The Role of the Cytoskeleton and Myosin-Vc in the Targeting of KCa3.1 to the Basolateral Membrane of Polarized Epithelial Cells.
The Role of the Cytoskeleton and Myosin-Vc in the Targeting of KCa3.1 to the Basolateral Membrane of Polarized Epithelial Cells.
复制标题
DOI：
10.3389/fphys.2016.00639
复制
发表时间：
2016
影响因子：
4
通讯作者：
Hamilton KL
中科院分区：
文献类型：
--
作者：
Farquhar RE;Rodrigues E;Hamilton KL
了解KCa3.1靶向极化上皮细胞的基底外侧膜（BLM）仍在出现。在这里，我们研究了细胞骨架（微管和微丝）和肌球蛋白-Vc（Myo-Vc）在Fischer大鼠甲状腺上皮细胞中靶向KCa3.1的作用。我们使用了具有免疫印迹（用于KCa3.1的BLM表达）、Ussing室（KCa3.1的功能性BLM表达）和siRNA实验的药理学方法。肌动蛋白细胞骨架抑制剂细胞松弛素D（10 μM，5 h）和latrunculin A（10 μM，5 h）分别使KCa 3.1对BLM的靶向作用降低了88 ± 4%和70 ± 5%。与对照细胞相比，微管抑制剂秋水仙碱（10 μM，5 h）使KCa 3.1靶向BLM的能力降低63 ± 7%，使1-EBIO刺激的KCa 3.1 K+电流降低46 ± 18%。与对照细胞相比，肌球蛋白轻链激酶抑制剂ML 9（10 μM，5 h）使通道靶向降低83 ± 2%，K+电流降低54 ± 8%。与对照细胞相比，用2，3-丁二酮单肟（10 mM，5 h）抑制Myo-V使通道对BLM的靶向减少58 ± 5%，并使KCa 3.1的刺激电流降低48 ± 12%。最后，使用针对Myo-Vc的siRNA，我们证明与对照细胞相比，Myo-Vc的敲低使KCa 3.1的BLM表达降低44 ± 7%，KCa 3.1 K+电流降低1.04 ± 0.14 μA。这些数据表明，微管和微丝细胞骨架和Myo-Vc是KCa 3.1靶向的关键。
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.
登录
查看更多内容
影响因子：
48
作者：
Chen, I;Howarth, M;Ting, AY
通讯作者：
Ting, AY
DOI：
10.1073/pnas.96.24.13726
发表时间：
1999-11-23
影响因子：
11.1
作者：
De La Cruz, EM;Wells, AL;Sweeney, HL
通讯作者：
Sweeney, HL
影响因子：
4
作者：
DURHAM, ACH
通讯作者：
DURHAM, ACH
影响因子：
4.2
作者：
Balut, Corina M.;Gao, Yajuan;Devor, Daniel C.
通讯作者：
Devor, Daniel C.
影响因子：
4.8
作者：
Gao, Yajuan;Balut, Corina M.;Devor, Daniel C.
通讯作者：
Devor, Daniel C.