Anoctamin‐1 Expression at the Mitochondrial Membrane of Pulmonary Artery Endothelial Cells

Abstract

Anoctamin‐1 (Ano1) is a calcium‐activated chloride channel that can regulate cell proliferation and cell cycle in endothelial cells (ECs). According to a recent proteomics study, Ano1 interacts with mitochondrial proteins in addition to plasma‐membrane proteins. However, the expression levels of Ano1 and its functions in other cellular compartments remain unclear. We previously reported that Ano1 is overexpressed in pulmonary artery ECs isolated from patients with idiopathic pulmonary arterial hypertension (PAH) and that Ano1 activation promotes apoptosis of ECs from patients with idiopathic PAH, likely via depolarization of mitochondrial membrane potential and increased mitochondrial reactive oxygen species generation. Therefore, we hypothesized that Ano1 forms channels at the inner mitochondrial membrane (IMM) in addition to the plasma membrane, which may contribute to a hyperproliferative phenotype of ECs under PAH. Here, we determined the mitochondrial expression of Ano1 and further investigated its role in the mitochondria.Rat lung microvascular ECs (RLMVECs) were used for biochemical and cell biological assays. HEK293T cells that stably overexpress Ano1 were generated and used for binding assays with mitochondrial inner membrane proteins. RLMVEC lysates were separated via centrifugation to obtain a mitochondria‐enriched fraction (Mito) and a cytosolic fraction (Cyto) containing other cellular organelles. Cyto was further separated by high‐speed centrifugation into a soluble fraction (Sol) and the insoluble particulate fraction (Insol) that is comprised of plasma and other organelle (e.g., Golgi and endoplasmic reticulum) membranes. The purity of each fraction and the structural integrity of isolated mitochondria in the Mito were assessed by immunoblotting of mitochondrial and non‐mitochondrial protein markers.The subcellular localization of Ano1 was determined by co‐immunostaining Ano1 and a mitochondria‐localized protein, translocase of the outer mitochondrial membrane 20 (TOM20), in RLMVECs. The mitochondrial localization of Ano1 was also confirmed by live cell imaging of HEK293T cells expressing GFP‐tagged Ano1. Biochemical assays using protein fractionation from RLMVECs showed Ano1 protein expression in the Mito as well as the Insol obtained from the Cyto. The data from these systems indicate that Ano1 is localized in both the plasma membrane and mitochondria. Moreover, using HEK293T cells stably overexpressing Ano1, we found by immunoprecipitation that Ano1 interacts with a mitochondrial fusion protein OPA1 which possesses a trans‐membrane domain at the IMM.These results indicate the existence of Ano1 protein in the mitochondria, especially at the IMM, as well as the plasma membrane. Since OPA1 is the critical regulator of mitochondrial bioenergetics and cristae formation, increased Ano1 and OPA1 interactions may be partly involved in the molecular mechanism underlying the hyperproliferation of pulmonary artery ECs during PAH.Support or Funding InformationJ.O.‐U. was supported by NIH/NHLBI R01HL136757B.S.J was supported by AHA 8CDA34110091R.C. is supported by NIH/NHLBI R01HL135236G.C is supported by R01HL128661This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.