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Modulation of KCa3.1 by ubiquitylation in a polarized epithelium (893.20)
Author(s) -
Lee ShihLiang,
Devor Daniel,
Hamilton Kirk
Publication year - 2014
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.28.1_supplement.893.20
Subject(s) - epithelium , microbiology and biotechnology , chemistry , ubiquitin , biophysics , biotinylation , internalization , biology , cell , biochemistry , genetics , gene
In this study, we examined the role of ubiquitylation in the retrograde pathway of KCa3.1, for the first time in a polarized epithelium. Previously, we have shown that UBEI‐41, an inhibitor of ubiquitinin‐activating enzyme, reduced ubiquitylation and internalization of KCa3.1 (Balut et al. FASEB J 25: 2938‐48, 2011) in a non‐polarized epithelium. We asked the question ‘Is KCa3.1 ubiquitylated at the basolateral membrane?’ In order to test this, we used our Fischer Thyroid epithelial cell line stably expressing KCa3.1‐BLAP along with Bir‐A‐KDEL (cells grown on filters) that allows the biotinylation of channels prior to leaving the Golgi. Initially, we determined the time course of KCa3.1 by labeling (at 4C) the surface channels with streptavidin and then incubating cells at 37C for 0, 1, 3, 5, 8 and 12 hours. The half‐life of KCa3.1 was 3 hrs (n=6). To confirm that KCa3.1 was ubiquityated in a polarized epithelium, we conducted a similar experiment, as above, in which the cells were incubated with UBEI‐41 (50 µM). UBEI‐41 increased the half‐life of KCa3.1 to 12 hrs (n=4). If, channels were ubiquitylated at the membrane, in the presence of UBE‐41, then we hypothesized that there would be an increase number of channels at the membrane. To test this hypothesis, we used the Ussing chamber to examine an increase in function of KCa3.1 in the presence of UBEI‐41. Indeed, after 1 hr, K current in the presence of UBEI‐41 was increased compared to the K current of cells that were not treated with UBEI‐41. These data suggest that KCa3.1 is ubiquitylated at the membrane prior to being internalized. Grant Funding Source : This work was supported by the Department of Physiology, Univ. of Otago (KLH) and NIH (DCD).