TMEM16F/ANO6, a Ca2+-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 [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 (IANO6,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 IANO6,w-c. Inside-out patch clamp recording of ANO6 (IANO6,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 IANO6,w-c, and also prevented the inactivation of IANO6,w-c. However, the addition of cytoD still accelerated both activation and inactivation of IANO6,w-c. We conclude that the actin cytoskeleton and intracellular ATP play major roles in the Ca2+-dependent activation and inactivation of IANO6,w-c, respectively.

Subtype-specific control of P2X receptor channel signaling by ATP and Mg2+.

The identity and forms of activating ligands for ion channels are fundamental to their physiological roles in rapid electrical signaling. P2X receptor channels are ATP-activated cation channels that serve important roles in sensory signaling and inflammation, yet the active forms of the nucleotide are unknown. In physiological solutions, ATP is ionized and primarily found in complex with Mg(2+). Here we investigated the active forms of ATP and found that the action of MgATP(2-) and ATP(4-) differs between subtypes of P2X receptors. The slowly desensitizing P2X2 receptor can be activated by free ATP, but MgATP(2-) promotes opening with very low efficacy. In contrast, both free ATP and MgATP(2-) robustly open the rapidly desensitizing P2X3 subtype. A further distinction between these two subtypes is the ability of Mg(2+) to regulate P2X3 through a distinct allosteric mechanism. Importantly, heteromeric P2X2/3 channels present in sensory neurons exhibit a hybrid phenotype, characterized by robust activation by MgATP(2-) and weak regulation by Mg(2+). These results reveal the existence of two classes of homomeric P2X receptors with differential sensitivity to MgATP(2-) and regulation by Mg(2+), and demonstrate that both restraining mechanisms can be disengaged in heteromeric channels to form fast and sensitive ATP signaling pathways in sensory neurons.