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

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.