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

• 批准号: 8860199
• 负责人: Alessio Accardi
• 金额: $ 51.4万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 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
• 来源: https://reporter.nih.gov/project-details/8860199
• 关键词: Animals; Anions; Apoptosis; Apoptotic; Binding; Binding Sites; Biochemical; Biological Assay; Blood Platelets; Blood coagulation; Cations; Cell Line; Cell membrane; Cell physiology; Cell surface; Cells; Chimera organism; Data; Defect; Dependence; Disease; Elements; Etiology; Family; Goals; Health; Hemorrhage; Impairment; Inherited; Ion Channel; Ion Transport; Ions; Knockout Mice; Light; Lipids; Measurement; Measures; Mediating; Molecular; Mutation; Osteogenesis; Pathway interactions; Phagocytes; Phenotype; Phosphatidylserines; Phospholipids; Physiologic calcification; Physiological; Process; Proteins; Regulation; Reporting; Research Personnel; Role; Scott syndrome; Site; Surface; System; Testing; Transmembrane Domain; Work; base; design; insight; lipid transport; member; mutant; protein function; reconstitution; research study

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）通常在质膜的内部小叶中隔离，其表面暴露会触发活化血小板的血液，并标记凋亡细胞的吞噬细胞以实现吞噬细胞清除。 PS暴露是由Ca2+依赖性的脂质串联酶部分介导的，该脂质串联在质膜上翻转脂质。尽管它们在细胞生理学中的重要性，但串联酶的分子身份已避免了研究人员数十年。最近，TMEM16F是Ca2+活化的Cl-通道的TMEM16家族的成员，对Ca2+依赖性PS暴露至关重要。 TMEM16F中的突变导致Scott综合征，Scott综合征是一种与血小板中脂质有缺陷相关的遗传出血障碍。 TMEM16F无效小鼠除了显示其他缺陷外，还概括了这种疾病，例如骨矿化降低。 尽管TMEM16F对于血小板中的磷脂扰乱很重要，但其在此过程中的作用尚不清楚和有争议：它被认为是散布酶，离子通道或具有Scramblase和通道活性的双重功能蛋白。我们的长期目标是阐明TMEM16F的脂质争夺的分子碱基及其对Ca2+的调节。这些见解将使我们能够了解斯科特综合征的病因以及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 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争夺和电生理测量相结合。我们最近成功地表达，净化和功能重构TMEM16蛋白，以证明其内在的拼写酶和通道活动。我们还表明，TMEM16F在细胞中的表达确实会导致离子转运和脂质争夺。因此，我们有一个强大的初步数据平台来支持我们的方法。 我们了解脂质炒作和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受调节的跨贝脂转运的基本机制，这一过程在任何系统中都不理解。