Anoctamins in Volume Regulation, Proliferation and Apoptotic Cell Death

The family of anoctamins consists of ten different proteins (called ANO1-10 or TMEM16A-K). A variety of functions have been attributed to anoctamins such as that of Ca2+ activated Cl- channels controlling Cl- secretion, cell volume, cell migration and proliferation, and that of phospholipid scramblases contributing to apoptotic cell death. Some anoctamin members are overexpressed in tumors and have been linked to developmental defects. Mutations in anoctamins can cause genetic diseases and cancer. However, it is unknown how anoctamins fulfill these various functions. Before ANO1 was identified as a protein forming a chloride channel, it was known as the cancer marker DOG1. DOG1/ANO1 is expressed in gastrointestinal stromal tumors (GIST) and particularly in head and neck squamous cell carcinoma (HNSCC), and is not detected at very high levels in other tissues. We found that ANO1 strongly augments cell proliferation, cell migration and metastasis. We also found that ANO1 expression is controlled by histone deacetylases (HDAC), corresponding well to the known role of HDAC in HNSCC. As ANO1 did not enhance proliferation in every cell type, its function is perhaps modulated by cell-specific factors. In contrast to ANO1, we found that ANO6, by operating as a Ca2+ activated membrane phospholipid scramblase, supports cellular apoptosis and necroptosis rather than promoting proliferation. Earlier findings from our laboratory and other groups indicated that ANO6 is a Cl- channel, a phospholipid scramblase and a nonselective cation channel activated by intracellular Ca2+ and pro-apoptotic stimuli. Moreover, previous studies characterized ANO6 as an outwardly rectifying Cl- channel (ORCC) and volume-regulated anion channel (VRAC) that is activated during apoptotic cell death. In the experiments presented here I found a major role of ANO6 for both regulatory volume decrease (RVD) as well as apoptotic volume decrease (AVD). RVD and AVD were examined in four different cell types under physiological conditions, i.e. in the presence of physiological intracellular and extracellular bath solutions and at 37 °C. Moreover swelling activated whole cell currents and volume regulation was assessed in freshly isolated intestinal epithelial cells from wild type and anoctamin 6 knockout mice. The data indicate that ANO6 generates a VRAC and is activated during RVD by membrane stretch, Ca2+ influx through TRP channels and activation of phospholipase A2 (PLA2), which generates membrane lysophospholipids (LPL). Accumulation of LPL in the plasma membrane enhances membrane tension, which possibly induces a conformational change of ANO6, thereby opening the Cl- conductive pore and causing RVD. Remarkably, RVD was largely reduced in intestinal epithelial cells from ANO6 knockout mice. The results also demonstrate that ANO6 is activated by low intracellular Cl-, which suggests that ANO6 operates as an osmosensor. ANO6 was further demonstrated to participate in the innate immune response in macrophages. We could demonstrate that in macrophages Ca2+ influx through P2X7 channels leads to initial cell shrinkage, phospholipid scrambling, cell membrane blebbing and ultimately cell death. Cell migration and phagocytic activity was found to be largely reduced in Ano6-/- macrophages. We proposed that ANO6 plays a central role for the immune defense realized by macrophages. The data also indicate that P2X7-induced cell death in macrophages is due to activation of apoptosis as well as necrosis. Another member of the anoctamin family, ANO10, was also demonstrated to be expressed in macrophages and to participate in innate immune response. In collaboration with the Max Planck institute of experimental Medicine in Göttingen we showed that the genomic ANO10 mutation R263H increases serum titers of antibodies directed against Lyme disease causing Borrelia. We suggest that ANO10-R263H enhances susceptibility towards Borrelia infection and attenuates clearance of spirochetes after tick bites. ANO10-R263H caused a reduced VRAC activity and RVD. Loss of ANO10 expression in macrophages compromised phagocytosis and clearance of Borrelia, attenuated volume regulation and reduced apoptotic cell death. Taken together the results demonstrate that anoctamins participate in the regulation of proliferation and cell death and play important roles in cancer and innate immune defense