Division of Cardiology, The Johns Hopkins University School of Medicine, Baltimore, MD, USA.
Circ Res. 2012 Aug 3;111(4):446-54. doi: 10.1161/CIRCRESAHA.112.266445. Epub 2012 Jul 17.
Activation of the mitochondrial ATP-sensitive potassium channel (mitoK(ATP)) has been implicated in the mechanism of cardiac ischemic preconditioning, yet its molecular composition is unknown.
To use an unbiased proteomic analysis of the mitochondrial inner membrane to identify the mitochondrial K(+) channel underlying mitoK(ATP).
Mass spectrometric analysis was used to identify KCNJ1(ROMK) in purified bovine heart mitochondrial inner membrane and ROMK mRNA was confirmed to be present in neonatal rat ventricular myocytes and adult hearts. ROMK2, a short form of the channel, is shown to contain an N-terminal mitochondrial targeting signal, and a full-length epitope-tagged ROMK2 colocalizes with mitochondrial ATP synthase β. The high-affinity ROMK toxin, tertiapin Q, inhibits mitoK(ATP) activity in isolated mitochondria and in digitonin-permeabilized cells. Moreover, short hairpin RNA-mediated knockdown of ROMK inhibits the ATP-sensitive, diazoxide-activated component of mitochondrial thallium uptake. Finally, the heart-derived cell line, H9C2, is protected from cell death stimuli by stable ROMK2 overexpression, whereas knockdown of the native ROMK exacerbates cell death.
The findings support ROMK as the pore-forming subunit of the cytoprotective mitoK(ATP) channel.
线粒体三磷酸腺苷敏感性钾通道（mitoKATP）的激活与心脏缺血预处理的机制有关，但它的分子组成尚不清楚。
利用线粒体内膜的无偏蛋白组学分析来鉴定mitoKATP 的线粒体 K(+)通道。
质谱分析用于鉴定纯化的牛心线粒体内膜中的 KCNJ1（ROMK），并证实 ROMK mRNA 存在于乳鼠心室肌细胞和成人心脏中。该通道的短形式 ROMK2 含有一个 N 端线粒体靶向信号，全长表位标记的 ROMK2 与线粒体 ATP 合酶 β 共定位。高亲和力的 ROMK 毒素，tertiapin Q，抑制分离线粒体和二辛可宁碱通透细胞中的 mitoK(ATP)活性。此外，短发夹 RNA 介导的 ROMK 敲低抑制了线粒体铊摄取的 ATP 敏感、二氮嗪激活部分。最后，心脏来源的细胞系 H9C2 通过稳定的 ROMK2 过表达来保护免受细胞死亡刺激，而天然 ROMK 的敲低则加剧了细胞死亡。
这些发现支持 ROMK 作为保护性 mitoK(ATP)通道的孔形成亚基。
