The ubiquitous TMEM16F Ca 2+ -activated channel and scramblase is the primary mediator of phosphatidylserine externalization in activated platelets, lung alveoli, and microglia. It plays vital roles in physiological processes like blood coagulation, membrane fusion, and brain immune surveillance. The molecular mechanisms underlying TMEM16F activation remain poorly understood. No conformation explaining its dual activity is known. Here, we obtained high-resolution cryogenic electron-microscopy structures of TMEM16F in liposomes where it functions as a channel and scramblase. In high activity conditions, TMEM16F adopts two conformations, the canonical Ca 2+ -bound closed state and one where the upward rotation of the cytosolic ferredoxin domain leads to the disengagement of TM4 from TM3 resulting in an X-shaped groove which forms a transmembrane pore and locally thins the membrane. Using mutagenesis, functional assays, and molecular dynamics simulations we show that this active conformation mediates non-selective ion flux and lipid scrambling. Strikingly, the two substrates take physically distinct pathways: ions move within the protein-delimited pore whereas lipids move outside the X-shaped groove. Our findings provide a complete picture of TMEM16F Ca 2+ -dependent gating, with implications for other family members. They also demonstrate that imaging membrane proteins in a native-like environment can allow sampling of active states inaccessible in non-native conditions.
Feng et al. (2026) studied this question.