Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA.
Am J Physiol Cell Physiol. 2013 Jan 1;304(1):C38-48. doi: 10.1152/ajpcell.00109.2012. Epub 2012 Sep 26.
The aquaporin-2 (AQP2) water channel relocates mainly to the apical plasma membrane of collecting duct principal cells after vasopressin (VP) stimulation. AQP2 transport to this membrane domain is assumed to be a direct route involving recycling of intracellular vesicles. However, basolateral plasma membrane expression of AQP2 is observed in vivo in principal cells. Here, we asked whether there is a transcytotic pathway of AQP2 trafficking between apical and basolateral membranes. We used MDCK cells in which AQP2 normally accumulates apically after VP exposure. In contrast, both site-specific biotinylation and immunofluorescence showed that AQP2 is strongly accumulated in the basolateral membrane, along with the endocytic protein clathrin, after a brief cold shock (4°C). This suggests that AQP2 may be constitutively targeted to basolateral membranes and then retrieved by clathrin-mediated endocytosis at physiological temperatures. Rab11 does not accumulate in basolateral membranes after cold shock, suggesting that the AQP2 in this location is not associated with Rab11-positive vesicles. After rewarming (37°C), basolateral AQP2 staining is diminished and it subsequently accumulates at the apical membrane in the presence of VP/forskolin, suggesting that transcytosis can be followed by apical insertion of AQP2. This process is inhibited by treatment with colchicine. Our data suggest that the cold shock procedure reveals the presence of microtubule-dependent AQP2 transcytosis, which represents an indirect pathway of apical AQP2 delivery in these cells. Furthermore, our data indicate that protein polarity data obtained from biotinylation assays, which require cells to be cooled to 4°C during the labeling procedure, should be interpreted with caution.
水通道蛋白-2（AQP2）在血管加压素（VP）刺激后主要向集合管主细胞的顶质膜重新分布。AQP2 向该膜域的转运被认为是一种直接途径，涉及细胞内囊泡的再循环。然而，在体内主细胞中观察到 AQP2 在基底外侧质膜上的表达。在这里，我们询问是否存在 AQP2 从顶膜到基底外侧膜的跨细胞运输途径。我们使用 MDCK 细胞，其中 AQP2 在 VP 暴露后通常在顶质膜上积累。相比之下，在用冷休克（4°C）处理后，两种特异性生物素化和免疫荧光均显示 AQP2 强烈积累在基底外侧膜上，与内吞蛋白网格蛋白一起。这表明 AQP2 可能被持续靶向基底外侧膜，然后在生理温度下通过网格蛋白介导的内吞作用被回收。Rab11 在冷休克后不会在基底外侧膜上积累，这表明该位置的 AQP2 与 Rab11 阳性囊泡无关。在复温（37°C）后，基底外侧 AQP2 染色减少，随后在 VP/forskolin 的存在下积累在顶质膜上，表明跨细胞运输后可以进行 AQP2 的顶端插入。该过程可被秋水仙碱处理抑制。我们的数据表明，冷休克程序揭示了微管依赖性 AQP2 易位的存在，这代表了这些细胞中 AQP2 顶端递呈的间接途径。此外，我们的数据表明，从生物素化测定获得的蛋白质极性数据，这些测定在标记过程中需要将细胞冷却至 4°C，应该谨慎解释。
