1.A.17.1.4
Anoctamin-6 (ANO6: TMEM16F) Ca²⁺-dependent phospholipid scramblase (flippase) (Suzuki et al., 2010; Chauhan et al. 2016). Defects cause Scott syndrome, and promote assembly of the tenase and prothrombinase complexes involved in blood coagulation (Fujii et al. 2015). It is an essential component of the outwardly rectifying chloride channel (Martins et al., 2011; Keramidas and Lynch 2012).  It has also been reported to be an anion channel with delayed Ca²⁺ activation (Adomaviciene et al. 2013) as well as a Ca²⁺-activated cation channel with activity that is required for lipid scrambing (Yang et al. 2012).  However, Suzuki et al. (2013) showed that TMEM16F is a Ca²⁺-dependent phospholipid scramblase that exposes phosphatidylserine (PS) to the cell surface but lacks calcium-dependent chloride channel activity (see also Segawa et al. 2011).  TMEM16C, 16D, 16G and 16J also have Ca²⁺-dependent scramblase activities but not channel activity (Suzuki et al. 2013).  The pore region suggested to be resonsible for Cl^- transport in TMEM16A is also responsible for phospholipid scramblase activity (Suzuki et al. 2014).  Anoctamin-6 (Ano6) plays an essential role in C2C12 myoblast proliferation, probably by regulating the ERK/AKT signaling pathway (Zhao et al. 2014).  It regulates baeline phosphatidyl serine exposure and cell viability in human embryonic kidney cells (Schenk et al. 2016). A single TMEM16F molecule transports phospholipids nonspecifically between the membrane bilayers dependent on Ca²⁺. Thermodynamic analyses indicated that TMEM16F transports 4.5 x 10⁴ lipids per second at 25 degrees C, with an activation free energy of 47 kJ/mol, suggesting a channel-dependent, facilitated diffusion,"stepping-stone" mechanism (Watanabe et al. 2018). TMEM16F plays roles in platelet activation during blood clotting, bone formation, and T cell activation. Activation of TMEM16F by Ca²⁺ ionophores triggers large-scale surface membrane expansion in parallel with phospholipid scrambling (Bricogne et al. 2019). With continued ionophore application,TMEM16F-expressing cells undergo extensive shedding of ectosomes which incorporate The T cell co-receptor PD-1. Cells lacking TMEM16F fail to expand the surface membrane in response to elevated cytoplasmic Ca²⁺and instead undergo endocytosis with PD-1 internalization. This suggests a new role for TMEM16F as a regulator of Ca²⁺-activated membrane trafficking (Bricogne et al. 2a019). The inner activation gate consists of  three hydrophobic residues, F₅₁₈, Y₅₆₃ and I₆₁₂, in the middle of the phospholipid permeation pathway. Disrupting the inner gate profoundly alters phospholipid permeation. Lysine substitutions of F518 and Y563 lead to constitutively active CaPLSases that bypass Ca²⁺-dependent activation. An analogous lysine mutation to TMEM16F-F518 in TMEM16A (L543K) is sufficient to confer CaPLSase activity to the Ca²⁺-activated Cl^- channel (CaCC) (Le et al. 2019).  ANO6, by virtue of its scramblase activity, may play a role as a regulator of the ADAM-network in the plasma membrane. TMEM16F inhibition limits pain-associated behavior and improves motor function by promoting microglia M2 polarization in mice (Zhao and Gao 2019). Polyphenols do not inhibit the phospholipid scramblase activity of TMEM16F (Le et al. 2020). TMEM16F is a ubiquitously expressed Ca²⁺-activated phospholipid scramblase that also functions as a largely non-selective ion channel with open, closed and intermediate conformations (Jia et al. 2022). An inner activation gate consists of F518, Y563, and I612, and  charged mutations of the inner gate residues leads to constitutively active mammalian (m)TMEM16F scrambling. Lysine substitution of F518 and Y563 leads to spontaneous opening of the permeation pore in the Ca²⁺-bound state of mTMEM16F. Dilation of the pore exposes hydrophilic patches in the upper pore region, greatly increases the pore hydration level, and enables lipid scrambling (Jia et al. 2022).