Anoctamin 6 (ANO6/TMEM16F) is a recently identified membrane protein that has both phospholipid scramblase activity and anion channel function activated by relatively high [Ca2+](i). In addition to the low sensitivity to Ca2+, the activation of ANO6 Cl- conductance is very slow (>3-5 min to reach peak level at 10 mu M [Ca2+](i)), with subsequent inactivation. In a whole-cell patch clamp recording of ANO6 current (I-ANO6,I-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,I-w-c Inside-out patch clamp recording of ANO6 (I-ANO6,I-i-o) showed immediate activation by raising [Ca2+](i). We also found that intracellular ATP (3 mM MgATP in pipette solution) decelerated the activation of I-ANO6,I-w-c, and also prevented the inactivation of I-ANO6,I-w-c. However, the addition of cytoD still accelerated both activation and inactivation of I-ANO6,I-w-c. We conclude that the actin cytoskeleton and intracellular ATP play major roles in the Ca2+-dependent activation and inactivation of I-ANO6,I-w-c, respectively. (C) 2018 Elsevier Inc. All rights reserved.