Since Anoctamin 1 (ANO1) has been identified as a calcium-activated chloride channel (CaCC), a lot of effort has been made to establish the relationship between ANO1 and the previously known calcium-activated chloride currents involved in diverse physiological processes. ANO1 is suggested to mediate the epithelial secretion of several tissues, the pace of interstitial cells of Cajal, the contraction of the reproductive tracts and blood vessels, and the excitability of peripheral neurons. Though ANO1 takes parts in these important physiological processes, little is known about its regulation by posttranslational modifications, especially ubiquitination that controls the turnover and activity of numerous ion channels. The first part of my thesis focuses on the modulation of ANO1...[ Read more ]

Since Anoctamin 1 (ANO1) has been identified as a calcium-activated chloride channel (CaCC), a lot of effort has been made to establish the relationship between ANO1 and the previously known calcium-activated chloride currents involved in diverse physiological processes. ANO1 is suggested to mediate the epithelial secretion of several tissues, the pace of interstitial cells of Cajal, the contraction of the reproductive tracts and blood vessels, and the excitability of peripheral neurons. Though ANO1 takes parts in these important physiological processes, little is known about its regulation by posttranslational modifications, especially ubiquitination that controls the turnover and activity of numerous ion channels. The first part of my thesis focuses on the modulation of ANO1 by ubiquitination. By using the yeast-two hybrid assay, Tripartite Motif Containing 23 (TRIM23) was identified as a binding partner of ANO1. The physical interaction between TRIM23 and ANO1, specifically the carboxyl-terminus of ANO1, was confirmed through GST-pull down and co-immunoprecipitation assays. In addition, mapping experiments indicated the ADP-ribosylation factor (ARF) domain of TRIM23 is responsible for recognizing ANO1 carboxyl-terminus. Furthermore, both in-vivo and in-vitro ubiquitination assays showed that ANO1 is ubiquitinated by TRIM23. Knockout of TRIM23 in breast cancer cell line ZR-75-1 increased the protein expression but reduced the ubiquitination of endogenous ANO1. By promoting ANO1 degradation, TRIM23 level can inhibit the proliferation of breast cancer cells. Our findings suggest that ANO1 is ubiquitinated and degraded by TRIM23 E3 ligases and this previous unrecognized regulatory mechanism plays a role in tumorigenesis.

The second part of my thesis study concerns the role of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in regulatory volume decrease (RVD) of epithelia cells. CFTR is a ligand-gated anion channel in various epithelia, with diverse mutations associated with the severe recessive genetic disease cystic fibrosis. CFTR G551D mutation impairs ATP hydrolysis and thereby makes CFTR refractory to cAMP stimulation. Both wild type (wt-) and G551D CFTR have been implicated in RVD, but the underlying mechanism remains incompletely understood. In my thesis research, I showed that the channel activity of both wild type and G551D CFTR is directly stimulated by mechanical perturbation induced by cell swelling at the single-channel, cellular, and tissue levels. Hypotonicity activated CFTR single channels in cell-attached membrane patches and wt-CFTR-mediated short-circuit current (Isc) in Calu-3 cells, and this was independent of calcium and protein kinase A signaling. Genetic ablation but not G551D mutation of CFTR attenuated the hypotonicity- and stretch-induced short-circuit current in mouse duodena. These results indicate CFTR involvement in volume regulation and suggest that the mechanosensitivity of G551D CFTR might underlie the mild phenotypes resulting from this mutation.