Drosophila Subdued is a moonlighting transmembrane protein 16 (TMEM16) that transports ions and phospholipids

Abstract

Transmembrane protein 16 (TMEM16) family members play numerous important physiological roles, ranging from controlling membrane excitability and secretion to mediating blood coagulation and viral infection. These diverse functions are largely due to their distinct biophysical properties. Mammalian TMEM16A and TMEM16B are Ca 2؉ -activated Cl ؊ channels (CaCCs), whereas mammalian TMEM16F, fungal afTMEM16, and nhTMEM16 are moonlighting (multifunctional) proteins with both Ca 2؉ -activated phospholipid scramblase (CaPLSase) and Ca 2؉ -activated, nonselective ion channel (CAN) activities. To further understand the biological functions of the enigmatic TMEM16 proteins in different organisms, here, by combining an improved annexin V-based CaPLSase-imaging assay with inside-out patch clamp technique, we thoroughly characterized Subdued, a Drosophila TMEM16 ortholog. We show that Subdued is also a moonlighting transport protein with both CAN and CaPLSase activities. Using a TMEM16F-deficient HEK293T cell line to avoid strong interference from endogenous CaPLSases, our functional characterization and mutagenesis studies revealed that Subdued is a bona fide CaPLSase. Our finding that Subdued is a moonlighting TMEM16 expands our understanding of the molecular mechanisms of TMEM16 proteins and their evolution and physiology in both Drosophila and humans.

Molecular underpinning of intracellular pH regulation on TMEM16F

“…Having shown that pH i can regulate TMEM16F ion channel activity, we next sought to examine whether pH i can also influence TMEM16F lipid scrambling activity. In order to quantify TMEM16F lipid scrambling in different pH i , we established a modified fluorescence imaging assay (Le, Le and Yang, 2019; T. Le et al ., 2019) to overcome the difficulties in precisely controlling pH i and Ca 2+ i (Fig. 2A).…”

“…3A), TMEM16F-F518K and TMEM16F-Y563K (Fig. 3D) (Peters et al ., 2018; T. Le et al ., 2019). As shown in Fig.…”

“…HEK293T cell line with stable expression of C-terminally eGFP-tagged mTMEM16F was a gift from Dr. Min Li. The TMEM16F and BeWo deficient (KO) HEK293T cell line were generated using CRISPR-Cas9 and have been validated in our recent studies (Le, Le and Yang, 2019; T. Le et al ., 2019; Zhang et al ., 2020). All our mutagenesis studies were conducted in the TMEM16F-KO HEK293T cells.…”

TMEM16F phospholipid scramblase mediates trophoblast fusion and placental development

“…With more in-depth understanding of TMEM16 CaPLSases 20 and caspase-activated lipid scramblases 27 , we now understand that a viable cell with high CaPLSase expression can effectively externalize PS to cell surface 41 without triggering apoptosis 30,42,43 . Our findings thus support that under physiological conditions, cell-cell fusion preferentially happen between healthy cells instead of involving apoptotic cells.…”

“…Instead, the human trophoblasts exhibited robust CaPLSase activity measured by an optimized phospholipid scrambling assay (Figure S1A) 30 . We stimulated both BeWo and primary human term placental trophoblasts with ionomycin, a Ca 2+ ionophore (Figures 1F, S1B and Movies S1), or GSK-1016790A 31 , a potent and specific agonist of TRPV4 Ca 2+ -permeable channel (Figures 1G, S1D and Movie S2).…”