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

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]. In addition to the low sensitivity to Ca, the activation of ANO6 Cl conductance is very slow (>3-5 min to reach peak level at 10 μM [Ca]), with subsequent inactivation. In a whole-cell patch clamp recording of ANO6 current (I), 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. Inside-out patch clamp recording of ANO6 (I) showed immediate activation by raising [Ca]. We also found that intracellular ATP (3 mM MgATP in pipette solution) decelerated the activation of I, and also prevented the inactivation of I. However, the addition of cytoD still accelerated both activation and inactivation of I. We conclude that the actin cytoskeleton and intracellular ATP play major roles in the Ca-dependent activation and inactivation of I, respectively.