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

TMEM 16家族的膜整合蛋白的成员是Ca 2+依赖性磷脂乱序酶。 由于对TMEM 16的脂质扰乱机制仍然知之甚少，因此解释TMEM 16的能力仍然很低。 它们在人体生理学中的功能，并设计有针对性的药理学干预措施， 操纵这些蛋白质的活性是有限的。我们的目标是通过确定 TMEM 16扰码酶如何响应于Ca 2+结合而被激活，它们如何以及出于什么目的被激活， 重塑细胞膜，以及它们如何受到这些膜的特定成分的影响。我们 解决这一机械目标与综合战略相结合的实验与结构，功能， 和计算方法。为了了解这些蛋白质是如何在体内调节的，我们将重点关注 神经酰胺作为第一类被发现抑制TMEM 16乱序酶功能的分子， 与内皮细胞中PS的过度暴露相关。我们的第一个目标是确定Ca 2 +- 使用低温电子显微镜组合的TMEM 16乱序酶的依赖门控机制 （cryoEM）、分子动力学（MD）模拟和功能测定。这些实验将揭示 钙离子结合位点和脂质门控结构元件之间的变构偶联机制 通路我们的第二个目标是确定TMEM 16扰码如何相互作用，并改变结构， 它们周围的膜环境支持它们的功能。使用结构测定， cryoEM我们将可视化afTMEM 16复合物与膜的各种物理化学性质 和组合物。结合MD模拟和功能测定， 将确定膜蛋白相互作用和膜的能量和分子决定因素 重塑以及它们在争夺中的作用。我们的第三个目标是确定的机制和体内作用， 神经酰胺调节TMEM 16乱序酶，使用功能测定来鉴定神经酰胺的分子决定簇。 神经酰胺抑制，以及确定其作用机制的结构和计算实验，以及 特异性神经酰胺在TMEM 16 F体内调节中的作用。

Members of the TMEM16 family of integral membrane proteins are Ca2+-dependent phospholipid scramblases. Because mechanisms of lipid scrambling by the TMEM16s remain poorly understood, the ability to interpret their function in human physiology and to design targeted pharmacological interventions that would selectively manipulate the activity of these proteins, is limited. Our goal is to overcome these limitations by determining how the TMEM16 scramblases are activated in response to Ca2+ binding, how and for what purpose they remodel cellular membranes, and how they are affected by specific components of these membranes. We address this mechanistic goal with an integrated strategy combining experimentation with structural, functional, and computational approaches. To understand how these proteins are modulated in vivo we will focus on ceramides as the first class of molecules found to inhibit the function of TMEM16 scramblases and to be associated in vivo with excessive exposure of PS in endothelial cells. Our 1st aim is to determine the Ca2+- dependent gating mechanism of the TMEM16 scramblases using a combination of cryo-electron microscopy (cryoEM), molecular dynamics (MD) simulations and functional assays. These experiments will reveal the allosteric coupling mechanism between the Ca2+ binding site and the structural elements gating the lipid pathway. Our 2nd aim is to determine how the TMEM16 scramblases interact with, and alter the structure of, their surrounding membrane environment in support of their function. Using structure determination with cryoEM we will visualize afTMEM16 complexes with membranes with a variety of physicochemical properties and compositions, in different functional states. In combination with MD simulations and functional assays we will identify the energetic and molecular determinants for membrane-protein interactions and membrane remodeling, and their role in scrambling. Our 3rd aim is to determine the mechanism and in vivo role of ceramide regulation of TMEM16 scramblases using functional assays to identify the molecular determinants of ceramide inhibition, and structural and computational experiments to determine their mechanism of action, and the role of specific ceramides in the in vivo regulation of TMEM16F.