Ion permeation, lipid flipping, and membrane remodeling by TMEM16 proteins

项目概要/摘要 钙激活的氯离子通道（CaCC）和其他TMEM 16家族成员形成离子通道和/或 帮助协调大量细胞过程的脂质乱序酶。人类表达了10种不同的 标记为TMEM 16 A-K（跳跃I）的旁系同源物在整个身体中表达，并且它们有助于多种 包括血液凝固、关节炎性信号抑制、疼痛控制 通过伤害感受神经元，并调节多个大脑区域的神经元兴奋性-仅举一例。 几个这个家庭如何参与如此多不同的生理过程仍然是一个有趣的开放问题 问题创始成员（TMEM 16 A）在2008年被3个实验室（包括Jan实验室）克隆， 可能阐明上面列出的生物学作用，但也带来了解剖生物物理学的能力。 这些蛋白质的特性。在接下来的几年里，简实验室采用了诱变筛选， 电生理学和小分子筛选，以揭示离子传导，脂质扰乱和门控 除了解决高分辨率cryo-EM结构外，TMEM 16 A和F的特性（与 Cheng实验室）和TMEM 16 F（双扰频酶/离子通道）的结构。 与此同时，Grabe实验室是第一个以原子细节展示nhTMEM 16（一种真菌乱序酶）如何翻转的实验室。 通过诱导膜的大规模变形，使亲水性格罗夫附近的双层变薄， 有助于极性头基从一个小叶传递到另一个小叶。尽管取得了这些进展， 关于这些蛋白质的功能，我们打算在这里回答。第一，磷脂酰丝氨酸（PS） 通过TMEM 16 F暴露于质膜的外小叶是启动细胞凋亡的关键信号事件。 血小板依赖性凝血和微囊泡（MV）的产生;然而，没有人已经证明， TMEM 16在生理条件下在原子水平上翻转带负电荷的PS分子， TMEM 16 s的特异性知之甚少，并且已经提出乱序酶也可能 除了Grabe实验室揭示的模式外，还通过“走出凹槽”模式完成脂质翻转。 第二，我们假设Cl-传导是通过Cl-中的一个专用孔与膜屏蔽而发生的。 TMEM 16 A具有选择性CaCC，但尽管存在许多TMEM 16 A结构，但尚未显示出这一点。我们也 假设乱序酶表现出依赖于脂质选择性，因为离子在 蛋白质-膜界面。总之，我们的研究将揭示与TMEM 16 家庭成员执行一系列不同的生物现象。

Project Summary/Abstract Calcium activated Chloride Channels (CaCCs) and other TMEM16 family members form ion channels and/or lipid scramblases that help orchestrate a large number of cellular processes. Humans express 10 different paralogs labeled TMEM16A-K (skipping I) that are expressed throughout the body, and they aid in diverse phenomena including coagulation of the blood, suppression of inflammatory signals in the joints, control of pain through nociceptive neurons, and modulating neuronal excitability in multiple brain regions – just to name a few. How this family can be involved in so many different physiological processes remains an intriguing open question. The founding member (TMEM16A) was cloned by 3 labs (including the Jan lab) in 2008 making it possible to elucidate the biological roles listed above, but also ushering in the ability to dissect the biophysical properties of these proteins. In the following years, the Jan lab employed mutagenesis screens, electrophysiology, and small molecule screening to uncover the ion conduction, lipid scrambling, and gating properties of TMEM16A and F in addition to solving high resolution cryo-EM structures (in collaboration with the Cheng lab) of TMEM16A (a Cl- channel) and structures of TMEM16F (a dual scramblase/ion channel). Meanwhile, the Grabe lab was the first to show in atomic detail how nhTMEM16 (a fungal scramblase) flips lipids by inducing large-scale deformations in the membrane that thin the bilayer near a hydrophilic grove that aids polar headgroups passing from one leaflet to the other. Despite these advances, fundamental questions about the function of these proteins remain that we intend to answer here. First, phosphatidylserine (PS) exposure to the outer leaflet of the plasma membrane via TMEM16F is the key signaling event that initiates platelet-dependent coagulation and microvesicle (MV) production; however, no one has demonstrated how a TMEM16 flips a negatively charged PS molecule at the atomic level under physiological conditions, the lipid specificity of TMEM16s is poorly understood, and it has been suggested that scramblases may also accomplish lipid flipping via an “out of the groove” mode in addition to the one revealed by the Grabe lab. Second, we hypothesize that Cl- conduction occurs via a dedicated pore shielded from the membrane in Cl- selective CaCC, but despite the existence of many TMEM16A structures, this has not been shown. We also hypothesize that scramblases exhibit selectivity that is lipid-dependent because ions co-permeate with lipids at the protein-membrane interface. Together, our studies will reveal basic mechanisms related to how TMEM16 family members carry out a diverse set of biological phenomena.