Ca2+-dependent lipid scrambling and ion transport by TMEM16 proteins

描述(申请人提供)：磷脂酰丝氨酸(PS)通常隔离在质膜的内叶，其表面暴露于激活的血小板触发血液凝结，并标记凋亡细胞进行吞噬清除。PS暴露在一定程度上是由依赖于钙离子的脂类扰乱酶介导的，这种扰乱酶将脂类翻转到质膜上。尽管它们在细胞生理学中很重要，但几十年来，研究人员一直无法确定这些扰乱酶的分子身份。最近，TMEM16F被证明在钙依赖的PS暴露中起重要作用，它是TMEM16家族的一员。TMEM16F基因突变会导致Scott综合征，这是一种遗传性出血性疾病，与血小板中有缺陷的脂质扰乱有关。TMEM16F基因缺失的小鼠除了表现出其他缺陷外，还表现出这种疾病，例如骨骼矿化减少。尽管TMEM16F对血小板中磷脂的扰乱很重要，但它在这一过程中的作用仍不清楚，也存在争议：它被认为是扰乱酶、离子通道或具有扰乱酶和通道活性的双功能蛋白。我们的长期目标是阐明TMEM16F扰乱脂质的分子基础以及钙离子对其的调节。这些见解将使我们了解Scott综合征的病因以及TMEM16F在这种疾病和其他过程中的作用。为了实现我们的总体目标，我们建议确定以下结构基础 TMEM16蛋白中的离子和脂质运输，确定TMEM16F的功能和生理作用，阐明TMEM16蛋白中钙敏感的分子基础。我们的方法是将对纯化蛋白质的生化分析与细胞中相同蛋白质的脂质扰乱和电生理测量结合起来。我们最近成功地表达、纯化和功能重组了TMEM16蛋白，以证明其内在的扰乱酶和通道活性。我们还发现，TMEM16F在细胞中的表达确实导致了离子转运和脂质扰乱。因此，我们有一个强大的初步数据平台来支持我们的方法。我们提出的了解脂质扰乱和TMEM16蛋白的生理功能的建议具有非常重要的意义，因为它将识别 斯科特综合征的基础，并阐明了调节跨双层脂质运输的基本机制，这一过程在任何系统中都不被理解。

DESCRIPTION (provided by applicant): Phosphatidylserine (PS) is normally sequestered in the inner leaflet of the plasma membrane and its surface exposure triggers blood clotting by activated platelets and marks apoptotic cells for phagocytic clearance. PS exposure is mediated in part by Ca2+-dependent lipid scramblases that flip lipids across the plasma membrane. Despite their importance in cell physiology, the molecular identity of the scramblases has eluded researchers for decades. Recently, TMEM16F, a member of the TMEM16 family of Ca2+-activated Cl- channels, was shown to be important for Ca2+-dependent PS exposure. Mutations in TMEM16F cause Scott syndrome, an inherited bleeding disorder associated with defective lipid scrambling in platelets. TMEM16F-null mice recapitulate this disorder, in addition to displaying other defects, such as decreased bone mineralization. Although TMEM16F is important for phospholipid scrambling in platelets, its role in the process remains unclear and controversial: it has been claimed to be a scramblase, an ion channel, or a dual function protein with both scramblase and channel activity. Our long-term goal is to elucidate the molecular bases of lipid scrambling by TMEM16F and its regulation by Ca2+. These insights will allow us to understand the etiology of Scott syndrome and the role of TMEM16F in this disease as well as in other processes. To achieve our overall goal we propose to identify the structural basis for ion and lipid transport in TMEM16 proteins, determine the function and physiological role of TMEM16F and elucidate the molecular basis of Ca2+ sensing in TMEM16 proteins. Our approach is to combine biochemical assays on purified proteins with lipid scrambling and electrophysiological measurements of the same proteins in cells. We recently succeeded in expressing, purifying and functionally reconstituting TMEM16 proteins to demonstrate their intrinsic scramblase and channel activities. We also showed that expression of TMEM16F in cells indeed leads to ion transport and lipid scrambling. Thus, we have a strong platform of preliminary data to support our approach. Our proposal to understand lipid scrambling and the physiological functions of TMEM16 proteins is highly significant, as it will identify the molecular basis of Scott syndrome and elucidate the fundamental mechanism of regulated transbilayer lipid transport, a process that is not understood in any system.