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 [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 μM [Ca²⁺]_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 [Ca²⁺]_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 Ca²⁺-dependent activation and inactivation of I_ANO6,w-c, respectively.

Keywords: Actin cytoskeleton; Anoctamin 6; Calcium activated Cl(−) current; Magnesium ATP; TMEM16F.