Modulation of Retrograde Trafficking of KCa3.1 in a Polarized Epithelium.
Modulation of Retrograde Trafficking of KCa3.1 in a Polarized Epithelium.
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DOI：
10.3389/fphys.2017.00489
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发表时间：
2017
影响因子：
4
通讯作者：
Hamilton KL
中科院分区：
文献类型：
--
作者：
Lee BS;Devor DC;Hamilton KL
在上皮细胞中，中间电导的钙激活钾通道(KCa3.1)定位于基底膜(BLM)，该通道在吸收和分泌过程中发挥多种作用。越来越多的研究表明，KCa3.1的膜驻留群体可能在疾病的临床表现中起关键作用。在这项研究中，我们利用稳定表达KCa3.1的Fisher大鼠甲状腺细胞系，研究了调节KCa3.1降解的关键分子成分。利用免疫印迹、Ussing小室和药理学方法，我们证明了KCa3.1是专门针对BLM的，提供了KCa3.1降解的完整时间过程以及在泛素化和去泛素化药物抑制剂存在下KCa3.1的降解时间过程，以加深我们对KCa3.1逆行转运的理解。我们提供了KCa3.1的完整降解图谱，并且这种降解是通过泛素依赖的途径进行的。UBEI-41抑制E1泛素激活酶使细胞内化通道的能力减弱，表现为BLM表面表达增加，通过DCEBIO敏感的K+电流测量，导致通道功能增强。此外，PR-619或亮肽素/胃抑素分别处理细胞也证实了脱泛素酶的参与和溶酶体的降解，显著降低了膜KCa3.1的降解速率。此外，我们提供了第一个证据，证明KCa3.1通道在BLM中没有去泛素化。这些数据进一步确定了KCa3.1的逆行贩运，并可能为疾病的治疗方法提供一条途径。
In epithelia, the intermediate conductance, Ca2+-activated K+ channel (KCa3.1) is targeted to the basolateral membrane (BLM) where this channel plays numerous roles in absorption and secretion. A growing body of research suggests that the membrane resident population of KCa3.1 may be critical in clinical manifestation of diseases. In this study, we investigated the key molecular components that regulate the degradation of KCa3.1 using a Fisher rat thyroid cell line stably expressing KCa3.1. Using immunoblot, Ussing chamber, and pharmacological approaches, we demonstrated that KCa3.1 is targeted exclusively to the BLM, provided a complete time course of degradation of KCa3.1 and degradation time courses of the channel in the presence of pharmacological inhibitors of ubiquitylation and deubiquitylation to advance our understanding of the retrograde trafficking of KCa3.1. We provide a complete degradation profile of KCa3.1 and that the degradation is via an ubiquitin-dependent pathway. Inhibition of E1 ubiquitin activating enzyme by UBEI-41 crippled the ability of the cells to internalize the channel, shown by the increased BLM surface expression resulting in an increased function of the channel as measured by a DCEBIO sensitive K+ current. Additionally, the involvement of deubiquitylases and degradation by the lysosome were also confirmed by treating the cells with PR-619 or leupeptin/pepstatin, respectively; which significantly decreased the degradation rate of membrane KCa3.1. Additionally, we provided the first evidence that KCa3.1 channels were not deubiquitylated at the BLM. These data further define the retrograde trafficking of KCa3.1, and may provide an avenue for therapeutic approach for treatment of disease.
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影响因子：
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