| Dynamin- and Rab5-dependent endocytosis of a Ca2+ -activated K+ channel, KCa2.3. | |
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MedLine Citation:
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PMID: 22952906 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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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. |
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Authors:
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Yajuan Gao; Claudia A Bertuccio; Corina M Balut; Simon C Watkins; Daniel C Devor |
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Publication Detail:
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Type: Journal Article; Research Support, N.I.H., Extramural Date: 2012-08-28 |
Journal Detail:
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Title: PloS one Volume: 7 ISSN: 1932-6203 ISO Abbreviation: PLoS ONE Publication Date: 2012 |
Date Detail:
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Created Date: 2012-09-06 Completed Date: 2013-02-07 Revised Date: 2013-04-02 |
Medline Journal Info:
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Nlm Unique ID: 101285081 Medline TA: PLoS One Country: United States |
Other Details:
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Languages: eng Pagination: e44150 Citation Subset: IM |
Affiliation:
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Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA. |
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| MeSH Terms | |
Descriptor/Qualifier:
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Animals Caveolae / drug effects, metabolism, ultrastructure Dynamins / metabolism* Endocytosis* Endosomes / metabolism, ultrastructure HEK293 Cells Humans Membrane Microdomains / drug effects, metabolism Mice Models, Biological Potassium Channels, Calcium-Activated / metabolism*, ultrastructure Protein Transport rab5 GTP-Binding Proteins / metabolism* |
| Grant Support | |
ID/Acronym/Agency:
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HL092157/HL/NHLBI NIH HHS |
| Chemical | |
Reg. No./Substance:
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0/Potassium Channels, Calcium-Activated; EC 3.6.5.2/rab5 GTP-Binding Proteins; EC 3.6.5.5/Dynamins |
| Comments/Corrections | |
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