Ion channels are required to generate the electrical activity that drives contractility in the gastrointestinal tract. In the past application cycle we ave shown that human gastrointestinal interstitial cells of Cajal (ICC) express Ano1, a calcium-activated chloride (Cl-) channel belonging to the anoctamin family. We have also shown that Ano1 generates a current that due to alternative splicing has different electrophysiological properties in motility diseases such as gastroparesis. A central feature of the regulation of rhythmic smooth muscle contractile activity in the gastrointestinal tract is a Ca2+ signal that spreads from ICC to ICC in a highly coordinated fashion. The mechanism that underlies the synchronization of this Ca2+ signal is unknown. Understanding the mechanism is important as coordinated contractile activity is required for normal gastrointestinal function. The central hypothesis of this proposal is that Ano1 is required for synchronization of the ICC Ca2+ signal and subsequent coordinated smooth muscle contractility. We also hypothesize that we have identified the promoter region for Ano1 and that variants of Ano1 result in functional changes including the risk of developing delayed gastric emptying. We will test the central hypothesis in 2 specific aims. In SA1 we will determine the mechanisms by which Ano1 regulates coordinated gastrointestinal motility. In SA2 we will determine the regulation of Ano1 expression in health and disease including the promoter that drives Ano1 expression and the transcription factors involved. The SA are supported by preliminary data which show that absence of Ano1 expression is associated with loss of synchronization of the ICC Ca2+ signal, that knockdown of Ano1 in organotypic cultures disrupts Ca2+ signal synchronization, that smooth muscle contractility is uncoordinated when Ano1 expression is altered, that contrary to the published sequence for human Ano1, there is a completely missed exon that is 93 Kb away from the previously labeled exon 1 and that a polymorphism in this new exon is associated with gastroparesis. We also identified the presence of other anoctamins in ICC that when co-expressed with Ano1 results in cyclic electrical activity. An area proximal to the new exon 0 has properties suggesting it is the promoter for Ano1 and we identified relevant transcriptional factors including STAT6 and GLI1 that drive Ano1 transcription. We will use conditional knockout mice, Ca2+ imaging, spatiotemporal mapping, patch clamp techniques, microelectrode recordings, immunohistochemistry, Western blots, RNA seq, RT-PCR, single cell PCR, quantitative PCR, lentivirus RNA knock down techniques, and organotypic and single cell cultures to investigate the central hypothesis. The significance of this work is that, as a result f the work done in the previous application cycles and the preliminary data presented in this proposal, we can significantly advance our understanding, at a cellular and molecular level, of the coordinated interaction between ICC and smooth muscle contractile activity and the contribution of Ano1 to normal and abnormal human physiology and risk of developing disease.

Public Health Relevance

Gastrointestinal motility disorders are common and associated with considerable morbidity and mortality. Several cell types contribute to normal gastrointestinal motility including interstitial cells of Cajal (ICC), cells that pace the intestin and express a calcium-activated chloride channel called Ano1. This proposal will investigate the role of Ano1 in the control of ICC function, including how Ano1 is made, how Ano1 coordinates smooth muscle contractility and how different forms of Ano1 are linked to motility disorders, with the aim of changing the function of Ano1 to restore normal gastrointestinal motility in disease states.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK057061-17
Application #
9096067
Study Section
Clinical, Integrative and Molecular Gastroenterology Study Section (CIMG)
Program Officer
Hamilton, Frank A
Project Start
2000-04-01
Project End
2018-06-30
Budget Start
2016-07-01
Budget End
2017-06-30
Support Year
17
Fiscal Year
2016
Total Cost
Indirect Cost
Name
Mayo Clinic, Rochester
Department
Type
DUNS #
006471700
City
Rochester
State
MN
Country
United States
Zip Code
55905
Bianco, F; Eisenman, S T; Colmenares Aguilar, M G et al. (2018) Expression of RAD21 immunoreactivity in myenteric neurons of the human and mouse small intestine. Neurogastroenterol Motil 30:e13429
Treichel, Anthony J; Farrugia, Gianrico; Beyder, Arthur (2018) The touchy business of gastrointestinal (GI) mechanosensitivity. Brain Res 1693:197-200
Gibbons, Simon J (2018) Not just there to fill space: profound observations on interstitial cells of Cajal in the gastric fundus. J Physiol 596:1535-1536
Saravanaperumal, Siva Arumugam; Gibbons, Simon J; Malysz, John et al. (2018) Extracellular Cl- regulates electrical slow waves and setting of smooth muscle membrane potential by interstitial cells of Cajal in mouse jejunum. Exp Physiol 103:40-57
Rajan, Elizabeth; Al-Bawardy, Badr; Gostout, Christopher J et al. (2018) Endoscopic muscle biopsy sampling of the duodenum and rectum: a pilot survival study in a porcine model to detect myenteric neurons. Gastrointest Endosc 87:600-606
Strege, Peter R; Gibbons, Simon J; Mazzone, Amelia et al. (2017) EAVK segment ""c"" sequence confers Ca2+-dependent changes to the kinetics of full-length human Ano1. Am J Physiol Gastrointest Liver Physiol 312:G572-G579
Angeli, T R; Du, P; Paskaranandavadivel, N et al. (2017) High-resolution electrical mapping of porcine gastric slow-wave propagation from the mucosal surface. Neurogastroenterol Motil 29:
Malysz, John; Gibbons, Simon J; Saravanaperumal, Siva A et al. (2017) Conditional genetic deletion of Ano1 in interstitial cells of Cajal impairs Ca2+transients and slow waves in adult mouse small intestine. Am J Physiol Gastrointest Liver Physiol 312:G228-G245
Gibbons, Simon J; Grover, Madhusudan; Choi, Kyoung Moo et al. (2017) Repeat polymorphisms in the Homo sapiens heme oxygenase-1 gene in diabetic and idiopathic gastroparesis. PLoS One 12:e0187772
Eisenman, S T; Gibbons, S J; Verhulst, P-J et al. (2017) Tumor necrosis factor alpha derived from classically activated ""M1"" macrophages reduces interstitial cell of Cajal numbers. Neurogastroenterol Motil 29:

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