Localization of the nucleic acid channel regulatory subunit, cytosolic malate dehydrogenase.
Localization of the nucleic acid channel regulatory subunit, cytosolic malate dehydrogenase.
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核酸通道调节亚基、胞质苹果酸脱氢酶的定位。
DOI：
10.1007/s00232-008-9133-5
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发表时间：
2008
期刊：
影响因子：
--
通讯作者：
Klotman,PaulE
中科院分区：
文献类型：
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作者：
Hanss,Basil;Leal-Pinto,Edgar;Teixeira,Avelino;Tran,Baohuong;Lee,Chun-Hui;Henderson,ScottC;Klotman,PaulE
NACh是在大鼠肾脏近端小管顶膜中发现的核酸传导通道。它是由至少两种蛋白质组成的异源多聚体复合物：45-kDa孔形成亚基和36-kDa调节亚基。调节亚基赋予离子选择性并影响门控动力学。调节亚基已被确定为胞质苹果酸脱氢酶（cMDH）。cMDH在文献中被描述为不与质膜相关的可溶性蛋白质。然而，cMDH作为NACh的调节亚基的作用需要它存在于质膜上。为了解决这一矛盾，开始研究以确定是否可以在质膜上发现cMDH。在进行定位研究之前，鉴定了表达NACh的合适的模型系统。在LLC-PK 1细胞中鉴定了一个通道，该细胞系来源于猪近端小管，对核酸具有选择性，电导率约为10 pS。硫酸乙酰肝素或l-苹果酸盐可剂量依赖性阻断。这些特征与已报道的大鼠肾脏NACh相似，表明NACh存在于LLC-PK 1细胞中。因此，LLC-PK 1细胞被用作cMDH免疫定位的模型系统。免疫荧光和免疫电镜均显示cMDH存在于LLC-PK 1细胞的质膜上。这一发现支持先前的功能数据，描述了cMDH作为NACh的调节亚基的作用。
NACh is a nucleic acid–conducting channel found in apical membrane of rat kidney proximal tubules. It is a heteromultimeric complex consisting of at least two proteins: a 45-kDa pore-forming subunit and a 36-kDa regulatory subunit. The regulatory subunit confers ion selectivity and influences gating kinetics. The regulatory subunit has been identified as cytosolic malate dehydrogenase (cMDH). cMDH is described in the literature as a soluble protein that is not associated with plasma membrane. Yet a role for cMDH as the regulatory subunit of NACh requires that it be present at the plasma membrane. To resolve this conflict, studies were initiated to determine whether cMDH could be found at the plasma membrane. Before performing localization studies, a suitable model system that expressed NACh was identified. A channel was identified in LLC-PK1cells, a line derived from pig proximal tubule, that is selective for nucleic acid and has a conductance of approximately 10 pS. It exhibits dose-dependent blockade by heparan sulfate orl-malate. These characteristics are similar to what has been reported for NACh from rat kidney and indicate that NACh is present in LLC-PK1cells. LLC-PK1cells were therefore used as a model system for immunolocalization of cMDH. Both immunofluorescence and immunoelectron microscopy demonstrated cMDH at the plasma membrane of LLC-PK1cells. This finding supports prior functional data that describe a role for cMDH as the regulatory subunit of NACh.