The TMEM16 Family of Ion Channels and Lipid Scramblases

项目摘要/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在 2008年。哺乳动物TMEM16家族有10个成员，结果令人惊讶地多样化，有家族 作为钙激活的离子通道和/或钙激活的脂质扰乱酶的成员。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，如 神经元兴奋性和体温调节的调节。事实上，哺乳动物TMEM16家族的一些成员 成员与人类疾病有关，如发热性癫痫和神经退行性变 斯科特综合症，一种出血性疾病。因此，进行分子和细胞生物学研究具有重要意义。 调查，以了解这些TMEM16家族成员的功能机制。 为了研究钙激活的氯离子通道是如何工作的，我们解决了无钙和 与钙结合的TMEM16A，并进行了结构启发的定点突变，以鉴定10孔- 衬里残留物对阴离子选择性很重要，而7个孔隙衬里残留物位于孔道狭窄附近，对阴离子选择性很重要 频道选通。然后我们展示了TMEM16B调制动作电位波形和放电模式 多个脑区。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 我们研究了TMEM16F的冷冻-EM结构，并进行了结构分析. 激发的定点突变为TMEM16F中离子渗透的不同途径提供证据 和脂肪的混杂。 为了确定TMEM16家族成员的生理重要性，我们建立了基因敲除(KO)小鼠来 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 (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)。为了监测大脑中不同细胞类型的内源性TMEM16C，我们修改了分裂 利用CRISPR介导的敲入将FLAG标签与GFP第11β链融合的GFP方法 GFP到TMEM16C的C末端，并以Cre依赖的方式表达GFP 1-10(GFP的其余部分) 在特定细胞类型中。这种方法将允许将荧光标记的内源蛋白质可视化为 以及鉴定它们的相关蛋白，以便更好地了解它们的生理功能。

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.