Characterization of the Oligomeric Structure of the Ca2+-activated Cl- Channel Ano1/TMEM16A
Characterization of the Oligomeric Structure of the Ca2+-activated Cl- Channel Ano1/TMEM16A
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DOI：
10.1074/jbc.m110.174847
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发表时间：
2011-01-14
影响因子：
4.8
通讯作者：
Tarran, Robert
中科院分区：
文献类型：
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作者：
Sheridan, John T.;Worthington, Erin N.;Tarran, Robert
最近发现Anoctamin(ANO)/TMEM16A家族成员是钙激活氯通道(CACC)的重要亚单位。例如，Ano1在包括呼吸道上皮细胞在内的多种组织中都有高表达，在这些组织中，它作为跨上皮氯离子分泌的顶端管道，帮助调节肺液的动态平衡和粘液清除。然而，对该蛋白在质膜中的齐聚作用知之甚少。因此，利用mCherry和EGFP标记的Ano1构建物，我们进行了基于生化和Forster共振能量转移(FRET)的实验来确定Ano1的四级结构。FRET和免疫共沉淀研究表明，标记的Ano1亚单位在到达质膜之前直接相关。这种联系没有被细胞内钙离子的变化所改变，这表明这是一种固定的相互作用。为了确定Ano1的寡聚结构，我们进行了化学交联、非变性PAGE和电迁移率分析，结果表明Ano1以二聚体的形式存在。这些数据首次探索了Ano1的四级结构。了解Ano1的寡聚体性质是开发可用于治疗囊性纤维化的治疗药物的关键一步。
Members of the Anoctamin (Ano)/TMEM16A family have recently been identified as essential subunits of the Ca2+-activated chloride channel (CaCC). For example, Ano1 is highly expressed in multiple tissues including airway epithelia, where it acts as an apical conduit for transepithelial Cl- secretion and helps regulate lung liquid homeostasis and mucus clearance. However, little is known about the oligomerization of this protein in the plasma membrane. Thus, utilizing mCherry- and eGFP-tagged Ano1 constructs, we conducted biochemical and Forster resonance energy transfer (FRET)-based experiments to determine the quaternary structure of Ano1. FRET and co-immunoprecipitation studies revealed that tagged Ano1 subunits directly associated before they reached the plasma membrane. This association was not altered by changes in cytosolic Ca2+, suggesting that this is a fixed interaction. To determine the oligomeric structure of Ano1, we performed chemical cross-linking, non-denaturing PAGE, and electromobility shift assays, which revealed that Ano1 exists as a dimer. These data are the first to probe the quaternary structure of Ano1. Understanding the oligomeric nature of Ano1 is an essential step in the development of therapeutic drugs that could be useful in the treatment of cystic fibrosis.