Connexin 43 hemichannels regulate mitochondrial ATP generation, mobilization, and mitochondrial homeostasis against oxidative stress.
Connexin 43 hemichannels regulate mitochondrial ATP generation, mobilization, and mitochondrial homeostasis against oxidative stress.
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DOI：
10.7554/elife.82206
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发表时间：
2022-11-08
期刊：
影响因子：
7.7
通讯作者：
Jiang JX
中科院分区：
文献类型：
--
作者：
Zhang J;Riquelme MA;Hua R;Acosta FM;Gu S;Jiang JX
氧化应激是导致骨细胞死亡和骨丢失的主要危险因素。以往的研究主要集中在细胞表面表达的Cx43通道的功能。在这里，我们报告了一个新的作用，线粒体Cx43（mtCx 43）和半通道（HC）在调节线粒体稳态和功能的骨骨细胞在氧化应激。在小鼠长骨骨细胞-Y 4细胞中，Cx43向线粒体的移位在H2 O2诱导的氧化应激下增加。H2 O2增加mtCx 43水平，同时升高mtCx 43 HC活性，通过染料摄取测定。通过CRISPR-Cas9慢病毒系统的Cx43敲低（KD）导致线粒体功能受损，主要表现为ATP产生减少。Cx43 KD降低了细胞内活性氧化物水平和线粒体膜电位。此外，活细胞成像结果表明质子通量依赖于mtCx 43 HC，因为其活性被靶向Cx43 C末端的抗体特异性抑制。通过Förster共振能量转移和蛋白质下拉试验证实了mtCx 43和ATP合酶亚基F（ATP 5 J2）的共定位和相互作用。总之，我们的研究表明，mtCx 43 HC调节线粒体ATP的产生介导的K+，H+，和ATP跨线粒体内膜和线粒体ATP合酶的相互作用，有助于维持线粒体氧化还原水平，以响应氧化应激。
Oxidative stress is a major risk factor that causes osteocyte cell death and bone loss. Prior studies primarily focus on the function of cell surface expressed Cx43 channels. 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. In murine long bone osteocyte-Y4 cells, the translocation of Cx43 to mitochondria was increased under H2O2-induced oxidative stress. H2O2 increased the mtCx43 level accompanied by elevated mtCx43 HC activity, determined by dye uptake assay. Cx43 knockdown (KD) by the CRISPR-Cas9 lentivirus system resulted in impairment of mitochondrial function, primarily manifested as decreased ATP production. Cx43 KD had reduced intracellular reactive oxidative species levels and mitochondrial membrane potential. 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. 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. 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.