Browsing by Author "Yang, Huanghe"

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.

The TMEM16 protein family comprises two novel classes of structurally conserved but functionally distinct membrane transporters that function as Ca2+-dependent Cl- channels (CaCCs) or dual functional Ca2+-dependent ion channels and phospholipid scramblases. The TMEM16A and TMEM16B CaCCs conduct Cl- across the membrane and regulate transepithelial fluid transport, smooth muscle contraction, neuronal excitability and sensory signal transduction. Most other TMEM16 members are scramblases that mediate the flip-flop of phospholipids across the membrane to allow phosphatidylserine externalization, which is essential in a plethora of important processes such as blood coagulation, bone development and viral and cell fusion. Activation of TMEM16 proteins requires the binding of intracellular Ca2+ to two highly conserved orthosteric binding sites in transmembrane helices (TM) 6-8 opens the permeation pathway formed by TMs 3-7. However, prolonged Ca2+-dependent simulation of TMEM16 channels results in current desensitization or rundown, the mechanism of which is unclear. In addition, recent structural studies of TMEM16 scramblases revealed yet another Ca2+ binding site in TM2 and TM10 whose functional relevance remains unknown. By combining electrophysiology, systematic mutagenesis and molecular dynamics simulation, we first demonstrate that the phosphatidylinositol-(4,5)-bisphosphate (or PI(4,5)P2) plays a critical role in TMEM16A’s Ca2+-dependent activation and desensitization. We identify key basic residues at the cytosolic interface of transmembrane segments (TMs) 3–5 as the putative PI(4,5)P2-binding site, which is supported by our molecular dynamics studies. These studies reveal that TMEM16A is constituted of two functionally distinct modules: a Ca2+-binding module formed by TMs 6–8 and a PI(4,5)P2-binding regulatory module formed by TMs 3–5, which mediate channel activation and desensitization, respectively. Next, we show that Ca2+ binds with high affinity to the putative third Ca2+ site in TM2 and TM10 of TMEM16A to enhance channel activation. Our cadmium (Cd2+) metal bridging experiments further show that the third Ca2+ site’s conformational states can profoundly influence TMEM16A’s opening. Taken together, our studies not only establish the molecular bases for the PI(4,5)P2-dependent regulation of TMEM16A and the long-range allosteric gating mechanism via the third Ca2+ binding site; they also provide functional insight into the structural organization of TMEM16 ion channels and lipid scramblases.