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. 2020 Apr 1;6(14):eaay4472. doi: 10.1126/sciadv.aay4472

Fig. 5. Kv7.1 and Kv7.1-KCNE1 complexes follow an unconventional secretory pathway.

Fig. 5

(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.