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 Ca-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. 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. Ca 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 Ca signal imaged fields of view was as follows: vehicle controls>>>cKO(synchronized)>cKO(desynchronized). Maintenance of Ca 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 Ca transients and electrical activity depending on the level of Ano1 expression. Partial Ano1 loss results in Ca transients and slow waves displaying reduced duration, while complete and widespread absence of Ano1 in ICC-MY causes lack of slow wave and desynchronized Ca transients. The Ca-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 Ca transients in myenteric ICC but Ca 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.

The cells and conductance mediating cholinergic neurotransmission in the murine proximal stomach

“…; Malysz et al . ). In the murine stomach, pacemaker activity, attributed to ICC in the plane of the myenteric plexus (ICC‐MY), is resolved in the gastric corpus and antrum but typically not observed in the fundus (Burns et al .…”

Extracellular Cl− regulates electrical slow waves and setting of smooth muscle membrane potential by interstitial cells of Cajal in mouse jejunum

“…) and are altered in Ano1 conditional knockout mice (Malysz et al . ). Slow waves are also altered in NKCC1 knockout mice, as well as after treatment of wild‐type (WT) tissues with the NKCC1 inhibitor, bumetanide (Wouters et al .…”

Ca2+ signalling behaviours of intramuscular interstitial cells of Cajal in the murine colon

“…All populations of ICC in the GI tract express the Ca 2+ -activated Cl − channel, Ano1 (Gomez-Pinilla et al 2009;Hwang et al 2009), and activation of Ano1 is fundamental to the physiological functions of ICC. Pharmacological antagonism or genetic ablation of Ano1 causes loss of pacemaker activity and defects in post-junctional neuronal responses (Hwang et al 2009(Hwang et al , 2016(Hwang et al , 2019Singh et al 2014;Malysz et al 2016;Cobine et al 2017;Sung et al 2018). Ano1 channels are responsible for the generation of spontaneous transient inward currents (STICs) and depolarizations known as unitary potentials or spontaneous transient depolarizations (STDs) that are thought to be a fundamental transduction mechanism in ICC (Edwards et al 1999;Kito & Suzuki, 2003).…”