The calcium-sensitive large-conductance potassium channel (BK/Maxi K) is present in the inner mitochondrial membrane of rat brain
The calcium-sensitive large-conductance potassium channel (BK/Maxi K) is present in the inner mitochondrial membrane of rat brain
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DOI：
10.1016/j.neuroscience.2006.01.061
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发表时间：
2006-01-01
期刊：
影响因子：
3.3
通讯作者：
Haddad, G. G.
中科院分区：
文献类型：
--
作者：
Douglas, R. M.;Lai, J. C. K.;Haddad, G. G.
已知大电导电压和钙敏感通道在中枢神经元的质膜中表达;然而，最近的数据表明，大电导电压和钙敏感通道也可能存在于线粒体膜中。为了确定大电导电压和钙敏感性通道的亚细胞定位和分布，通过Ficoll-蔗糖密度梯度离心获得的大鼠脑组分通过Western印迹，免疫细胞化学和免疫金电子显微镜检查。免疫印迹研究表明，存在一致的信号的α亚基的大电导电压和钙敏感通道的线粒体部分。双标记免疫荧光还表明，大电导电压和钙敏感性通道存在于线粒体中，并与线粒体特异性蛋白质共定位，如内膜23，腺嘌呤核苷酸转运蛋白，细胞色素c氧化酶或复合物IV-亚基1和线粒体内膜蛋白的易位酶，但不与钙连接蛋白，内质网标记共定位。离散的亚细胞组分的Western印迹表明，细胞色素c氧化酶或复合物IV-亚基1只在线粒体组分中表达，而肌动蛋白，乙酰胆碱酯酶，钙粘蛋白，钙连接蛋白，58 kDa高尔基蛋白，乳酸脱氢酶和微管相关蛋白1没有，证明线粒体组分的纯度。线粒体颗粒的电子显微镜检查表明线粒体内的金颗粒标记，表明线粒体内膜中存在大电导电压和钙敏感通道。这些研究为线粒体中存在大电导电压和钙敏感通道提供了具体的形态学证据：我们的研究结果证实了最近神经胶质瘤和心脏细胞中线粒体大电导电压和钙敏感通道的电生理证据。(c)2006年IBRO。由爱思唯尔有限公司出版。保留所有权利。
Large-conductance voltage- and calcium-sensitive channels are known to be expressed in the plasmalemma of central neurons; however, recent data suggest that large-conductance voltage- and calcium-sensitive channels may also be present in mitochondrial membranes. To determine the subcellular localization and distribution of large-conductance voltage- and calcium-sensitive channels, rat brain fractions obtained by Ficoll-sucrose density gradient centrifugation were examined by Western blotting, immunocytochemistry and immuno-gold electron microscopy. Immunoblotting studies demonstrated the presence of a consistent signal for the alpha subunit of the large-conductance voltage- and calcium-sensitive channel in the mitochondrial fraction. Double-labeling immunofluorescence also demonstrated that large-conductance voltage- and calcium-sensitive channels are present in mitochondria and co-localize with mitochondrial-specific proteins such as the translocase of the inner membrane 23, adenine nucleotide translocator, cytochrome c oxidase or complex IV-subunit 1 and the inner mitochondrial membrane protein but do not co-localize with calnexin, an endoplasmic reticulum marker. Western blotting of discrete subcellular fractions demonstrated that cytochrome c oxidase or complex IV-subunit 1 was only expressed in the mitochondrial fraction whereas actin, acetylcholinesterase, cadherins, calnexin, 58 kDa Golgi protein, lactate dehydrogenase and microtubule-associated protein 1 were not, demonstrating the purity of the mitochondrial fraction. Electron microscopic examination of the mitochondrial pellet demonstrated gold particle labeling within mitochondria, indicative of the presence of large-conductance voltage- and calcium-sensitive channels in the inner mitochondrial membrane. These studies provide concrete morphological evidence for the existence of large-conductance voltage- and calcium-sensitive channels in mitochondria: our findings corroborate the recent electrophysiological evidence of mitochondrial large-conductance voltage- and calcium-sensitive channels in glioma and cardiac cells. (c) 2006 IBRO. Published by Elsevier Ltd. All rights reserved.