Document Detail


Dynamin- and Rab5-dependent endocytosis of a Ca2+ -activated K+ channel, KCa2.3.
MedLine Citation:
PMID:  22952906     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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:
Yajuan Gao; Claudia A Bertuccio; Corina M Balut; Simon C Watkins; Daniel C Devor
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural     Date:  2012-08-28
Journal Detail:
Title:  PloS one     Volume:  7     ISSN:  1932-6203     ISO Abbreviation:  PLoS ONE     Publication Date:  2012  
Date Detail:
Created Date:  2012-09-06     Completed Date:  2013-02-07     Revised Date:  2013-07-11    
Medline Journal Info:
Nlm Unique ID:  101285081     Medline TA:  PLoS One     Country:  United States    
Other Details:
Languages:  eng     Pagination:  e44150     Citation Subset:  IM    
Affiliation:
Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.
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MeSH Terms
Descriptor/Qualifier:
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:
HL092157/HL/NHLBI NIH HHS
Chemical
Reg. No./Substance:
0/Potassium Channels, Calcium-Activated; EC 3.6.5.2/rab5 GTP-Binding Proteins; EC 3.6.5.5/Dynamins
Comments/Corrections

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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