Ca 2+ ‐Activated Cl‐ Channel Anoctamin‐1 Interacts with Mitochondrial Fusion Protein OPA1

Anoctamin-1 (Ano1) forms a Ca2+-activated chloride channel found at the plasma membrane (PM) of several different cell types, including endothelial cells (ECs). We previously showed Ano1 overexpression in pulmonary artery ECs isolated from patients with idiopathic pulmonary arterial hypertension (PAH), which may be associated with hyperproliferative phenotype of pulmonary artery ECs in PAH. However, the detailed molecular mechanisms explaining how Ano1 modulates proliferative signaling are still not clear. In addition, recent proteomics studies showed that Ano1 may interact with inner mitochondrial membrane (IMM) proteins in addition to plasma membrane (PM)-localized proteins. However, the expression levels of Ano1 and its functions in other cellular compartments including mitochondria are not fully understood. Here, we report that Ano1 is expressed in the mitochondria in pulmonary artery ECs and may be involved in EC proliferation by interacting with an IMM protein, optic atrophy 1 (OPA1) which is a critical regulator of mitochondrial bioenergetics and cristae formation. First, mitochondrial localization of Ano1 in addition to PM was found by protein fractionation and immunostaining of Ano1 in rat lung microvascular ECs (RLMVECs). We also confirmed mitochondrial Ano1 expression by live cell imaging of HEK293T cells expressing GFP-tagged Ano1. Next, a co-immunoprecipitation assay showed that overexpressed Ano1 interacts with OPA1 in HEK293T cells, indicating that Ano1 is expressed at IMM. We also confirmed that endogenous Ano1 interacts with OPA1 in mouse embryonic fibroblasts. Overexpression of Ano1 in HEK293T cells showed higher proliferation rate compared to controls (cells stably expressing GFP) at rest, but showed higher cell death rates in response to oxidative stress compared to control compared to control cells. Lastly, we found that treatment of a small molecule Ano1 activator Eact promotes apoptosis in pulmonary artery ECs from patients with idiopathic PAH, which likely occurs via depolarization of mitochondrial membrane potential by opening of mitochondrial Ano1. In summary, we found that 1) Ano1 is expressed in both the IMM and the PM, 2) Ano1 and OPA1 physically interact at IMM, 3) overexpression of Ano1 does increase proliferation, but Ano1 activation promotes cellular apoptosis. Interactions between Ano1 and OPA1 may provide a molecular mechanism underlying the hyperproliferation of pulmonary artery ECs during PAH. In addition, Ano1 activation may serve as a new therapeutic tool in PAH associated with hyperproliferative ECs.