Investigating the Molecular Mechanisms of TMEM16F – a Ca2+ Activated Phospholipid Scramblase and Ion Channel

Abstract

Transmembrane protein 16 (TMEM16) is a novel family of transmembrane proteins that function either as ion channels, lipid scramblases or both. In mammals, the majority of TMEM16 members are Ca2+-dependent phospholipid scramblases (CaPLSases) that catalyze bidirectional movement of phospholipids across the membrane bilayer. Interestingly, some of these TMEM16 CaPLSases can also conduct ions, making them multifunctional (moonlighting) transporters. These moonlighting TMEM16 members have been linked to various physiological and pathological conditions, such as blood coagulation, ataxia, muscle dystrophy, cell-cell fusion and viral infection.To further understand their biology and design therapeutics to treat the related diseases, it is urgent to unveil the structures, machineries as well as pharmacological profiles of the multifunctional TMEM16 proteins. However, studying TMEM16 proteins has been challenging due to their unique structural topologies and biophysical properties. Despite the recent progress in the structure and function understanding of the TMEM16 family, how the moonlighting TMEM16s gate and distinguish different permeating substrates remain open questions. To resolve these unknowns and contribute to a more comprehensive understanding of the multifunctional TMEM16 proteins, this dissertation focuses on investigating the molecular mechanisms of TMEM16F – the first identified moonlighting member of the TMEM16 family. We first developed a sensitive and reliable fluorescence microscopy-based scrambling assay that can be either used alone to assess TMEM16F CaPLSase activity or combined with electrophysiology to simultaneously examine TMEM16F CaPLSase and ion channel components (Chapter 2). Next, by applying our optimized scrambling assay together with computational simulation, mutagenesis screening and electrophysiology approaches, we uncovered the gating mechanism of TMEM16F and revealed the differences in protein conformation between TMEM16 -CaPLSases and -ion channels (Chapter 3). Furthermore, during our drug screening to identify antagonists for TMEM16F CaPLSase, we made a surprising discovery about the potential pitfalls of using fluorescence-based assay that could cause false positive results and challenge the identification of bona fide inhibitors for the CaPLSases (Chapter 4). Finally, our discovery of Subdued – a TMEM16 fly homolog – as a new moonlighting protein with similar biophysical properties to those of TMEM16F further expands our knowledge about the diversity and relationship among TMEM16 members (Chapter 5). In summary, this dissertation advances the current understanding of the molecular underpinning and diverse functions of the TMEM16 family in general, and TMEM16F in particular.