Research ArticleCELL BIOLOGY

The unconventional biogenesis of Kv7.1-KCNE1 complexes

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Science Advances  01 Apr 2020:
Vol. 6, no. 14, eaay4472
DOI: 10.1126/sciadv.aay4472
  • 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 immunoblotted (IB) against Kv7.1 (left) and KCNE1 (right). SM, starting material; SN, supernatant; +AB, presence of antibodies; −AB, absence of antibodies during the immunoprecipitation. (J) Coexpression of Kv7.1 and KCNE1 in COS-7 cells recapitulates IKs currents. COS-7 cells were cotransfected with Kv7.1 and KCNE1 for 24 and 48 hours. The cells were held at −40 mV, and 5.5-s depolarizing pulses was applied from −80 to +60 mV. (K) Maximal intensity of currents at +60 mV. White bar, cells transfected for 24 hours; black bar, currents recorded after 48 hours of expression. The values are the means ± SEM of six to eight cells. **P < 0.01 versus 24 hours, Student’s t test. (L to P) Expression of Kv7.1 and KCNE1 in HEK-293 cells. Cells were cotransfected with Kv7.1-CFP and KCNE1-YFP for 48 hours, and representative confocal images were acquired. (L) Kv7.1, red; (M) KCNE1, green; (N) PM marker, blue; (O) merged signals. White represents triple colocalization. Scale bar, 10 μm. (P) Cellular colocalization of Kv7.1-CFP and KCNE1-YFP. The PM staining was used to create a mask to analyze the Kv7.1-KCNE1 colocalization. Whole cell, the entire cell was analyzed. Without (W/O) PM, whole-cell colocalization values were subtracted from those at the PM. The values are the means ± SEM of >25 cells. *P < 0.05 and ***P < 0.01 versus PM, Student’s t test. A.U., arbitrary units.

  • 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 versus entire cell (n = 31 to 36, Student’s t test). (S) Coimmunoprecipitation of Kv7.1 with KCNE1 using anti-KCNE1 antibodies (IP: KCNE1) in CUPs and whole-cell lysates entire cells (EC; entire cells) from cultured cells. Immunoblotting was performed with antibodies against Kv7.1 (IB: Kv7.1, 100 kDa, arrow) and KCNE1 (IB: KCNE1, 37 kDa, arrow). −, immunoprecipitation in absence of anti-KCNE1 antibodies. (T) Coimmunoprecipitation (co-IP) analysis of Kv7.1 with KCNE1 in entire cells (black bar) or CUPs (white bar). The values show the means ± SEM. *P < 0.05 CUP versus entire cell (n = 3, Student’s t test).

  • 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 (IB: Kv7.1) and anti-KCNE1 (IB: KCNE1) antibodies. Biotin, biotinylated pull down proteins. (I) Densitometric analysis of biotinylated Kv7.1 and KCNE1. KCNE1 (black), Kv7.1 (white), and Kv7.1 + KCNE1 (light and dark gray, respectively). The bars represent the means ± SEM. ***P < 0.001 for KCNE1 in the absence of Kv7.1 versus KCNE1 in the presence of Kv7.1 (n = 3, Student’s t test). (J to R) Representative results of Kv7.1-CFP and KCNE1-YFP FRET experiments in ER-containing CUPs (mCUPs). (J to L) Prebleaching images of KCNE1-YFP (J), Kv7.1-CFP (K), and ER-DsRed (L). (M and N) Postbleaching images of KCNE1-YFP (M) and Kv7.1-CFP (N). The bleached area is highlighted with a white square. (O) Merged prebleaching image (J to L). (Q) Magnified view of the bleached area in the merged image that enables the PM to be distinguished from the ER. (P and R) FRET ratio images. The calibration bar ranges from 0.8 (blue) to 1.4 (red). Scale bar, 10 μm. (S) FRET was measured independently in the PM (black bars) and the ER (white bars) within the same cell. The values indicate the means ± SEM. ***P < 0.001 for Kv7.1-KCNE1 in the PM versus the ER (n = 40, Student’s t test).

  • 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 HEK-293 cells. (K) KCNE1 staining of Golgi cisternae as indicated by the black arrowheads. Representative ER structures are circled. (L) Kv7.1 was located in ER-like structures, indicated by the white arrowheads, and was absent from the vicinity of the Golgi. (M) KCNE1 (indicated by the black arrowheads) disappeared from the Golgi cisternae and was located in ER-like structures in the presence of Kv7.1 (indicated by the white arrowheads). Kv7.1, 12-nm gold particles; KCNE1, 18-nm gold particles. Scale bars, 1 μm.

  • 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 in the absence (C) or the presence of Sar1H79G. Immunoblotting was performed against Kv7.1 (IB: Kv7.1) and KCNE1 (IB: KCNE1). (L) Coimmunoprecipitation of Kv7.1 with KCNE1 using anti-KCNE1 antibodies (IP: KCNE1) in whole-cell lysates from HEK-293 cells cotransfected with Kv7.1-CFP and KCNE1-YFP and treated without (C) or with BFA. Immunoblotting was performed against Kv7.1 (IB: Kv7.1) and KCNE1 (IB: KCNE1). −, immunoprecipitation in the absence of antibodies. (M) Analysis of KCNE1 glycosylation in the presence of tunicamycin. HEK-293 cells were transfected with KCNE1-YFP and Kv7.1-CFP + KCNE1-YFP and treated (+) or not treated (−) with tunicamycin (5 μg/ml). Cell extracts were immunoprecipitated and immunoblotted with anti-Kv7.1 and anti-KCNE1 antibodies. (N) Analysis of KCNE1 glycosylation in the presence of a cocktail of glycosidases (Gase). HEK-293 cells were transfected with KCNE1-YFP and Kv7.1-CFP + KCNE1-YFP and treated with EndoH, peptide N-glycosidase F (PNGase F), and PNGase β1 to PNGase β4 galactosidase (Gase). Cell extracts from Kv7.1-CFP + KCNE1-YFP–transfected HEK-293 cells were immunoprecipitated with anti-Kv7.1 antibodies and immunoblotted with anti-Kv7.1 and anti-KCNE1 antibodies. Cell extracts from HEK-293 cells transfected with KCNE1 were immunoprecipitated with anti-KCNE1 antibodies and 50 μg of immunoprecipitate treated with Gase and immunoblotted with anti-Kv7.1 and anti-KCNE1 antibodies. Note that in both cases, heavy KCNE1 bands (>50 kDa) disappeared in the presence of either Kv7.1 or Gase.

  • 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, red; (Cc) ER, blue; (Cd) Merged image. Scale bars, 10 μm. The white rectangle indicates the magnified area in (Ce) and (Cf). White, triple colocalization. The arrows indicate Kv2.1 at ER-PM junctions. (Cf) 3D surface render. ER, blue; PM, red; Kv2.1, green. The white arrow points to an ER-PM contact site where Kv2.1 is localized. Scale bar, 1.5 μm. (Da to Df) Kv7.1-KCNE1 complexes localized at ER-PM junctions in 3D surface renders. (Da to Dd) Maximal projection of the z stack of the whole-cell volume. (Da) Kv7.1-CFP, red; (Db) KCNE1-YFP, green; (Dc) ER, blue; (Dd) merge. Scale bars, 10 μm. The white rectangle indicates the magnified area in (De) and (Df). (De) White, triple colocalization. The arrows highlight Kv7.1-KCNE1 at ER-PM junctions. (Df) 3D surface render. ER, blue; Kv7.1, red; KCNE1, green. The white arrow points to a Kv7.1-KCNE1 complex localized at a cortical ER projection juxtaposed with the PM. Scale bar, 2 μm. (Ea to Ei) Results of FRET experiments between Kv7.1-CFP and KCNE1-YFP in mCUPs. (Ea and Eb) Prebleaching images showing KCNE1-YFP in green (Ea) and Kv7.1-CFP in red (Eb). (Ec and Ed) Postbleaching images of KCNE1-YFP (Ec) and Kv7.1-CFP (Ed). The bleached area is highlighted with a white square. (Ee) ER-DsRed, blue. (Ef) Merged image of (Ea) to (Ee). (Eh) Magnified view of the bleached area in the merged image that enables the PM (green line) to be distinguished from the ER (red line). Kv7.1 and KCNE1 were highly colocalized in discrete ER-related structures identified as ER-PM junctions (yellow line). (Eg and Ei) Original and magnified FRET ratio images, respectively. The calibration bars range from 0.8 (blue) to 1.4 (red). Scale bar, 10 μm. (F) Plot of individual FRET measurements in the PM (black), ER-PM junctions (gray), and the ER (white) in mCUPs. (G) FRET measurements in the PM (black), positive ER-PM junctions (+, dark gray), negative ER-PM junctions (−, light gray), and the ER (white) within the same cell. The bars represent the means ± SEM (n = 20 to 38). ***P < 0.001 for Kv7.1-KCNE1 in ER-PM+ versus the ER (n = 20 to 38, Student’s t test).

Supplementary Materials

  • Supplementary Materials

    The unconventional biogenesis of Kv7.1-KCNE1 complexes

    Anna Oliveras, Clara Serrano-Novillo, Cristina Moreno, Alicia de la Cruz, Carmen Valenzuela, Christian Soeller, Núria Comes, Antonio Felipe

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