The TMEM16 Family of Ion Channels and Lipid Scramblases

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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.