Premium
Basolateral trafficking of KCa3.1 in a polarized epithelium
Author(s) -
Winter Barclay J,
Balut Corina M,
Butterworth Michael B,
Gao Yajuan,
Devor Daniel C,
Hamilton Kirk L
Publication year - 2011
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.25.1_supplement.860.13
Subject(s) - epithelial polarity , kdel , microbiology and biotechnology , apical membrane , chemistry , biology , membrane , biochemistry , endoplasmic reticulum , golgi apparatus
In this study, we examined the trafficking of the intermediate‐conductance, Ca 2+ ‐activated K + channel (KCa3.1) using novel biotin‐ligase acceptor peptide (BLAP) – BirA‐KDEL immunofluorescence (IF) technology. Fischer rat thyroid (FRT) cells were transfected with KCa3.1‐BLAP and BirA‐KDEL and grown on filters as a monolayer. To define the membrane location of KCa3.1‐BLAP, we labeled cell monolayers with streptavidin‐Alexa 546 on either the apical or basolateral side of the monolayers and incubated cells for 0 or 2 hr. Only basolateral labeling was evident demonstrating that KCa3.1‐BLAP was targeted to the basolateral membrane. Using IF, we determined which part of KCa3.1 was responsible for the basolateral trafficking of the channel. Various C‐terminal constructs of KCa3.1 were engineered to the neural growth factor receptor (NGFR) that is an apically targeted receptor. WT‐NGFR trafficked to the apical membrane while NGFR‐137‐KCa3.1 (entire C‐terminus of KCa3.1) and NGFR‐C137‐D370STOP‐KCa3.1 trafficked to the basolateral membrane, however, NGFR‐C59‐KCa3.1 (last 59 amino acids of KCa3.1) trafficked to the apical membrane. Our results demonstrate, for the first time in a polarized epithelium, that KCa3.1 is trafficked to the basolateral membrane and that the amino acids between the end of S6 and D370 of KCa3.1 are required for basolateral targeting of this channel. This work was funded by Lotteries Health and Department of Physiology (KLH) and NIH (DCD).