Farquhar Rachel E, Rodrigues Ely, Hamilton Kirk L
Department of Physiology, Otago School of Medical Sciences, University of Otago Dunedin, New Zealand.
Department of Medicine, Otago School of Medical Sciences, University of Otago Dunedin, New Zealand.
Front Physiol. 2017 Jan 4;7:639. doi: 10.3389/fphys.2016.00639. eCollection 2016.
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.
对于KCa3.1定位于极化上皮细胞基底外侧膜（BLM）的机制仍在不断探索中。在此，我们研究了细胞骨架（微管和微丝）及肌球蛋白-Vc（Myo-Vc）在费希尔大鼠甲状腺上皮细胞中KCa3.1定位过程中的作用。我们采用了药理学方法，结合免疫印迹法（用于检测KCa3.1在BLM上的表达）、尤斯灌流小室法（用于检测KCa3.1在BLM上的功能表达）以及RNA干扰实验。肌动蛋白细胞骨架抑制剂细胞松弛素D（10 μM，5小时）和拉春库林A（10 μM，5小时）分别使KCa3.1向BLM的定位减少了88±4%和70±5%。微管抑制剂秋水仙碱（10 μM，5小时）使KCa3.1向BLM的定位减少了63±7%，与对照细胞相比，使1-EBIO刺激的KCa3.1钾电流降低了46±18%。肌球蛋白轻链激酶抑制剂ML9（10 μM，5小时）使该通道的定位减少了83±2%，与对照细胞相比，使钾电流降低了54±8%。用2,3-丁二酮单肟（10 mM，5小时）抑制Myo-V，与对照细胞相比，使该通道向BLM的定位减少了58±5%，并使KCa3.1的刺激电流降低了48±12%。最后，使用针对Myo-Vc的RNA干扰技术，我们证明与对照细胞相比，敲低Myo-Vc可使KCa3.1在BLM上的表达减少44±7%，并使KCa3.1钾电流降低1.04±0.14 μA。这些数据表明，微管和微丝细胞骨架以及Myo-Vc对于KCa3.1的定位至关重要。
