Regulation of TMEM16A/ANO1 and TMEM16F/ANO6 ion currents and phospholipid scrambling by Ca2+ and plasma membrane lipid

Key points TMEM16 proteins can operate as Ca2+‐activated Cl− channels or scramble membrane phospholipids, which are both highly relevant mechanisms during disease. Overexpression of TMEM16A and TMEM16F were found to be partially active at 37°C and at resting intracellular Ca2+ concentrations. We show that TMEM16 Cl− currents and phospholipid scrambling can be activated by modification of plasma membrane phospholipids, through reactive oxygen species and phospholipase A2. While phospholipids and Cl− ions are likely to use the same pore within TMEM16F, TMEM16A only conducts Cl− ions. Lipid regulation of TMEM16 proteins is highly relevant during inflammation and regulated cell death such as apoptosis and ferroptosis. Abstract TMEM16/anoctamin (ANO) proteins form Ca2+‐activated ion channels or phospholipid scramblases. We found that both TMEM16A/ANO1 and TMEM16F/ANO6 produced Cl− currents when activated by intracellular Ca2+, but only TMEM16F was able to expose phosphatidylserine to the outer leaflet of the plasma membrane. Mutations within TMEM16F or TMEM16A/F chimeras similarly changed Cl− currents and phospholipid scrambling, suggesting the same intramolecular pathway for Cl− and phospholipids. When overexpressed, TMEM16A and TMEM16F produced spontaneous Cl− currents at 37°C even at resting intracellular Ca2+ levels, which was abolished by inhibition of phospholipase A2 (PLA2). Connversely, activation of PLA2 or application of active PLA2, as well as lipid peroxidation induced by reactive oxygen species (ROS) using staurosporine or tert‐butyl hydroperoxide, enhanced ion currents by TMEM16A/F and in addition activated phospholipid scrambling by TMEM16F. Thus, TMEM16 proteins are activated by an increase in intracellular Ca2+, or independent of intracellular Ca2+, by modifications occurring in plasma and intracellular membrane phospholipids. These results may help to explain why regions distant to the TMEM16 pore and the Ca2+ binding sites control Cl− currents and phospholipid scrambling. Regulation of TMEM16 proteins through modification of membrane phospholipids occurs during regulated cell death such as apoptosis and ferroptosis. It contributes to inflammatory and nerve injury‐induced hypersensitivity and generation of pain and therefore provides a regulatory mechanism that is particularly relevant during disease.