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

TMEM16完整膜蛋白家族的成员是钙依赖的磷脂扰乱酶。 由于TMEM16的脂质扰乱机制仍然知之甚少，因此解释 它们在人体生理学中的作用，并设计有针对性的药物干预措施，选择性地 操纵这些蛋白质的活性，是有限的。我们的目标是通过确定 TMEM16加扰酶是如何响应钙结合而被激活的，它们是如何被激活的以及它们的目的是什么 重塑细胞膜，以及这些膜的特定成分如何影响它们。我们 通过将实验与结构、功能、 和计算方法。为了了解这些蛋白质在体内是如何调节的，我们将重点放在 神经酰胺作为第一类分子被发现抑制TMEM16扰乱酶的功能，并被 在体内与内皮细胞中PS的过度暴露有关。我们的第一个目标是确定钙离子- 结合低温电子显微镜研究TMEM16扰码的依赖选通机制 (低温电子显微镜)、分子动力学(MD)模拟和功能分析。这些实验将揭示 钙结合部位与脂质门控结构元件的变构偶联机制 路径。我们的第二个目标是确定TMEM16加扰酶如何与TMEM16相互作用，并改变其结构， 它们周围的膜环境支持着它们的功能。使用结构确定与 我们将可视化afTMEM16与具有各种物理化学性质的膜的复合体 和组合物，处于不同的功能状态。结合MD模拟和功能分析，我们 将确定膜-蛋白质相互作用和膜的能量和分子决定因素 重塑，以及它们在争先恐后中的作用。我们的第三个目标是确定其机制和在体内的作用 神经酰胺对TMEM16扰乱酶的调节作用 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TMEM16F体内调控中的作用

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.