title: Variomics screen identifies the re-entrant loop of the calcium-activated chloride channel ANO1 that facilitates channel activation.
abstract: 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.
authors.affiliation: From the Novartis Institutes for Biomedical Research, Cambridge, Massachusetts 02139.
authors.forename: Anke
authors.initials: A
authors.lastname: Bill
authors.affiliation: the Novartis Institutes for Biomedical Research, Horsham, West Sussex RH12 5AB, United Kingdom, and.
authors.forename: M Oana
authors.initials: MO
authors.lastname: Popa
authors.affiliation: the Novartis Institutes for Biomedical Research, Horsham, West Sussex RH12 5AB, United Kingdom, and.
authors.forename: Michiel T
authors.initials: MT
authors.lastname: van Diepen
authors.affiliation: From the Novartis Institutes for Biomedical Research, Cambridge, Massachusetts 02139.
authors.forename: Abraham
authors.initials: A
authors.lastname: Gutierrez
authors.affiliation: the Novartis Institutes for Biomedical Research, Horsham, West Sussex RH12 5AB, United Kingdom, and.
authors.forename: Sarah
authors.initials: S
authors.lastname: Lilley
authors.affiliation: the Novartis Institutes for Biomedical Research, Horsham, West Sussex RH12 5AB, United Kingdom, and.
authors.forename: Maria
authors.initials: M
authors.lastname: Velkova
authors.affiliation: the Novartis Institutes for Biomedical Research, Horsham, West Sussex RH12 5AB, United Kingdom, and.
authors.forename: Kathryn
authors.initials: K
authors.lastname: Acheson
authors.affiliation: the Novartis Institutes for Biomedical Research, Horsham, West Sussex RH12 5AB, United Kingdom, and.
authors.forename: Hedaythul
authors.initials: H
authors.lastname: Choudhury
authors.affiliation: From the Novartis Institutes for Biomedical Research, Cambridge, Massachusetts 02139.
authors.forename: Nicole A
authors.initials: NA
authors.lastname: Renaud
authors.affiliation: From the Novartis Institutes for Biomedical Research, Cambridge, Massachusetts 02139.
authors.forename: Douglas S
authors.initials: DS
authors.lastname: Auld
authors.affiliation: the Novartis Institutes for Biomedical Research, Horsham, West Sussex RH12 5AB, United Kingdom, and.
authors.forename: Martin
authors.initials: M
authors.lastname: Gosling
authors.affiliation: the Novartis Institutes for Biomedical Research, Basel CH-4002, Switzerland.
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Groot-Kormelink
authors.affiliation: From the Novartis Institutes for Biomedical Research, Cambridge, Massachusetts 02139, alex.gaither@novartis.com.
authors.forename: L Alex
authors.initials: LA
authors.lastname: Gaither
mesh_terms: D000818:Animals; D000075369:Anoctamin-1; D016466:CHO Cells; D018118:Chloride Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003412:Cricetulus; D057809:HEK293 Cells; D006801:Humans; D007473:Ion Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009363:Neoplasm Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D013329:Structure-Activity Relationship
chemical_list: C476213:ANO1 protein, human; D000075369:Anoctamin-1; D018118:Chloride Channels; D007473:Ion Channels; D009363:Neoplasm Proteins
keywords: ANO1; Chloride Channel; Ion Channel; Mutagenesis; Protein Structure; Site-directed Mutagenesis; TMEM16A; Variomics