Anterograde Trafficking of KCa3.1 in Polarized Epithelia Is Rab1- and Rab8-Dependent and Recycling Endosome-Independent
Figure 1
Localization and degradation of KCa3.1 in polarized epithelia cells.
A. BLAP-KCa3.1 was transduced into MDCK, Caco-2 or FRT cells grown to confluence on Transwell® filters. Basolateral (BL) and Apical (AP) plasma membrane KCa3.1 channels were specifically biotinylated using recombinant BirA and labeled with either streptavidin-Alexa555 (red) for IF localization or unconjugated streptavidin for IB. Apical membrane was co-labeled with wheat germ agglutinin (WGA)-Alexa488 (green) and nuclei were labeled with DAPI (blue) for IF localization. The top panels show a single confocal section through the mid-plane of the cells and the bottom panels show a z-stack. For the IB experiments, BL and AP membranes were independently labeled on separate Transwell filters. 30 μg of protein was loaded per lane. Data are representative of 3 separate experiments. KCa3.1 was localized exclusively to the BL membrane as assessed by both IF and IB. B. KCa3.1 was biotinylated at the BL membrane as above in MDCK and Caco-2 cells or biotinylated at the level of the ER in FRT cells stably expressing BirA-KDEl/KCa3.1-BLAP and its degradation evaluated over time. Note the different time points used for each cell line. Tubulin was used as a loading control. C. Quantification of the blots shown in B for at least 3 separate experiments. *P<0.05 with respect to T = 0.