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

TMEM16 蛋白的离子渗透、脂质翻转和膜重塑

• 批准号: 10531602
• 负责人: Michael Grabe
• 金额: $ 35.69万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-01 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10531602
• 关键词: Anions; Binding; Binding Sites; Biological; Biological Phenomena; Blood Coagulation Disorders; Blood Platelets; Blood coagulation; Brain region; Calcium; Cell membrane; Cell physiology; Cells; Charge; Chloride Channels; Coagulation Process; Collaborations; Cryoelectron Microscopy; Data; Dedications; Development; Disease; Electrophysiology (science); Event; Exhibits; Exposure to; Family; Family member; Functional disorder; Human; Immune response; Inflammatory; Inflammatory Arthritis; Ion Channel; Ions; Joints; Kinetics; Knowledge; Label; Life; Lipid Bilayers; Lipid Binding; Lipids; Malignant Neoplasms; Measures; Membrane; Membrane Proteins; Modeling; Molecular; Molecular Conformation; Muscular Dystrophies; Mutagenesis; Names; Neurons; Nociception; Nociceptors; Pain management; Pathway interactions; Permeability; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylserines; Physiological; Physiological Processes; Physiology; Play; Production; Property; Proteins; Protocols documentation; Publishing; Resolution; Role; Sampling; Scott syndrome; Signal Transduction; Site; Site-Directed Mutagenesis; Specificity; Stroke; Structure; Testing; Thinness; Tissues; Vesicle; biophysical properties; design; experimental study; hydrophilicity; insight; link protein; member; microvesicles; mutant; neuronal excitability; novel therapeutics; paralogous gene; protein function; screening; simulation; small molecule; structural determinants; targeted treatment; tumor progression; voltage

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.

项目摘要/摘要 钙激活的氯化物通道（CACC）和其他TMEM16家庭成员形成离子通道和/或 脂质的串联酶有助于协调大量的细胞过程。人类表达10个不同 标记为TMEM16A-K（跳过I）的旁系同源物，在整个身体中表达，它们有助于潜水员 现象包括血液凝结，关节中炎症信号的抑制，控制疼痛 通过伤害性神经元，并在多个大脑区域调节神经元令人兴奋 - 仅举一个 很少。这个家庭如何参与这么多不同的身体过程仍然是一个有趣的公开 问题。创始成员（TMEM16A）于2008年由3个实验室（包括Jan Lab）克隆 可以阐明上述生物学作用 这些蛋白质的特性。在接下来的几年中，Jan实验室采用了诱变筛选， 电生理学和小分子筛选，以发现离子传导，脂质争夺和门控 除了解决高分辨率冷冻结构之外，TMEM16A和F的属性（与 TMEM16A（Cl-通道）的Cheng Lab）和TMEM16F（双重拼写酶/离子通道）的结构。 同时，Grabe Lab是第一个显示原子详细信息的一个NHTMEM16（真菌拼写板）翻转 通过诱导的大规模变形在膜上脂质的脂质，该膜在亲水树枝附近的双层薄薄的脂质变形 AIDS极性头组从一个传单到另一个传单。尽管有这些进展，但基本问题 关于这些蛋白质的功能，我们打算在这里回答。首先，磷脂酰丝氨酸（PS） 通过TMEM16F暴露于质膜的外叶是启动的关键信号事件 血小板依赖性凝血和微泡（MV）产生；但是，没有人证明 TMEM16在生理条件下在原子水平上翻转带负电荷的PS分子，脂质 TMEM16S的特异性知之甚少，并且有人提出Scramblases也可能 除了Grabe Lab所揭示的脂质模式外，已完成的脂质通过“凹槽的凹槽”模式进行了翻转。 其次，我们假设Cl-传导是通过从Cl-中的膜上屏蔽的专用孔进行的 选择性CACC，但希望存在许多TMEM16A结构的存在，但尚未显示。我们也是 假设Scramblase暴露了脂质依赖性的选择性，因为离子与脂质在 蛋白质膜界面。总之，我们的研究将揭示与TMEM16的基本机制 家庭成员进行一系列生物学现象。