Anoctamin 1 (Tmem16A) Ca ^{2 +} -activated chloride channel stoichiometrically interacts with an ezrin–radixin–moesin network

Abstract

The newly discovered Ca 2+ -activated Cl − channel (CaCC), Anoctamin 1 (Ano1 or TMEM16A), has been implicated in vital physiological functions including epithelial fluid secretion, gut motility, and smooth muscle tone. Overexpression of Ano1 in HEK cells or Xenopus oocytes is sufficient to generate Ca 2+ -activated Cl − currents, but the details of channel composition and the regulatory factors that control channel biology are incompletely understood. We used a highly sensitive quantitative SILAC proteomics approach to obtain insights into stoichiometric protein networks associated with the Ano1 channel. These studies provide a comprehensive footprint of putative Ano1 regulatory networks. We find that Ano1 associates with the signaling/scaffolding proteins ezrin, radixin, moesin, and RhoA, which link the plasma membrane to the cytoskeleton with very high stoichiometry. Ano1, ezrin, and moesin/radixin colocalize apically in salivary gland epithelial cells, and overexpression of moesin and Ano1 in HEK cells alters the subcellular localization of both proteins. Moreover, interfering RNA for moesin modifies Ano1 current without affecting its surface expression level. Another network associated with Ano1 includes the SNARE and SM proteins VAMP3, syntaxins 2 and -4, and syntaxin-binding proteins munc18b and munc18c, which are integral to translocation of vesicles to the plasma membrane. A number of other regulatory proteins, including GTPases, Ca 2+ -binding proteins, kinases, and lipid-interacting proteins are enriched in the Ano1 complex. These data provide stoichiometrically prioritized information about mechanisms regulating Ano1 function and trafficking to polarized domains of the plasma membrane.calcium | interactome | cross-linker | apical targeting

Role of anoctamins in cancer and apoptosis

“…Our earlier findings suggested that Ano1 Cl 2 currents are controlled by the actin cytoskeleton [38]. This was supported by a subsequent report indicating that Ano1 associates with the signalling/scaffolding proteins ezrin, radixin, moesin and RhoA, which are known to connect plasma membrane proteins to the cytoskeleton [39]. Moreover, results from a two hybrid split ubiquitin screening suggested interaction of Ano1 with a number of proteins related to cell attachment and migration, such as zyxin, fibulin 1, S100A11, twinfilin and catenin (unpublished results from the Kunzelmann laboratory, K. Kunzelman 2012, figure 2).…”

Preassociated apocalmodulin mediates Ca 2+ -dependent sensitization of activation and inactivation of TMEM16A/16B Ca 2+ -gated Cl − channels

“…That the ChIMP method, which was previously developed in Ca V channels (39), is similarly effective in the unrelated TMEM16A/ 16B suggests that it may be generally applicable to the study and modulation of many different ion channel families. A recent quantitative proteomic approach revealed that TMEM16A is associated with a large network of intracellular proteins, many of which could be important for channel regulation (45). The ChIMP assay could be an invaluable tool for dissecting the functional relevance of other cytosolic proteins that putatively interact with TMEM16A intracellular domains.…”

Calcium-activated chloride channel ANO1 promotes breast cancer progression by activating EGFR and CAMK signaling

“…Furthermore, EGF stimulation has been shown to mediate calcium-activated chloride channel activation via activation of SRC (47) and to increase ANO1 expression in epithelial cells, suggesting a positive feedback mechanism between EGFR/SRC signaling and ANO1 (47,48). SRC recently has been shown to interact with ANO1 and can itself modulate the activation of EGFR, providing an additional explanation for the ANO1-dependent activation of EGFR signaling (49,50). EGFR is a known contributor to HNSCC, ESCC, and breast cancer tumorigenesis and is one the most important therapeutic targets in HNSCC.…”