Variomics Screen Identifies the Re-entrant Loop of the Calcium-activated Chloride Channel ANO1 That Facilitates Channel Activation

• Published in: The Journal of biological chemistry Vol. 290; no. 2; pp. 889 - 903
• Main Authors: Bill, Anke, Popa, M. Oana, van Diepen, Michiel T., Gutierrez, Abraham, Lilley, Sarah, Velkova, Maria, Acheson, Kathryn, Choudhury, Hedaythul, Renaud, Nicole A., Auld, Douglas S., Gosling, Martin, Groot-Kormelink, Paul J., Gaither, L. Alex
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 09.01.2015; American Society for Biochemistry and Molecular Biology
• Subjects: Animals; ANO1; Anoctamin-1; Cell Biology; Chloride Channel; Chloride Channels - chemistry; Chloride Channels - genetics; Chloride Channels - metabolism; CHO Cells; Cricetulus; HEK293 Cells; Humans; Ion Channel; Ion Channels - chemistry; Ion Channels - genetics; Ion Channels - metabolism; Mutagenesis; Mutagenesis, Site-Directed; Neoplasm Proteins - chemistry; Neoplasm Proteins - genetics; Neoplasm Proteins - metabolism; Protein Conformation; Protein Structure; Site-directed Mutagenesis; Structure-Activity Relationship; TMEM16A; Variomics; Chloride Channel; Site-directed Mutagenesis; ANO1; Mutagenesis; Variomics; Ion Channel; Protein Structure; TMEM16A
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M114.618140

The calcium-activated chloride channel ANO1 regulates multiple physiological processes. However, little is known about the mechanism of channel gating and regulation of ANO1 activity. Using a high-throughput, random mutagenesis-based variomics screen, we generated and functionally characterized ∼6000 ANO1 mutants and identified novel mutations that affected channel activity, intracellular trafficking, or localization of ANO1. Mutations such as S741T increased ANO1 calcium sensitivity and rendered ANO1 calcium gating voltage-independent, demonstrating a critical role of the re-entrant loop in coupling calcium and voltage sensitivity of ANO1 and hence in regulating ANO1 activation. Our data present the first unbiased and comprehensive study of the structure-function relationship of ANO1. The novel ANO1 mutants reported have diverse functional characteristics, providing new tools to study ANO1 function in biological systems, paving the path for a better understanding of the function of ANO1 and its role in health and diseases.The calcium-activated chloride channel ANO1 regulates multiple physiological processes. We identified residues that when mutated affected channel activity, intracellular trafficking, or localization and report the first structure-function map of ANO1. The re-entrant loop mediates calcium/voltage sensitivity and activation of ANO1. We provide new tools for studying ANO1 function in biological systems and its potential as a therapeutic target.