Anterograde trafficking of KCa3.1 in polarized epithelia is Rab1- and Rab8-dependent and recycling endosome-independent.
Anterograde trafficking of KCa3.1 in polarized epithelia is Rab1- and Rab8-dependent and recycling endosome-independent.
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DOI：
10.1371/journal.pone.0092013
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Devor DC
中科院分区：
文献类型：
--
作者：
Bertuccio CA;Lee SL;Wu G;Butterworth MB;Hamilton KL;Devor DC
介导的Ca2+激活的K+通道（KCa3.1）靶向极化上皮细胞的基底外侧（BL）膜，在跨上皮离子转运中起关键作用。然而，没有研究确定KCa 3.1在极化上皮中的顺行和逆行运输。在此，我们利用生物素连接酶受体肽（BTH）标记的KCa3.1来解决极化上皮细胞中的这些运输步骤，使用MDCK，Caco-2和FRT细胞。我们证明，KCa3.1是专门针对BL膜在这些细胞生长时，过滤器支持。内吞作用后，通过抑制溶酶体/蛋白酶体途径防止KCa 3.1降解。此外，KCa3.1的泛素化在BL膜的内吞作用后增加，PR-619（一种去泛素化酶抑制剂）阻止降解，表明KCa3.1被泛素化靶向降解。我们证明了KCa3.1在极化LLC-PK1细胞中靶向BL膜，该细胞缺乏AP-1复合物的μ 1B亚基，表明KCa3.1的BL靶向不依赖于μ 1B。由于Rab 1、2、6和8在ER/高尔基体出口和蛋白质向BL膜的运输中起作用，我们评估了这些Rab在KCa 3.1运输中的作用。在显性负性Rab1或Rab8的存在下，KCa 3.1细胞表面表达显著降低，而Rabs 2和Rabs 6没有影响。我们还将KCa3.1与Rab1和Rab8共免疫沉淀。这些结果表明，这些Rabs是KCa 3.1顺行运输所必需的。最后，我们确定KCa 3.1是否直接运输到BL膜或通过MDCK细胞中的再循环内体。对于这些研究，我们使用再循环内体消融或显性阴性RME-1构建体，并确定KCa 3.1直接运输到BL膜，而不是通过再循环内体。这些结果是第一个描述的顺行和逆行贩运KCa 3.1极化上皮细胞。
The intermediate conductance, Ca2+-activated K+ channel (KCa3.1) targets to the basolateral (BL) membrane in polarized epithelia where it plays a key role in transepithelial ion transport. However, there are no studies defining the anterograde and retrograde trafficking of KCa3.1 in polarized epithelia. Herein, we utilize Biotin Ligase Acceptor Peptide (BLAP)-tagged KCa3.1 to address these trafficking steps in polarized epithelia, using MDCK, Caco-2 and FRT cells. We demonstrate that KCa3.1 is exclusively targeted to the BL membrane in these cells when grown on filter supports. Following endocytosis, KCa3.1 degradation is prevented by inhibition of lysosomal/proteosomal pathways. Further, the ubiquitylation of KCa3.1 is increased following endocytosis from the BL membrane and PR-619, a deubiquitylase inhibitor, prevents degradation, indicating KCa3.1 is targeted for degradation by ubiquitylation. We demonstrate that KCa3.1 is targeted to the BL membrane in polarized LLC-PK1 cells which lack the μ1B subunit of the AP-1 complex, indicating BL targeting of KCa3.1 is independent of μ1B. As Rabs 1, 2, 6 and 8 play roles in ER/Golgi exit and trafficking of proteins to the BL membrane, we evaluated the role of these Rabs in the trafficking of KCa3.1. In the presence of dominant negative Rab1 or Rab8, KCa3.1 cell surface expression was significantly reduced, whereas Rabs 2 and 6 had no effect. We also co-immunoprecipitated KCa3.1 with both Rab1 and Rab8. These results suggest these Rabs are necessary for the anterograde trafficking of KCa3.1. Finally, we determined whether KCa3.1 traffics directly to the BL membrane or through recycling endosomes in MDCK cells. For these studies, we used either recycling endosome ablation or dominant negative RME-1 constructs and determined that KCa3.1 is trafficked directly to the BL membrane rather than via recycling endosomes. These results are the first to describe the anterograde and retrograde trafficking of KCa3.1 in polarized epithelia cells.
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影响因子：
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