Transmembrane protein 16F (TMEM16F) is an enigmatic Ca 2+ -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 Ca 2+ -dependent activation. Strikingly, an analogous lysine mutation to TMEM16F-F518 in TMEM16A (L543K) is sufficient to confer CaPLSase activity to the Ca 2+ -activated Cl − channel (CaCC). The identification of an inner activation gate can help elucidate the gating and permeation mechanism of TMEM16 CaPLSases and channels.

SUMMARY Ca2+-activated ion channels shape membrane excitability and Ca2+ dynamics in response to cytoplasmic Ca2+ elevation. Compared to the Ca2+-activated K+ channels known as BK and SK channels, the physiological importance of Ca2+-activated Cl− channels (CaCCs) in neurons has been largely overlooked. Here we report that CaCCs coexist with BK and SK channels in inferior olivary (IO) neurons that send climbing fibers to innervate cerebellar Purkinje cells for the control of motor learning and timing. Ca2+ influx through the dendritic high-threshold voltage-gated Ca2+ channels activates CaCCs, which contribute to membrane repolarization of IO neurons. Loss of TMEM16B expression resulted in the absence of CaCCs in IO neurons, leading to markedly diminished action potential firing of IO neurons in TMEM16B knockout mice. Moreover, these mutant mice exhibited severe cerebellar motor learning deficits. Our findings thus advance the understanding of the neurophysiology of CaCCs and the ionic basis of IO neuron excitability.

Cell-cell fusion or syncytialization is fundamental to the reproduction, development, and homeostasis of multicellular organisms. In addition to various cell type–specific fusogenic proteins, cell surface externalization of phosphatidylserine (PS), a universal eat-me signal in apoptotic cells, has been observed in different cell fusion events. Nevertheless, the molecular underpinnings of PS externalization and cellular mechanisms of PS-facilitated cell-cell fusion are unclear. Here, we report that TMEM16F, a Ca2+-activated phospholipid scramblase (CaPLSase), plays an essential role in placental trophoblast fusion by translocating PS to cell surface independent of apoptosis. The placentas from the TMEM16F knockout mice exhibit deficiency in trophoblast syncytialization and placental development, which lead to perinatal lethality. We thus identified a new biological function of TMEM16F CaPLSase in trophoblast fusion and placental development. Our findings provide insight into understanding cell-cell fusion mechanism of other cell types and on mitigating pregnancy complications such as miscarriage, intrauterine growth restriction, and preeclampsia.