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

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 ?M, 5 h) and latrunculin A (10 ?M, 5 h) reduced the targeting of KCa3.1 to the BLM by 88 ± 4 and 70 ± 5%, respectively. Colchicine (10 ?M, 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 ?M, 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 ?A compared with control cells. These data suggest that the microtubule and microfilament cytoskeleton and Myo-Vc are critical for the targeting of KCa3.1.

SUBMITTER: Farquhar RE 

PROVIDER: S-EPMC5209343 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature