TMEM16F/ANO6, a Ca2+-activated anion channel, is negatively regulated by the actin cytoskeleton and intracellular MgATP

Abstract Anoctamin 6 (ANO6/TMEM16F) is a recently identified membrane protein that has both phospholipid scramblase activity and anion channel function activated by relatively high [Ca 2+ ] i . In addition to the low sensitivity to Ca 2+ , the activation of ANO6 Cl − conductance is very slow (>3–5 min to reach peak level at 10 μM [Ca 2+ ] i ), with subsequent inactivation. In a whole-cell patch clamp recording of ANO6 current (I ANO6,w-c ), disruption of the actin cytoskeleton with cytochalasin-D (cytoD) significantly accelerated the activation kinetics, while actin filament-stabilizing agents (phalloidin and jasplakinolide) commonly inhibited I ANO6,w-c . Inside-out patch clamp recording of ANO6 (I ANO6,i-o ) showed immediate activation by raising [Ca 2+ ] i . We also found that intracellular ATP (3 mM MgATP in pipette solution) decelerated the activation of I ANO6,w-c , and also prevented the inactivation of I ANO6,w-c . However, the addition of cytoD still accelerated both activation and inactivation of I ANO6,w-c . We conclude that the actin cytoskeleton and intracellular ATP play major roles in the Ca 2+ -dependent activation and inactivation of I ANO6,w-c , respectively.