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

TMEM16 蛋白的 Ca2 依赖性脂质扰乱和离子传输

基本信息

批准号：
8728513
负责人：
Alessio Accardi
金额：
$ 57.42万
依托单位：
WEILL MEDICAL COLL OF CORNELL UNIV
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-06-04 至 2018-02-28
项目状态：
已结题

项目摘要

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.

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叶中，其表面暴露会引发活化血小板的血液凝固，并标记凋亡细胞以进行吞噬清除。 PS 暴露部分由 Ca2+ 依赖性脂质扰乱酶介导，这些脂质扰乱酶可翻转脂质穿过质膜。尽管它们在细胞生理学中很重要，但数十年来研究人员一直未能弄清楚它们的分子身份。最近，TMEM16F（Ca2+激活的Cl-通道TMEM16家族的成员）被证明对于Ca2+依赖性PS暴露很重要。 TMEM16F 突变会导致斯科特综合征，这是一种与血小板中脂质扰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其他缺陷（例如骨矿化减少）外，还重现了这种疾病。尽管 TMEM16F 对于血小板中的磷脂扰乱很重要，但其在该过程中的作用仍不清楚且存在争议：它被声称是扰乱酶、离子通道或同时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 脂质扰乱的分子基础及其 Ca2+ 的调节。这些见解将使我们能够了解 Scott 综合征的病因以及 TMEM16F 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： TMEM16 蛋白中的离子和脂质转运，确定 TMEM16F 的功能和生理作用，并阐明 TMEM16 蛋白中 Ca2+ 传感的分子基础。我们的方法是将纯化蛋白质的生化测定与细胞中相同蛋白质的脂质加扰和电生理学测量结合起来。我们最近成功表达、纯化和功能重建 TMEM16 蛋白，以证明其内在的扰乱酶和通道活性。我们还表明，细胞中 TMEM16F 的表达确实会导致离子转运和脂质扰乱。因此，我们拥有强大的初步数据平台来支持我们的方法。我们关于了解脂质扰乱和 TMEM16 蛋白的生理功能的建议非常重要，因为它将识别分子斯科特综合征的基础，并阐明了调节跨双层脂质转运的基本机制，这是任何系统中尚不了解的过程。