In a previous study we have shown that KCa3.1 is rapidly internalized from the plasma membrane and has a short half-life in HEK293 and endothelial HMEC-1 cells (Biophys. J. 2008 94: 529). The aim of the present work was to investigate the molecular mechanisms controlling this fast degradation of KCa3.1. Using the Biotin-acceptor-KCa3.1 construct, recently engineered in our lab, the channel was fluorescently labeled at the cell surface and the cells were incubated at 37°C for different periods of time. The fate of the endocytosed channels was addressed by confocal microscopy.After 5 h incubation at 37 °C, almost all protein was degraded, as demonstrated by a very low fluorescence level inside the cells. However, when the same treatment was applied in the presence of lysosomal proteases inhibitors leupeptin/pepstatin, we observed an accumulation of the channel inside the cells, suggesting that lysosomes are involved in KCa3.1 degradation.Next, we addressed the possible role of the endosomal sorting complex required for transport (ESCRT) components in this process. We have investigated the role of TSG101 (a member of ESCRT-I complex) and SKD1/VPS4 (ESCRT-III). Cells were doubly transfected with Biotin-KCa3.1 and either the wild type construct or a dominant negative form of SKD1/VPS4 (E235Q) and TSG101, respectively. For SKD1E235Q and mutant TSG101 cells, we observed a lack of channel degradation, as compared to control cells.These results show for the first time the role of ESCRT family proteins in targeting KCa3.1 for lysosomal degradation in HEK and HMEC-1 cells.This work was supported by AHA Grant 0825542D.