The TMEM16 Family of Ion Channels and Lipid Scramblases

TMEM16 离子通道和脂质扰乱系列

• 批准号: 10397634
• 负责人: LILY Y JAN
• 金额: $ 53.13万
• 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: 2021-05-01 至 2029-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10397634
• 关键词: Action Potentials; Anions; Biological; Blood Coagulation Disorders; Brain; Brain region; Calcium; Calcium Binding; Calcium Channel; Cations; Cells; Chloride Channels; Clustered Regularly Interspaced Short Palindromic Repeats; Cryoelectron Microscopy; Family; Family member; Febrile Convulsions; Integral Membrane Protein; Investigation; Ion Channel; Ions; Knock-in; Knockout Mice; Learning; Lipids; Mediating; Molecular; Monitor; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Nociceptors; Pathway interactions; Pattern; Physiologic Thermoregulation; Physiological; Potassium Channel; Proteins; Rest; Scott syndrome; Site-Directed Mutagenesis; Sodium; Spinal Ganglia; Spinocerebellar Ataxias; Structure; Visualization; Work; cell type; constriction; extracellular vesicles; human disease; member; mouse model; neuronal excitability; neurotransmission; pain sensitivity

Project Summary/Abstract To initiate molecular characterization of the CaCC calcium-activated chloride channels that have been found in multiple neuronal types since 1980s, we first showed that CaCC is formed by TMEM16A or TMEM16B in 2008. The mammalian TMEM16 family with ten members turns out to be surprisingly diverse, with family members acting as calcium-activated ion channels and/or calcium-activated lipid scramblase. The TMEM16 family members provide a variety of activities in central neurons to serve important functions such as modulation of neuronal excitability and thermoregulation. Indeed, some of the mammalian TMEM16 family members have been associated with human diseases such as febrile seizure and neurodegeneration as well as Scott syndrome, a bleeding disorder. Therefore, it will be important to conduct molecular and cell biological investigations to learn about the mechanisms that underlie the functions of these TMEM16 family members. To ask how the calcium-activated chloride channel works, we solved cryo-EM structures of calcium-free and calcium-bound TMEM16A and carried out structure-inspired site-directed mutagenesis to identify 10 pore- lining residues important for anion selectivity and 7 pore-lining residues near pore constrictions important for channel gating. We then showed that TMEM16B modulates action potential waveform and firing patterns in multiple brain regions. To ask how TMEM16F fulfils the dual functions of calcium-activated ion channel and calcium-activated lipid scramblase, we examined cryo-EM structures of TMEM16F and conducted structure- inspired site-directed mutagenesis to provide evidence for separate pathways in TMEM16F for ion permeation and lipid scrambling. To establish the physiological importance of TMEM16 family members, we generated knockout (KO) mice to show that they provide mouse models for human diseases, such as the bleeding disorder Scott syndrome (TMEM16F), febrile seizure (TMEM16C), and the progressive neurodegenerative disease spinocerebellar ataxia (TMEM16K). To monitor endogenous TMEM16C in various cell types in the brain, we modified the split GFP approach by using CRISPR mediated knock-in to fuse the FLAG tag along with the 11th beta strand of GFP to the C-terminus of TMEM16C, and expressing GFP1-10 (the rest of GFP) in a Cre-dependent manner in specific cell types. This approach will allow visualization of fluorescently tagged endogenous proteins as well as identification of their associated proteins for better understanding of their physiological functions.

项目概要/摘要 启动已发现的 CaCC 钙激活氯离子通道的分子表征 自 20 世纪 80 年代以来，在多种神经元类型中，我们首次证明 CaCC 是由 TMEM16A 或 TMEM16B 形成的 2008. 拥有 10 名成员的哺乳动物 TMEM16 家族具有惊人的多样性， 充当钙激活离子通道和/或钙激活脂质扰乱酶的成员。 TMEM16 家庭成员在中枢神经元中提供多种活动来服务重要功能，例如 神经元兴奋性和体温调节的调节。事实上，一些哺乳动物 TMEM16 家族 成员还与热性惊厥和神经退行性疾病等人类疾病有关 斯科特综合症，一种出血性疾病。因此，开展分子和细胞生物学研究非常重要。 调查以了解这些 TMEM16 家族成员功能的机制。 为了探究钙激活氯离子通道的工作原理，我们解决了无钙和 钙结合 TMEM16A 并进行结构启发的定点诱变以鉴定 10 个孔 对阴离子选择性很重要的内衬残基和对孔收缩很重要的 7 个孔内衬残基 通道选通。然后我们证明 TMEM16B 可以调节动作电位波形和放电模式 多个大脑区域。询问TMEM16F如何实现钙激活离子通道和钙激活离子通道的双重功能 钙激活脂质扰乱酶，我们检查了 TMEM16F 的冷冻电镜结构并进行了结构- 激发定点诱变，为 TMEM16F 离子渗透的单独途径提供证据 和脂质扰乱。 为了确定 TMEM16 家族成员的生理重要性，我们生成了基因敲除 (KO) 小鼠 表明他们为人类疾病提供了小鼠模型，例如出血性疾病斯科特综合症 (TMEM16F)、热性惊厥 (TMEM16C) 和进行性神经退行性疾病脊髓小脑 共济失调（TMEM16K）。为了监测大脑中各种细胞类型的内源性 TMEM16C，我们修改了分裂 GFP 方法通过使用 CRISPR 介导的基因敲入将 FLAG 标签与 11 条 β 链融合 GFP 连接到 TMEM16C 的 C 末端，并以 Cre 依赖性方式表达 GFP1-10（GFP 的其余部分） 在特定的细胞类型中。这种方法将允许荧光标记的内源蛋白质的可视化 以及鉴定其相关蛋白质，以更好地了解其生理功能。