Ca(2+) Sensitivity of Anoctamin 6/TMEM16F Is Regulated by the Putative Ca(2+)-Binding Reservoir at the N-Terminal Domain

Anoctamin 6/TMEM16F (ANO6) is a dual-function protein with Ca(2+)-activated ion channel and Ca(2+)-activated phospholipid scramblase activities, requiring a high intracellular Ca(2+) concentration (e.g., half-maximal effective Ca(2+) concentration [EC(50)] of [Ca(2+)](i) > 10 μM), and strong and sustained depolarization above 0 mV. Structural comparison with Anoctamin 1/TMEM16A (ANO1), a canonical Ca(2+)-activated chloride channel exhibiting higher Ca(2+) sensitivity (EC(50) of 1 μM) than ANO6, suggested that a homologous Ca(2+)-transferring site in the N-terminal domain (Nt) might be responsible for the differential Ca(2+) sensitivity and kinetics of activation between ANO6 and ANO1. To elucidate the role of the putative Ca(2+)-transferring reservoir in the Nt (Nt-CaRes), we constructed an ANO6-1-6 chimera in which Nt-CaRes was replaced with the corresponding domain of ANO1. ANO6-1-6 showed higher sensitivity to Ca(2+) than ANO6. However, neither the speed of activation nor the voltage-dependence differed between ANO6 and ANO6-1-6. Molecular dynamics simulation revealed a reduced Ca(2+) interaction with Nt-CaRes in ANO6 than ANO6-1-6. Moreover, mutations on potentially Ca(2+)-interacting acidic amino acids in ANO6 Nt-CaRes resulted in reduced Ca(2+) sensitivity, implying direct interactions of Ca(2+) with these residues. Based on these results, we cautiously suggest that the net charge of Nt-CaRes is responsible for the difference in Ca(2+) sensitivity between ANO1 and ANO6.