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Characterization of the trafficking of a Ca2+‐activated K+ channel (KCa3.1) in polarized epithelial cells
Author(s) -
Bertuccio Claudia Alejandra,
Devor Daniel
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.913.20
Subject(s) - microbiology and biotechnology , endocytosis , leupeptin , apical membrane , chemistry , hek 293 cells , biophysics , cell culture , cell polarity , membrane , cell , biology , biochemistry , receptor , enzyme , genetics , protease
While the intermediate conductance, Ca 2+ ‐activated K + channel (KCa3.1) is targeted to the basolateral membrane in several polarized epithelial cells, an alternatively spliced form has been shown to traffic to the apical membrane in colonic epithelia. Thus, the aim of this work was to study how KCa3.1 traffics to the plasma membrane (PM) in polarized epithelial cells. We expressed B iotin L igase A cceptor P eptide (BLAP)‐tagged KCa3.1 in polarized MDCK and CACO‐2 cells grown on Transwell filters and labeled PM channels with streptavidin. KCa3.1 was localized exclusively to the basolateral PM in both cell types and was shown to be functional in Ussing chamber studies by activation with DCEBIO and inhibition with TRAM‐34. Degradation of PM KCa3.1 had a half‐life of ~5 hrs in MDCK cells and ~10 hrs in CACO‐2 cells; being inhibited by leupeptin/pepstatin, indicative of lysosomal degradation. Further, lactacystin inhibited degradation of endocytosed KCa3.1 suggesting a role for proteaosmes in this degradation process. We further demonstrated that KCa3.1 is ubiquitylated following endocytosis in polarized epithelial cells. Finally, in HEK cells we demonstrate a role for Rab1 and Rab8 in the PM expression of KCa3.1 and that both Rab1 and Rab8 coimmunoprecipitate with KCa3.1. (Supported by NIH HL092157)