The TMEM16 Family of Ion Channels and Lipid Scramblases

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，我们首次表明CaCC由TMEM 16 A或TMEM 16 B形成， 2008.哺乳动物TMEM 16家族有10个成员，其多样性令人惊讶， 充当钙激活离子通道和/或钙激活脂质乱序酶的成员。TMEM16 家族成员在中枢神经元中提供各种活动，以发挥重要功能， 调节神经元兴奋性和体温调节。事实上，一些哺乳动物TMEM 16家族 成员也与人类疾病如热性惊厥和神经变性有关 斯科特综合征，一种出血性疾病因此，进行分子和细胞生物学研究将是非常重要的。 研究以了解这些TMEM 16家族成员的功能机制。 为了了解钙激活的氯离子通道是如何工作的，我们解决了无钙和 钙结合的TMEM 16 A，并进行结构启发的定点诱变，以确定10个孔， 内衬残留物对阴离子选择性很重要，7孔收缩附近的孔内衬残留物对 通道选通然后，我们表明TMEM 16 B调节动作电位波形和放电模式， 多个大脑区域为了了解TMEM 16 F是如何实现钙激活离子通道的双重功能， 钙激活的脂质乱序酶，我们检查了TMEM 16 F的冷冻-EM结构，并进行了结构- 启发定点诱变，为TMEM 16 F中离子渗透的单独途径提供证据 和脂质紊乱。 为了确定TMEM 16家族成员的生理重要性，我们产生敲除（KO）小鼠， 表明它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征 TMEM 16 F、热性惊厥（TMEM 16 C）和进行性神经退行性疾病脊髓小脑 共济失调（TMEM 16 K）。为了监测脑中各种细胞类型中的内源性TMEM 16 C，我们修改了分裂 GFP方法通过使用CRISPR介导的敲入将FLAG标签沿着与第11条β链融合， GFP至TMEM 16 C的C末端，并以Cre依赖性方式表达GFP 1 -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性蛋白质的可视化， 并鉴定其相关蛋白，以更好地了解其生理功能。

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.