Distribution and Characterization of ANO10 in Mouse Small Intestine

Background: Anoctamin 10 (Ano10) belongs to the class of proteins that includes Ca2+ activated Cl-channels such as Ano1, which is expressed in interstitial cells of Cajal (ICC). Mutations in Ano10 lead to spinocerebellar ataxia and immunological defects. Evidence suggests that Ano10 is not a Ca2+ -activated Cl-channel but rather an intracellular protein that may regulate intracellular Ca2+. The aim of this study was to characterize the distribution of Ano10 in the mouse small intestine in order to determine the cell types in which Ano10 may contribute to cellular physiology. Methods: Small intestinal tissues from adult Balb/c (n=3) mice were obtained for both cryosections (10 μm thickness) and whole mount preparations. Tissues were immunolabeled using antibodies raised against Ano10 (rabbit polyclonal), a marker of ICC - Kit (goat polyclonal), a pan-neuronal marker - HuC/D (human, serum derived), and a macrophage marker - F4/80 (rat monoclonal). Secondary antibody controls were performed by exposing samples to secondary antibody in the absence of primary antibody to test for non-specific staining. Double labeling of Ano10 with other cell specific markers was performed on 5 cryosections and 2 whole mounts for each mouse. The distribution of immunoreactivity (IR) was determined by confocal microscopic imaging. Results: Ano10-IR was found to be present in neuronal cell bodies outside of the nucleus. Ano10-IR was present in 100% of HuC/D-positive neurons of the myenteric and submucosal plexuses. Ano10-IR was not found in ICC of the myenteric region; however, there was colocalization of Ano10-IR with Kit-IR on ICC of the deep muscularis plexus (ICC-DMP). Ano10-IR also colocalized with F4/80-IR present on resident macrophages in the region of the myenteric plexus. 100% of F4/80-IR colocalized with Ano10-IR. Conclusions: Immuno-reactivity for Ano10 is present in all types of myenteric and submucosal neurons and in all resident macrophages. Ano10-IR was present in a subset of ICC, specifically ICC-DMP but not ICC in the myenteric plexus. Given the putative role of Ano10 as a regulator of intracellular Ca2+, Ano10 may play a role both in regulation of gastrointestinal motility via neurons and ICC-DMP, as well as immunological responses via resident macrophages. Grant support: NIH DK057061, P01DK68055, and P30DK084567