INTRODUCTION: The Ca2+ -activated Cl- channel Anoctamin-1 (Ano1) regulates multiple cell functions including cell proliferation, survival, and migration. We previously reported that overexpression of Ano1 is associated with hyperproliferation of pulmonary artery endothelial cells isolated from patients with idiopathic pulmonary arterial hypertension. We also showed that Ano1 is expressed not only in the plasma membrane (PM), but also in the mitochondria. However, the physiological and pathological roles of mitochondrial Ano1 have not been fully investigated. AIM: To investigate the role of mitochondria-localized Ano1 on the regulation of mitochondrial dynamics and reactive oxygen species (ROS) generation. METHODS: Stable overexpression and knockdown of Ano1 were employed in HEK293T cells and mouse embryonic fibroblasts (MEFs), respectively. Cells were used for biochemical and cell biological assays. RESULTS: First, mitochondrial localization of Ano1 in addition to the PM was confirmed by live cell imaging of HEK293T cells expressing GFP-tagged Ano1 and immunostaining of endogenous Ano1 in MEFs. Next, a co-immunoprecipitation assay showed that overexpressed Ano1 in HEK293T cells was associated with optic atrophy 1 (OPA1) which is an inner mitochondrial membrane (IMM) protein and a critical regulator of mitochondrial fusion, cristae formation, and bioenergetics. We also confirmed the interaction between endogenous Ano1 and OPA1 using wild-type and OPA1-knockout MEFs. Overexpression of wild-type Ano1 in HEK293T cells facilitated proliferation, but a pore-dead mutant (Ano1-R621E) did not. Importantly, Ano1 overexpressing HEK293T cells have higher cell death rates in response to oxidative stress compared to control cells. To further understand the role of endogenous Ano1 expression on mitochondrial functions, we next employed MEFs stably overexpressing shRNA targeting Ano1. Quantifying mitochondrial morphology from live MEFs expressing mitochondria-matrix targeted GFP revealed that Ano1-knockdown (Ano1-KD) MEFs contained more elongated mitochondria compared to cells stably expressing control shRNA. Importantly, we also found higher expression of mitofusin 2 and long-form OPA1 in Ano1-KD MEFs compared to control cells. The expression levels of other fission/fusion proteins were not altered by Ano1-KD. Lastly, significantly lower levels of mitochondrial superoxide in Ano1-KD cells and a tendency towards lower cellular oxidative levels in Ano1-KD cells compared to controls were observed by live cell imaging with mitochondrial superoxide-sensitive dye MitoSOX Red and protein carbonyl quantification in the whole cell lysates, respectively. SUMMARY: Ano1, expressed in both the IMM and the PM, is involved in the maintenance of mitochondrial morphology and ROS, possibly by modulating fusion protein expression levels. Future studies will include the development of genetically engineered mitochondria-targeted Ano1 to more precisely dissect the roles of IMM-localized Ano1 in mitochondrial functions.
|Original language||English (US)|
|Journal||FASEB journal : official publication of the Federation of American Societies for Experimental Biology|
|State||Published - May 1 2022|
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