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

Abstract

TMEM16/anoctamin 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). Inversely, 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 understand, 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 particularly relevant during disease.

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