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

• Published in: Molecules and cells Vol. 44; no. 2; pp. 88 - 100
• Main Authors: Roh, Jae Won, Hwang, Ga Eun, Kim, Woo Kyung, Nam, Joo Hyun
• Format: Journal Article
• Language: English
• Published: Korean Society for Molecular and Cellular Biology 01.02.2021; 한국분자세포생물학회
• Subjects: 생물학
• ISSN: 1016-8478; 0219-1032
• DOI: 10.14348/molcells.2021.2203

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.