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Possible role of myosin 2 and myosin 5C in the anterograde trafficking of KCa3.1 in a polarized epithelium
Author(s) -
Farquhar Rachel,
Rodrigues Ely,
Hamilton Kirk L.
Publication year - 2016
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.30.1_supplement.1224.41
Subject(s) - myosin , microfilament , myosin light chain kinase , microbiology and biotechnology , colchicine , microtubule , biology , cytoskeleton , chemistry , cell , biochemistry , genetics
Recently, we reported that KCa3.1 is trafficked to the basolateral membrane (BLM) of epithelial cells by a Rab1‐ and Rab8‐dependent mechanism (Bertuccio et al. PlosONE 9:e92013, 2014). Here, we extend our work on the role of microtubule/microfilament function in the trafficking of KCa3.1 to the BLM of Fischer rat thyroid (FRT) epithelial cells. We used our FRT epithelial cell line stably expressing KCa3.1‐BLAP/Bir‐A‐KDEL (cells grown on filters), immunoblot and Ussing chamber experiments in this study. Our immunoblot approach was to streptavidin label channels that arrive at the BLM to determine the effects of inhibitors of microtubule/microfilament function on the trafficking of the KCa3.1. We examined the effect colchicine, a microtubule inhibitor (10 μM for 0, 3 and 5 hr); ML‐9 an inhibitor of myosin light chain kinase (10 μM for 0, 3, and 5 hr) and 2,3‐butanedione monoxime (BDM) an inhibitor of muscle and non‐muscle myosin 2 and myosin 5 (10 mM for 0, 3, 5 hr) on trafficking of KCa3.1 to the BLM. As determined by immunoblot, colchicine significantly reduced trafficking of KCa3.1 by 70±8% (at 5 hr, P≤0.05, n=5); ML‐9 decreased trafficking of the channel by 70±3% (5 hr, n=5) and BDM reduced trafficking of the channel by 47±11% (5 hr, P≤0.05, n=5) compared to controls. We examined the effects of the inhibitors on the functional expression of KCa3.1 by Ussing chamber experiments. By using 1‐EBIO (100 μM, an activator of KCa3.1) to stimulate current via KCa3.1, we determined that colchicine (5 hr treatment) decreased 1‐EBIO‐stimulated current by 40±20% (P≤0.05, n=5); ML‐9 (5 hr) reduced stimulated current by 60±10% (P≤0.05, n=5) and BDM (5 hr) decreased stimulated current by 46±8% (P≤0.05, n=5) compared the controls. These data suggest that myosin 2 and myosin 5 may be critical for the trafficking of KCa3.1 to BLM of polarized epithelial cells. Support or Funding Information This work was supported by UORG and Dean's Fund grants from the Univ. of Otago (KLH).