An inner activation gate controls TMEM16F phospholipid scrambling

Transmembrane protein 16F (TMEM16F) is an enigmatic Ca²⁺-activated phospholipid scramblase (CaPLSase) that passively transports phospholipids down their chemical gradients and mediates blood coagulation, bone development and viral infection. Despite recent advances in the structure and function understanding of TMEM16 proteins, how mammalian TMEM16 CaPLSases open and close, or gate their phospholipid permeation pathways remains unclear. Here we identify an inner activation gate, which is established by three hydrophobic residues, F518, Y563 and I612, in the middle of the phospholipid permeation pathway of TMEM16F-CaPLSase. Disrupting the inner gate profoundly alters TMEM16F phospholipid permeation. Lysine substitutions of F518 and Y563 even lead to constitutively active CaPLSases that bypass Ca²⁺-dependent activation. Strikingly, an analogous lysine mutation to TMEM16F-F518 in TMEM16A (L543K) is sufficient to confer CaPLSase activity to the Ca²⁺-activated Cl^- channel (CaCC). The identification of an inner activation gate can help elucidate the gating and permeation mechanism of TMEM16 CaPLSases and channels.