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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. Kit^CreERT2/+;Ano1^Fl/Fl mice 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 & NOTEWORTHY The 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.

SUBMITTER: Malysz J

PROVIDER: S-EPMC5401988 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Key pointsColonic intramuscular interstitial cells of Cajal (ICC-IM) exhibit spontaneous Ca2+ transients manifesting as stochastic events from multiple firing sites with propagating Ca2+ waves occasionally observed. Firing of Ca2+ transients in ICC-IM is not coordinated with adjacent ICC-IM in a field of view or even with events from other firing sites within a single cell. Ca2+ transients, through activation of Ano1 channels and generation of inward current, cause net depolarization of colonic muscles. Ca2+ transients in ICC-IM rely on Ca2+ release from the endoplasmic reticulum via IP3 receptors, spatial amplification from RyRs and ongoing refilling of ER via the sarcoplasmic/endoplasmic-reticulum-Ca2+ -ATPase. ICC-IM are sustained by voltage-independent Ca2+ influx via store-operated Ca2+ entry. Some of the properties of Ca2+ in ICC-IM in the colon are similar to the behaviour of ICC located in the deep muscular plexus region of the small intestine, suggesting there are functional similarities between these classes of ICC.AbstractA component of the SIP syncytium that regulates smooth muscle excitability in the colon is the intramuscular class of interstitial cells of Cajal (ICC-IM). All classes of ICC (including ICC-IM) express Ca2+ -activated Cl- channels, encoded by Ano1, and rely upon this conductance for physiological functions. Thus, Ca2+ handling in ICC is fundamental to colonic motility. We examined Ca2+ handling mechanisms in ICC-IM of murine proximal colon expressing GCaMP6f in ICC. Several Ca2+ firing sites were detected in each cell. While individual sites displayed rhythmic Ca2+ events, the overall pattern of Ca2+ transients was stochastic. No correlation was found between discrete Ca2+ firing sites in the same cell or in adjacent cells. Ca2+ transients in some cells initiated Ca2+ waves that spread along the cell at ∼100 µm s-1 . Ca2+ transients were caused by release from intracellular stores, but depended strongly on store-operated Ca2+ entry mechanisms. ICC Ca2+ transient firing regulated the resting membrane potential of colonic tissues as a specific Ano1 antagonist hyperpolarized colonic muscles by ∼10 mV. Ca2+ transient firing was independent of membrane potential and not affected by blockade of L- or T-type Ca2+ channels. Mechanisms regulating Ca2+ transients in the proximal colon displayed both similarities to and differences from the intramuscular type of ICC in the small intestine. Similarities and differences in Ca2+ release patterns might determine how ICC respond to neurotransmission in these two regions of the gastrointestinal tract.

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Conditional genetic deletion of Ano1 in interstitial cells of Cajal impairs Ca²⁺ transients and slow waves in adult mouse small intestine.

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Dataset Information

Conditional genetic deletion of Ano1 in interstitial cells of Cajal impairs Ca2+ transients and slow waves in adult mouse small intestine.

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OmicsDI is part of the ELIXIR infrastructure

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Conditional genetic deletion of Ano1 in interstitial cells of Cajal impairs Ca²⁺ transients and slow waves in adult mouse small intestine.