The ubiquitous transmembrane protein 16F (TMEM16F) Ca2+-activated channel and scramblase catalyzes phosphatidylserine externalization to enable blood coagulation, membrane fusion and brain immune surveillance. Despite its importance, the molecular mechanisms underlying TMEM16F activation remain poorly understood. Here, we obtained high-resolution cryo-electron microscopy structures of TMEM16F active in liposomes. In high-activity conditions, TMEM16F adopts two conformations, the canonical Ca2+-bound closed state and one where the upward rotation of the cytosolic domain leads to an X-shaped groove that forms a transmembrane pore and locally thins the membrane. Using mutagenesis, functional assays and molecular dynamics simulations, we show that the X-shaped groove is active and mediates nonselective ion flux and lipid scrambling through distinct pathways; ions move within the protein-delimited pore, whereas lipids skirt the X-shaped groove. Our findings provide a complete picture of TMEM16F Ca2+-dependent gating and demonstrate that imaging membrane proteins in a native-like environment can allow capturing otherwise inaccessible active states. Feng, Alvarenga et al. use cryo-electron microscopy to visualize the activation of the transmembrane protein 16F channel and scramblase in liposomes to show that it adopts a conformation that forms separate pathways for ions and lipids, thereby rationalizing its dual activity.
Feng et al. (Fri,) studied this question.