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.