HC opening is stimulated by many factors like low extracellular Ca 2+ concentration, oxidative stress, mechanical signals, and extracellular alkalinization ( Burra and Jiang, 2011 Decrock et al., 2011 Francis et al., 1999 Gault et al., 2014 Kar et al., 2012 Leithe et al., 2018 Schalper et al., 2010). However, pathophysiological conditions, such as injury or disease, drive their opening, leading to the augmentation of certain pathological conditions, such as increased inflammatory reactions ( Peng et al., 2022). Under normal physiological conditions, undocked Cx HCs remain mainly closed ( Sáez et al., 2010). Two HCs in adjacent cells can form a gap junction, which assists in cell-cell communication directly from the cytoplasm of adjoining cells, thus synchronizing cellular functions ( Iyyathurai et al., 2013). HCs function as a gate for molecular communication between intracellular and extracellular spaces, allowing the passage of molecules and the exchange of information across the membrane. Six Cx subunits form a hexamer as a hemichannel (HC), allowing molecules less than 1 kDa to pass through ( Goodenough and Paul, 2003 Jiang et al., 2007). The connexins (Cxs) family comprises 20 members in mice and 21 in humans ( Bedner et al., 2012 Söhl and Willecke, 2004). Together, our study suggests that mtCx43 HCs regulate mitochondrial ATP generation by mediating K +, H +, and ATP transfer across the mitochondrial inner membrane and the interaction with mitochondrial ATP synthase, contributing to the maintenance of mitochondrial redox levels in response to oxidative stress. The co-localization and interaction of mtCx43 and ATP synthase subunit F (ATP5J2) were confirmed by Förster resonance energy transfer and a protein pull-down assay. Additionally, live-cell imaging results demonstrated that the proton flux was dependent on mtCx43 HCs because its activity was specifically inhibited by an antibody targeting Cx43 C-terminus.
Cx43 KD had reduced intracellular reactive oxidative species levels and mitochondrial membrane potential. Cx43 knockdown (KD) by the CRISPR-Cas9 lentivirus system resulted in impairment of mitochondrial function, primarily manifested as decreased ATP production.
H 2O 2 increased the mtCx43 level accompanied by elevated mtCx43 HC activity, determined by dye uptake assay. In murine long bone osteocyte-Y4 cells, the translocation of Cx43 to mitochondria was increased under H 2O 2-induced oxidative stress. Here, we reported a new role of mitochondrial Cx43 (mtCx43) and hemichannels (HCs) in modulating mitochondria homeostasis and function in bone osteocytes under oxidative stress. Prior studies primarily focus on the function of cell surface expressed Cx43 channels. Oxidative stress is a major risk factor that causes osteocyte cell death and bone loss.
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