Dynamin- and Rab5-Dependent Endocytosis of a Ca²⁺-Activated K⁺ Channel, KCa2.3

Gao, Y and Bertuccio, CA and Balut, CM and Watkins, SC and Devor, DC (2012) Dynamin- and Rab5-Dependent Endocytosis of a Ca²⁺-Activated K⁺ Channel, KCa2.3. PLoS ONE, 7 (8).

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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 Ca2+-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. © 2012 Gao et al.

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Details

Item Type:	Article
Status:	Published
Creators/Authors:	Creators Email Pitt Username ORCID Gao, Y Bertuccio, CA cab199@pitt.edu CAB199 Balut, CM cmb89@pitt.edu CMB89 Watkins, SC simon.watkins@pitt.edu SWATKINS Devor, DC dd2@pitt.edu DD2
Contributors:	Contribution Contributors Name Email Pitt Username ORCID Editor Guerrero-Hernandez, Agustin UNSPECIFIED UNSPECIFIED UNSPECIFIED
Date:	28 August 2012
Date Type:	Publication
Journal or Publication Title:	PLoS ONE
Volume:	7
Number:	8
DOI or Unique Handle:	10.1371/journal.pone.0044150
Schools and Programs:	School of Medicine > Cell Biology and Molecular Physiology
Refereed:	Yes
Other ID:	NLM PMC3429460
PubMed Central ID:	PMC3429460
PubMed ID:	22952906
Date Deposited:	19 Oct 2012 21:20
Last Modified:	13 Jun 2021 02:55
URI:	http://d-scholarship.pitt.edu/id/eprint/15896

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