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=7 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 h) and latrunculin A (10 μM, 5 h) reduced the targeting of KCa3.1 to the BLM by 88 ± 4 and 70 ± 5%, respectively. Colchicine (10 μM, 5 h) 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 h), 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 h) 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.