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.

项目概要/摘要 钙激活氯离子通道 (CaCC) 和其他 TMEM16 家族成员形成离子通道和/或 脂质扰乱有助于协调人类表达 10 种不同的细胞过程。 标记为 TMEM16A-K（跳过 I）的旁系同源物在全身表达，它们有助于多种功能 包括血液凝固、抑制关节炎症信号、控制疼痛等现象 通过伤害性神经元，调节多个大脑区域的神经元兴奋性——仅举一个例子 这个家族如何参与如此多不同的生理过程仍然是一个有趣的问题。 创始成员（TMEM16A）是由 3 个实验室（包括 Jan 实验室）于 2008 年克隆的。 可能阐明上面列出的生物学作用，但也带来了剖析生物物理的能力 在接下来的几年里，Jan 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 除了解决高分辨率冷冻电镜结构之外，TMEM16A 和 F 的特性（与 Cheng 实验室）的 TMEM16A（Cl-通道）和 TMEM16F（双扰乱酶/离子通道）的结构。 与此同时，Grabe 实验室率先展示了 nhTMEM16（一种真菌扰乱酶）如何翻转的原子细节 通过诱导膜的大规模变形，使亲水树林附近的双层变薄，从而减少脂质的产生 尽管取得了这些进展，但仍存在一些基本问题。 关于这些蛋白质的功能，我们首先要回答的是磷脂酰丝氨酸（PS）。 通过 TMEM16F 暴露于质膜外层是启动的关键信号事件 然而，没有人证明血小板依赖性凝血和微泡（MV）的产生是如何发生的。 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 的特异性尚不清楚，有人认为扰乱酶也可能 除了 Grabe 实验室揭示的模式之外，还通过“out of thegroove”模式实现脂质翻转。 其次，我们发现 Cl- 传导是通过与 Cl- 膜屏蔽的专用孔发生的。 选择性 CaCC，但尽管存在许多 TMEM16A 结构，但我们还没有证明这一点。 浸泡后，扰乱酶表现出依赖脂质的选择性，因为离子与脂质在 我们的研究将共同​​揭示 TMEM16 的基本机制。 家庭成员进行一系列不同的生物现象。