AUTHOR=Farquhar Rachel E. , Rodrigues Ely , Hamilton Kirk L. 

TITLE=The Role of the Cytoskeleton and Myosin-Vc in the Targeting of KCa3.1 to the Basolateral Membrane of Polarized Epithelial Cells

JOURNAL=Frontiers in Physiology

VOLUME=Volume 7 - 2016

YEAR=2017

URL=https://www.frontiersin.org/journals/physiology/articles/10.3389/fphys.2016.00639

DOI=10.3389/fphys.2016.00639

ISSN=1664-042X

ABSTRACT=Understanding the targeting of KCa3.1 to the basolateral membrane (BLM) of polarized epithelial cells is still emerging. Here, we examined the role of the cytoskeleton (microtubules and microfilaments) and Myosin-Vc (Myo-Vc) in the targeting of KCa3.1 in Fischer rat thyroid epithelial cells. We used a pharmacological approach with immunoblot (for the BLM expression of KCa3.1), Ussing chamber (functional BLM expression of KCa3.1) and siRNA experiments. The actin cytoskeleton inhibitors cytochalasin D (10 µM, 5 hr) and latrunculin A (10 µM, 5 hr) reduced the targeting of KCa3.1 to the BLM by 88±4% and 70±5%, respectively. Colchicine (10 µM, 5 hr) a microtubule inhibitor reduced targeting of KCa3.1 to the BLM by 63±7% and decreased 1-EBIO-stimulated KCa3.1 K+ current by 46±18%, compared with control cells. ML9 (10 µM, 5 hr), an inhibitor of myosin light chain kinase, decreased targeting of the channel by 83±2% and reduced K+ current by 54±8% compared to control cells. Inhibiting Myo-V with 2,3-butanedione monoxime (10 mM, 5 hr) reduced targeting of the channel to the BLM by 58±5% and decreased the stimulated current of KCa3.1 by 48±12% compared with control cells.  Finally, using siRNA for Myo-Vc, we demonstrated that knockdown of Myo-Vc reduced the BLM expression of KCa3.1 by 44±7% and KCa3.1 K+ current by 1.04±0.14 μA compared with control cells. These data suggest that the microtubule and microfilament cytoskeleton and Myo-Vc are critical for the targeting of KCa3.1.