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

描述（由申请方提供）：磷脂酰丝氨酸（PS）通常被隔离在质膜的内小叶中，其表面暴露通过活化血小板触发凝血，并标记凋亡细胞进行吞噬清除。PS暴露部分由Ca 2+依赖性脂质乱序酶介导，所述脂质乱序酶使脂质翻转穿过质膜。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乱序酶的分子身份。最近，TMEM 16 F，TMEM 16家族的Ca 2+激活的Cl-通道的成员，被证明是重要的Ca 2+依赖性PS暴露。TMEM 16 F突变导致斯科特综合征，这是一种与血小板中脂质紊乱缺陷相关的遗传性出血性疾病。TMEM 16 F缺失小鼠重现了这种疾病，此外还显示出其他缺陷，如骨矿化减少。 尽管TMEM 16 F对于血小板中的磷脂扰乱是重要的，但其在该过程中的作用仍然不清楚且有争议：它已被声称是扰乱酶、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TMEM 16 F的脂质扰乱及其由Ca 2+调节的分子基础。这些见解将使我们能够了解斯科特综合征的病因以及TMEM 16 F在这种疾病以及其他过程中的作用。 为了实现我们的总体目标，我们建议确定以下结构基础： TMEM 16蛋白质中的离子和脂质转运，确定TMEM 16 F的功能和生理作用，并阐明TMEM 16蛋白质中Ca 2+感应的分子基础。我们的方法是结合联合收割机生化分析纯化的蛋白质与脂质扰乱和细胞中相同的蛋白质的电生理测量。我们最近成功地表达，纯化和功能重建TMEM 16蛋白，以证明其内在的扰码酶和通道活动。我们还表明，TMEM 16 F在细胞中的表达确实导致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。 我们提出的理解脂质混乱和TMEM 16蛋白的生理功能的建议是非常重要的，因为它将确定TMEM 16蛋白的分子结构。 斯科特综合征的基础，并阐明调节跨双层脂质转运的基本机制，这是一个过程，是不理解的任何系统。

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.