title: Conditional genetic deletion of Ano1 in interstitial cells of Cajal impairs Ca2+transients and slow waves in adult mouse small intestine.
abstract: Myenteric plexus interstitial cells of Cajal (ICC-MY) in the small intestine are Kit+electrical pacemakers that express the Ano1/TMEM16A Ca2+-activated Cl-channel, whose functions in the gastrointestinal tract remain incompletely understood. In this study, an inducible Cre-LoxP-based approach was used to advance the understanding of Ano1 in ICC-MY of adult mouse small intestine.KitCreERT2/+;Ano1Fl/Flmice were treated with tamoxifen or vehicle, and small intestines (mucosa free) were examined. Quantitative RT-PCR demonstrated ~50% reduction in Ano1 mRNA in intestines of conditional knockouts (cKOs) compared with vehicle-treated controls. Whole mount immunohistochemistry showed a mosaic/patchy pattern loss of Ano1 protein in ICC networks. Ca2+transients in ICC-MY network of cKOs displayed reduced duration compared with highly synchronized controls and showed synchronized and desynchronized profiles. When matched, the rank order for Ano1 expression in Ca2+signal imaged fields of view was as follows: vehicle controls>>>cKO(synchronized)>cKO(desynchronized). Maintenance of Ca2+transients' synchronicity despite high loss of Ano1 indicates a large functional reserve of Ano1 in the ICC-MY network. Slow waves in cKOs displayed reduced duration and increased inter-slow-wave interval and occurred in regular- and irregular-amplitude oscillating patterns. The latter activity suggested ongoing interaction by independent interacting oscillators. Lack of slow waves and depolarization, previously reported for neonatal constitutive knockouts, were also seen. In summary, Ano1 in adults regulates gastrointestinal function by determining Ca2+transients and electrical activity depending on the level of Ano1 expression. Partial Ano1 loss results in Ca2+transients and slow waves displaying reduced duration, while complete and widespread absence of Ano1 in ICC-MY causes lack of slow wave and desynchronized Ca2+transients.NEW & NOTEWORTHYThe Ca2+-activated Cl-channel, Ano1, in interstitial cells of Cajal (ICC) is necessary for normal gastrointestinal motility. We knocked out Ano1 to varying degrees in ICC of adult mice. Partial knockout of Ano1 shortened the widths of electrical slow waves and Ca2+transients in myenteric ICC but Ca2+transient synchronicity was preserved. Near-complete knockout was necessary for transient desynchronization and loss of slow waves, indicating a large functional reserve of Ano1 in ICC.
authors.affiliation: Enteric NeuroScience Program, Mayo Clinic, Rochester, Minnesota.
authors.forename: John
authors.initials: J
authors.lastname: Malysz
authors.affiliation: Enteric NeuroScience Program, Mayo Clinic, Rochester, Minnesota.
authors.forename: Simon J
authors.identifier: http://orcid.org/0000-0001-5311-5993
authors.initials: SJ
authors.lastname: Gibbons
authors.affiliation: Enteric NeuroScience Program, Mayo Clinic, Rochester, Minnesota.
authors.forename: Siva A
authors.initials: SA
authors.lastname: Saravanaperumal
authors.affiliation: Auckland Bioengineering Institute, University of Auckland, Auckland, New Zealand.
authors.forename: Peng
authors.initials: P
authors.lastname: Du
authors.affiliation: Enteric NeuroScience Program, Mayo Clinic, Rochester, Minnesota.
authors.forename: Seth T
authors.initials: ST
authors.lastname: Eisenman
authors.affiliation: Enteric NeuroScience Program, Mayo Clinic, Rochester, Minnesota.
authors.forename: Chike
authors.initials: C
authors.lastname: Cao
authors.affiliation: Sensory Research Center, CRI, College of Pharmacy, Seoul National University, Seoul, Republic of Korea; and.
authors.forename: Uhtaek
authors.initials: U
authors.lastname: Oh
authors.affiliation: Department of Internal Medicine II, Klinikum rechts der Isar, Technische Universität München, München, Germany.
authors.forename: Dieter
authors.initials: D
authors.lastname: Saur
authors.affiliation: Department of Internal Medicine II, Klinikum rechts der Isar, Technische Universität München, München, Germany.
authors.forename: Sabine
authors.initials: S
authors.lastname: Klein
authors.affiliation: Enteric NeuroScience Program, Mayo Clinic, Rochester, Minnesota.
authors.forename: Tamas
authors.initials: T
authors.lastname: Ordog
authors.affiliation: Enteric NeuroScience Program, Mayo Clinic, Rochester, Minnesota; farrugia.gianrico@mayo.edu.
authors.forename: Gianrico
authors.initials: G
authors.lastname: Farrugia
mesh_terms: D000818:Animals; D000075369:Anoctamin-1; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000235:genetics*; D018118:Chloride Channels / Q000235:genetics* / Q000378:metabolism; D056885:Interstitial Cells of Cajal / Q000166:cytology / Q000378:metabolism*; D007421:Intestine, Small / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D009130:Muscle, Smooth / Q000378:metabolism; D009197:Myenteric Plexus / Q000378:metabolism*
chemical_list: C531181:ANO1 protein, mouse; D000075369:Anoctamin-1; D018118:Chloride Channels; D002118:Calcium
keywords: Ca2+ transients; anoctamin; genetic knockout; ion channel; pacemaker; slow wave