Secreted CLCA1 modulates TMEM16A to activate Ca(2+)-dependent chloride currents in human cells.
Secreted CLCA1 modulates TMEM16A to activate Ca(2+)-dependent chloride currents in human cells.
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DOI：
10.7554/elife.05875
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发表时间：
2015-03-17
期刊：
影响因子：
7.7
通讯作者：
Brett TJ
中科院分区：
文献类型：
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作者：
Sala-Rabanal M;Yurtsever Z;Nichols CG;Brett TJ
钙激活氯离子通道调节器1 （CLCA1）激活钙依赖性氯离子电流；目标和机制都还不清楚。我们证明分泌的CLCA1以旁分泌方式激活HEK293T细胞中钙依赖性氯电流，内源性TMEM16A/Anoctamin1传导电流。暴露于外源性CLCA1会增加细胞表面的TMEM16A水平，细胞结合实验表明CLCA1与这些细胞表面的TMEM16A结合。总之，我们的数据表明CLCA1稳定了细胞表面的TMEM16A，从而增加了表面表达，从而导致钙依赖性氯电流增加。我们的研究结果确定了CLCA蛋白家族的第一个Cl -通道靶点，并确定CLCA1是TMEM16A活性的第一个分泌的直接调节剂，描绘了一个独特的增加电流的机制。这些结果表明CLCA和TMEM16蛋白在影响多种组织的生理和多种疾病（包括哮喘、COPD、囊性纤维化和某些癌症）的病理中发挥协同作用。DOI: http://dx.doi.org/10.7554/eLife.05875.001许多对我们的健康很重要的生物过程涉及离子进出我们细胞的运动。例如，氯离子从细胞中流出控制着黏液的产生，黏液排列在我们的气管和其他气道上。这种粘液有助于在污染和其他外来颗粒到达我们的肺部之前将它们困住，从而保护肺部免受伤害。然而，在某些疾病中，如囊性纤维化和哮喘，会产生过多的粘稠粘液；这会导致呼吸困难和感染风险增加。属于CLCA蛋白家族的蛋白质最初被认为是氯离子通过细胞膜的通道。后来的研究表明，这些蛋白质不是通道；相反，它们通过激活其他通道蛋白来触发氯离子穿过细胞膜的运动。然而，这些通道蛋白的身份尚不清楚，也不清楚CLCA蛋白如何激活这些通道。Sala-Rabanal， Yurtsever等人现在已经证明，CLCA蛋白家族的一个成员CLCA1从人体细胞中释放出来，当通道检测到钙离子时，它会导致附近的细胞释放氯离子。CLCA1引发的氯离子运动看起来与氯离子通过通道蛋白TMEM16A的方式非常相似，因此Sala-Rabanal， Yurtsever等人想知道这两种蛋白是否相互作用。TMEM16A是几年前发现的，但仍然是哺乳动物中唯一已知的钙依赖性氯离子通道。Sala-Rabanal， Yurtsever等研究表明，在细胞中加入CLCA1后，细胞表面膜上出现了更多的TMEM16A通道，从而增加了氯离子的流动。CLCA蛋白还与膜上的氯离子通道发生物理相互作用以稳定膜；在此之前，还没有其他蛋白质以这种方式调节离子通道。Sala-Rabanal， Yurtsever等人的发现提供了对CLCA蛋白和氯离子通道如何工作的更清晰的理解。已知这两种蛋白质都有助于气道疾病中粘液的过量产生；两者都与心血管疾病和某些癌症有关。因此，这些新发现可能也有助于研究人员瞄准这些蛋白质并开发治疗这些疾病的方法。DOI: http://dx.doi.org/10.7554/eLife.05875.002
Calcium-activated chloride channel regulator 1 (CLCA1) activates calcium-dependent chloride currents; neither the target, nor mechanism, is known. We demonstrate that secreted CLCA1 activates calcium-dependent chloride currents in HEK293T cells in a paracrine fashion, and endogenous TMEM16A/Anoctamin1 conducts the currents. Exposure to exogenous CLCA1 increases cell surface levels of TMEM16A and cellular binding experiments indicate CLCA1 engages TMEM16A on the surface of these cells. Altogether, our data suggest that CLCA1 stabilizes TMEM16A on the cell surface, thus increasing surface expression, which results in increased calcium-dependent chloride currents. Our results identify the first Cl− channel target of the CLCA family of proteins and establish CLCA1 as the first secreted direct modifier of TMEM16A activity, delineating a unique mechanism to increase currents. These results suggest cooperative roles for CLCA and TMEM16 proteins in influencing the physiology of multiple tissues, and the pathology of multiple diseases, including asthma, COPD, cystic fibrosis, and certain cancers. DOI: http://dx.doi.org/10.7554/eLife.05875.001 Many biological processes that are important for our health involve the movement of ions into, and out of, our cells. For example, the flow of chloride ions out of cells controls the production of the sticky mucus that lines our windpipe and other airways. This mucus helps trap pollution and other foreign particles before they reach our lungs, and thus protects the lungs from harm. However in some diseases—such as cystic fibrosis and asthma—excessive amounts of thick mucus are produced; this can lead to breathing difficulties and an increased risk of infection. Proteins belonging to the CLCA protein family were first thought to act as channels that allow chloride ions to flow through cell membranes. Later studies then revealed that these proteins are not channels; instead they trigger the movement of chloride ions across cell membranes by activating other channel proteins. However, the identity of these channel proteins was unknown, and it was unclear how CLCA proteins might activate these channels. Sala-Rabanal, Yurtsever et al. have now shown that a member of the CLCA protein family, called CLCA1, is released from human cells and causes nearby cells to release chloride ions when the channel detects calcium ions. The movement of chloride ions triggered by CLCA1 looked very similar to the way chloride ions flow through a channel protein called TMEM16A, and so Sala-Rabanal, Yurtsever et al. asked whether these two proteins interact. TMEM16A was discovered several years ago, but remains the only calcium-dependent chloride channel known in mammals. Sala-Rabanal, Yurtsever et al. showed that adding CLCA1 to cells caused more TMEM16A channels to appear in the cell surface membrane and thereby increased the flow of chloride ions. The CLCA protein also physically interacted with the chloride channel in the membrane to stabilize it; no other protein has been shown to regulate ion channels in this way before. The findings of Sala-Rabanal, Yurtsever et al. provide a much clearer understanding of how the CLCA protein and the chloride channel work. Both of these proteins are known to contribute to excess mucus production in airway diseases; and both have been linked to cardiovascular diseases and certain cancers. These new findings may therefore also help researchers to target these proteins and develop treatments for these diseases. DOI: http://dx.doi.org/10.7554/eLife.05875.002