taken (J and K and O and P). The bleached area is highlighted with a white square. (L and Q) FRET ratio images from the previous panels. The calibration bar indicates the FRET ratio ranging from 0.8 (blue) to 1.4 (red). Scale bars, 10 μm. (R) Energy transfer efficiencies. The values represent the means ± SEM of the FRET measured in entire cells (black) or in CUPs (white). ***P < 0.001 CUP…

Fig. 3. Kv7.1-KCNE1 assembly within the secretory pathway. (A to F) Representative images of KCNE1-YFP (A, green), Kv7.1-CFP (B, red), and KCNE1-YFP + Kv7.1-CFP (C, green and red) coexpressed with Akt-PH-pDsRed (A to C, PM marker in blue) and ER-DsRed (D to F, blue) in HEK-293 cells. PH, pleckstrin homology. (A and D) Cyan shows colocalization between green and blue. (B and E) Magenta shows colocalization between red and blue. (C and F) Yellow shows partial colocalization between green and red. White shows triple colocalization between green, red, and blue. Scale bar, 10 μm. (G) M coefficient analysis of KCNE1 (black), Kv7.1 (white), and Kv7.1 + KCNE1 (light and dark gray) overlapping the ER and membrane (PM) markers. The bars represent the means ± SEM. **P < 0.01 and ***P < 0.001 for KCNE1 in the absence of Kv7.1 versus KCNE1 in the presence of Kv7.1 (n = 19 to 40, Student’s t test). (H) Biotin assay of KCNE1 and Kv7.1 cell surface expression. HEK-293 cells were transfected with Kv7.1-CFP, KCNE1-YFP, and Kv7.1-CFP + KCNE1-YFP. After transfection, the cells were labeled with biotin and processed as described in Materials and Methods. Western blot analysis was performed with anti-Kv7.1…

Fig. 4. Kv7.1 and KCNE1 colocalization with post-ER compartments of the secretory pathway. (A to C) KCNE1 colocalization with post-ER subcellular compartment markers. (A) Cis-Golgi network (GM130), (B) TGN (TGN46), and (C) early endosomes (EEA1). Green, KCNE1-YFP; blue, subcellular compartments; cyan, colocalization between green and blue. (D to F) Kv7.1 colocalization with GM130 (D), TGN46 (E), and EEA1 (F). Red, Kv7.1-CFP; blue, subcellular compartments; magenta, colocalization between red and blue. (G to I) Kv7.1 + KCNE1 colocalization with GM130 (G), TGN46 (H), and EEA1 (I). KCNE1-YFP localization is shown in green, Kv7.1-CFP is shown in red, and the corresponding subcellular compartments are shown in blue. White shows triple colocalization between green, red, and blue. Yellow shows partial green and red colocalization. Scale bars, 10 μm. (J) The values represent the means ± SEM of the M overlap coefficient between KCNE1 (black), Kv7.1 (white), or Kv7.1 + KCNE1 (light and dark gray) and the corresponding subcellular compartment. *P < 0.05, **P < 0.01, and ***P < 0.001 (n = 10 to 34, Student’s t test). (K to M) Electron micrographs of KCNE1 (K), Kv7.1 (L), and Kv7.1 + KCNE1 (M) in…

Fig. 5. Kv7.1 and Kv7.1-KCNE1 complexes follow an unconventional secretory pathway. (A to I) Images of KCNE1-YFP (green, A to C), Kv7.1CFP (red, D to F), and Kv7.1-CFP + KCNE1-YFP complexes (red and green, G to I) colocalizing with ER-DsRed (blue) in the absence (A, D, and G) or the presence of either Sar1H79G (B, E, and H) or BFA (C, F, and I). Cyan, colocalization between KCNE1-YFP (green) and ER-DsRe (blue); magenta, colocalization between Kv7.1-CFP (red) and blue; and white, triple colocalization of green, red, and blue. (G to I) Partial colocalization between KCNE1 (green) and Kv7.1 (red) is also shown in yellow. Scale bars, 10 μm. (J) M coefficient values for the overlap of KCNE1 (black), Kv7.1 (white), and Kv7.1 + KCNE1 (light and dark gray) with ER marker in the absence (control) or the presence of either Sar1H79G or BFA. The values represent the means ± SEM. ***P < 0.001 for the ER overlap of KCNE1 in the presence of Sar1H79G and BFA versus that of KCNE1 under control conditions (n = 7 to 19, Student’s t test). (K) Coimmunoprecipitation of Kv7.1 with KCNE1 using anti-Kv7.1 antibodies (IP: Kv7.1) in whole-cell lysates from HEK-293 cells cotransfected with Kv7.1-CFP and KCNE1-YFP…

Fig. 6. ER-PM junctions are platforms for Kv7.1-KCNE1 complex assembly. HEK-293 cells were transfected with Kv2.1-YFP and Kv7.1-CFP + KCNE1-YFP and with ER-DsRed and Akt-PH-pDsRed as ER and PM markers, respectively. (Aa to Ae) Kv2.1 colocalization with the ER compartment. (Aa) Merged image of Kv2.1-YFP (green) and ER-DsRed (blue) staining. Cyan, colocalization. (Ab) ER-DsRed. (Ac) Mask of the subcellular distribution of the Kv2.1-ER colocalization signal. (Ad) Merge image of Aa and Ac. (Ae) Magnified view [white rectangle in (Ad)]. (Ba to Be) Kv7.1 and KCNE1 subunits colocalize at discrete sites in distal ER domains. (Ba) Kv7.1-CFP (red) and KCNE1-YFP (green) coexpression. Yellow, colocalization. (Bb) ER-DsRed expression is shown in blue. (Bc) Mask of the subcellular distribution of the Kv7.1-KCNE1 colocalization signal. (Bd) Merge of the images in Bb and Bc. (Be) Magnified view [white rectangle in (Bd)]. Kv7.1-KCNE1 colocalized with the ER marker (white) in discrete areas juxtaposed with the PM. Scale bars, 10 μm. (Ca to Cf) Kv2.1 localization at ER-PM junctions in 3D surface renders. (Ca to Cd) Maximal projection of the z stack of the whole-cell volume. (Ca) Kv2.1-YFP, green; (Cb) PM,…