title: Anterograde trafficking of KCa3.1 in polarized epithelia is Rab1- and Rab8-dependent and recycling endosome-independent.
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 μ1B 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.
authors.affiliation: Department of Cell Biology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.
authors.forename: Claudia A
authors.initials: CA
authors.lastname: Bertuccio
authors.affiliation: Department of Physiology, Otago School of Medical Sciences, University of Otago, Dunedin, Otago, New Zealand.
authors.forename: Shih-Liang
authors.initials: SL
authors.lastname: Lee
authors.affiliation: Department of Pharmacology and Toxicology, Medical College of Georgia, Georgia Regents University, Augusta, Georgia, United States of America.
authors.forename: Guangyu
authors.initials: G
authors.lastname: Wu
authors.affiliation: Department of Cell Biology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.
authors.forename: Michael B
authors.initials: MB
authors.lastname: Butterworth
authors.affiliation: Department of Physiology, Otago School of Medical Sciences, University of Otago, Dunedin, Otago, New Zealand.
authors.forename: Kirk L
authors.initials: KL
authors.lastname: Hamilton
authors.affiliation: Department of Cell Biology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.
authors.forename: Daniel C
authors.initials: DC
authors.lastname: Devor
mesh_terms: D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D016764:Cell Polarity; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism*; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels / Q000378:metabolism*; D021381:Protein Transport; D054875:Ubiquitination; D020691:rab GTP-Binding Proteins / Q000378:metabolism*; D020761:rab1 GTP-Binding Proteins / Q000378:metabolism*
chemical_list: D051660:Intermediate-Conductance Calcium-Activated Potassium Channels; C082377:RAB8A protein, human; D020691:rab GTP-Binding Proteins; D020761:rab1 GTP-Binding Proteins