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

描述（由申请人提供）：磷脂酰丝氨酸（PS）通常被隔离在质膜的内小叶中，其表面暴露会通过活化的血小板触发血液凝固，并标记凋亡细胞进行吞噬清除。PS暴露部分是由Ca2+依赖性脂质超燃酶介导的，该酶使脂质在质膜上翻转。尽管它们在细胞生理学中具有重要意义，但几十年来，研究人员一直没有弄清楚它们的分子身份。最近，TMEM16F是Ca2+激活Cl-通道TMEM16家族的成员，被证明对Ca2+依赖性PS暴露很重要。TMEM16F突变导致斯科特综合征，这是一种与血小板脂质紊乱缺陷相关的遗传性出血性疾病。tmem16f缺失的小鼠除了表现出骨矿化减少等其他缺陷外，还重现了这种疾病。尽管TMEM16F对血小板中磷脂的混乱很重要，但其在这一过程中的作用仍不清楚且存在争议：有人声称它是一种混乱酶、离子通道或具有混乱酶和通道活性的双重功能蛋白。我们的长期目标是阐明TMEM16F对脂质扰乱的分子基础及其受Ca2+的调控。这些见解将使我们能够了解斯科特综合征的病因学以及TMEM16F在该疾病以及其他过程中的作用。为了实现我们的总体目标，我们建议确定的结构基础

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.