Ca2+ Sensitivity of Anoctamin 6/TMEM16F Is Regulated by the Putative Ca2+-Binding Reservoir at the N-Terminal Domain

Anoctamin 6/TMEM16F (ANO6) is a dual-function protein with Ca²⁺-activated ion channel and Ca²⁺-activated phospholipid scramblase activities, requiring a high intracellular Ca²⁺ concentration (e.g., half-maximal effective Ca²⁺ concentration EC₅₀] of Ca²⁺]_i > 10 μM), and strong and sustained depolarization above 0 mV. Structural comparison with Anoctamin 1/TMEM16A (ANO1), a canonical Ca²⁺-activated chloride channel exhibiting higher Ca²⁺ sensitivity (EC₅₀ of 1 μM) than ANO6, suggested that a homologous Ca²⁺-transferring site in the N-terminal domain (Nt) might be responsible for the differential Ca²⁺ sensitivity and kinetics of activation between ANO6 and ANO1. To elucidate the role of the putative Ca²⁺-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 Ca²⁺ 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 Ca²⁺ interaction with Nt-CaRes in ANO6 than ANO6-1-6. Moreover, mutations on potentially Ca²⁺-interacting acidic amino acids in ANO6 Nt-CaRes resulted in reduced Ca²⁺ sensitivity, implying direct interactions of Ca²⁺ with these residues. Based on these results, we cautiously suggest that the net charge of Nt-CaRes is responsible for the difference in Ca²⁺ sensitivity between ANO1 and ANO6.