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Rab5‐dependent endocytosis of KCa2.3
Author(s) -
Gao Yajuan,
Devor Daniel C
Publication year - 2011
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.25.1_supplement.860.15
Subject(s) - dynamin , endocytosis , chemistry , endocytic cycle , biophysics , microbiology and biotechnology , biochemistry , cell , biology
We demonstrated that recycling of KCa2.3 is dependent upon Rab35/EPI64C and RME‐1. Herein, we report our initial studies on the mechanism by which KCa2.3 is retrieved from the plasma membrane. Using a biotin ligase acceptor peptide‐tagged KCa2.3 construct, we demonstrate that expression of a dominant active Rab5 (Q79L) results in the colocalization of endocytosed KCa2.3 with the Rab5‐induced vacuoles. Expression of a dominant negative Rab5 (N133I) resulted in a 2‐fold accumulation of plasma membrane KCa2.3. Also, DN Rab5 slows the endocytosis of KCa2.3, i.e., after 10 min., 19% of KCa2.3 is endocytosed in the presence of WT Rab5, whereas only 5% of KCa2.3 is endocytosed in the presence of DN Rab5. Co‐IP confirmed a close association between Rab5 and KCa2.3. Expression of Rabex‐5 (RabGEF1) GEF inactive mutants or ubiquitin binding mutants resulted in an increased steady‐state expression of KCa2.3 at the plasma membrane. As the Rab5‐dependent endocytic pathway can be either dynamin dependent or independent we determined whether dynamin was required for the endocytosis of KCa2.3. Overexpression of K44A dynamin resulted in an increase in steady‐state plasma membrane KCa2.3, relative to WT dynamin, consistent with an inhibition of endocytosis. In addition, co‐IP confirmed a close association between KCa2.3 and dynamin. In total, our results suggest that KCa2.3 endocytosis is both Rab5‐ and dynamin‐dependent.