title: Dynamin- and Rab5-dependent endocytosis of a Ca2+ -activated K+ channel, KCa2.3.
abstract: Regulation of the number of ion channels at the plasma membrane is a critical component of the physiological response. We recently demonstrated that the Ca(2+)-activated K(+) channel, KCa2.3 is rapidly endocytosed and enters a Rab35- and EPI64C-dependent recycling compartment. Herein, we addressed the early endocytic steps of KCa2.3 using a combination of fluorescence and biotinylation techniques. We demonstrate that KCa2.3 is localized to caveolin-rich domains of the plasma membrane using fluorescence co-localization, transmission electron microscopy and co-immunoprecipitation (co-IP). Further, in cells lacking caveolin-1, we observed an accumulation of KCa2.3 at the plasma membrane as well as a decreased rate of endocytosis, as assessed by biotinylation. We also demonstrate that KCa2.3 and dynamin II are co-localized following endocytosis as well as demonstrating they are associated by co-IP. Further, expression of K44A dynamin II resulted in a 2-fold increase in plasma membrane KCa2.3 as well as a 3-fold inhibition of endocytosis. Finally, we evaluated the role of Rab5 in the endocytosis of KCa2.3. We demonstrate that expression of a dominant active Rab5 (Q79L) results in the accumulation of newly endocytosed KCa2.3 on to the membrane of the Rab5-induced vacuoles. We confirmed this co-localization by co-IP; demonstrating that KCa2.3 and Rab5 are associated. As expected, if Rab5 is required for the endocytosis of KCa2.3, expression of a dominant negative Rab5 (S34N) resulted in an approximate 2-fold accumulation of KCa2.3 at the plasma membrane. This was confirmed by siRNA-mediated knockdown of Rab5. Expression of the dominant negative Rab5 also resulted in a decreased rate of KCa2.3 endocytosis. These results demonstrate that KCa2.3 is localized to a caveolin-rich domain within the plasma membrane and is endocytosed in a dynamin- and Rab5-dependent manner prior to entering the Rab35/EPI64C recycling compartment and returning to the plasma membrane.
authors.affiliation: Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.
authors.forename: Yajuan
authors.initials: Y
authors.lastname: Gao
authors.forename: Claudia A
authors.initials: CA
authors.lastname: Bertuccio
authors.forename: Corina M
authors.initials: CM
authors.lastname: Balut
authors.forename: Simon C
authors.initials: SC
authors.lastname: Watkins
authors.forename: Daniel C
authors.initials: DC
authors.lastname: Devor
mesh_terms: D000818:Animals; D021941:Caveolae / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D034281:Dynamins / Q000378:metabolism*; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism / Q000648:ultrastructure; D057809:HEK293 Cells; D006801:Humans; D021962:Membrane Microdomains / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D008954:Models, Biological; D024681:Potassium Channels, Calcium-Activated / Q000378:metabolism* / Q000648:ultrastructure; D021381:Protein Transport; D020696:rab5 GTP-Binding Proteins / Q000378:metabolism*
chemical_list: D024681:Potassium Channels, Calcium-Activated; D020696:rab5 GTP-Binding Proteins; D034281:Dynamins