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research
Anterograde trafficking of KCa3.1 in polarized epithelia is Rab1- And Rab8-Dependent and recycling endosome-independent
Authors
A Al-Hazza
A Duffield
+67 more
A Schwab
A Wulf
AC Gerlach
Agustin Guerrero-Hernandez
AH Hutagalung
AL Ang
AL Ang
C Barmeyer
C Dong
C Dong
C Gorbea
CA Syme
CE Futter
CE Futter
Claudia A. Bertuccio
CM Babbey
CM Balut
CM Balut
CM Balut
CM Filipeanu
Daniel C. Devor
DC Devor
DC Devor
DC Devor
DC Devor
DC Devor
E Orzech
EJ Tisdale
FE Reyes-Turcu
G Griffiths
G Wu
GA Farr
Guangyu Wu
H Fölsch
H Ohno
HM Jones
HM Jones
I Pilecka
J Cancino
JL Seachrist
JS Yoo
Kirk L. Hamilton
LA Simms
M Albaqumi
M Jeffers
Michael B. Butterworth
MM Hammad
O Martinez
O Staub
P van Kerkhof
PA Rufo
R Warth
S Singh
SD Fuller
Shih-Liang Lee
SM Millard
SX Lin
TM Ishii
TR Muth
WJ Joiner
WJ Joiner
WJ Joiner
X Zhang
X Zhuang
Y Gao
Y Gao
Z Galcheva-Gargova
Publication date
14 March 2014
Publisher
'Public Library of Science (PLoS)'
Doi
Cite
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on
PubMed
Abstract
The intermediate conductance, Ca2+-activated K+ channel (KCa3.1) targets to the basolateral (BL) membrane in polarized epithelia where it plays a key role in transepithelial ion transport. However, there are no studies defining the anterograde and retrograde trafficking of KCa3.1 in polarized epithelia. Herein, we utilize Biotin Ligase Acceptor Peptide (BLAP)-tagged KCa3.1 to address these trafficking steps in polarized epithelia, using MDCK, Caco-2 and FRT cells. We demonstrate that KCa3.1 is exclusively targeted to the BL membrane in these cells when grown on filter supports. Following endocytosis, KCa3.1 degradation is prevented by inhibition of lysosomal/proteosomal pathways. Further, the ubiquitylation of KCa3.1 is increased following endocytosis from the BL membrane and PR-619, a deubiquitylase inhibitor, prevents degradation, indicating KCa3.1 is targeted for degradation by ubiquitylation. We demonstrate that KCa3.1 is targeted to the BL membrane in polarized LLC-PK1 cells which lack the m1B subunit of the AP-1 complex, indicating BL targeting of KCa3.1 is independent of μ1B. As Rabs 1, 2, 6 and 8 play roles in ER/Golgi exit and trafficking of proteins to the BL membrane, we evaluated the role of these Rabs in the trafficking of KCa3.1. In the presence of dominant negative Rab1 or Rab8, KCa3.1 cell surface expression was significantly reduced, whereas Rabs 2 and 6 had no effect. We also co-immunoprecipitated KCa3.1 with both Rab1 and Rab8. These results suggest these Rabs are necessary for the anterograde trafficking of KCa3.1. Finally, we determined whether KCa3.1 traffics directly to the BL membrane or through recycling endosomes in MDCK cells. For these studies, we used either recycling endosome ablation or dominant negative RME-1 constructs and determined that KCa3.1 is trafficked directly to the BL membrane rather than via recycling endosomes. These results are the first to describe the anterograde and retrograde trafficking of KCa3.1 in polarized epithelia cells. © 2014 Bertuccio et al
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