Molecular mechanisms of Bartter syndrome caused by mutations in the BSND gene
Molecular mechanisms of Bartter syndrome caused by mutations in the BSND gene
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DOI：
10.1007/s00418-003-0535-2
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发表时间：
2003-06-01
影响因子：
2.3
通讯作者：
Uchida, S
中科院分区：
文献类型：
--
作者：
Hayama, A;Rai, T;Uchida, S
Barttin是BSND的基因产物，被鉴定为负责Bartter综合征的第四个基因。已证明barttin与CLC-K氯离子通道的共表达显著诱导CLC-K电流的表达。然而，barttin如何与哺乳动物细胞中的CLC-K通道相互作用以及barttin的突变如何导致Bartter综合征仍然是未知的。在试图澄清的CLC-K2细胞定位的barttin表达的影响，我们检查了表达的CLC-K2氯离子通道和barttin，单独和组合，在哺乳动物细胞中的瞬时和稳定的表达系统。此外，我们产生了几个稳定的细胞系表达突变barttin，以澄清的后果，以前报告的barttin突变在巴特综合征。在免疫细胞化学中，CLC-K2在barttin表达不存在的情况下保留在高尔基体中，但当barttin存在时传递到质膜。Barttin与CLC-K2共沉淀，表明蛋白质-蛋白质相互作用。致病突变barttin，特别是R8 L，保留在细胞内，但其结合能力CLC-K2被保留。这导致CLC-K2在细胞内细胞器中与barttin的保留，以及质膜定位的损失。CLC-K2蛋白的稳定性也通过与barttin共表达而显著增加。这些结果阐明了barttin通过蛋白质-蛋白质相互作用决定CLC-K2的细胞定位。因此，CLC-K2的错误定位被确定为Bartter综合征的突变型Barttin的分子发病机制。
Barttin, a gene product of BSND, was identified as a fourth gene responsible for Bartter syndrome. The co-expression of barttin with CLC-K chloride channels has been demonstrated to dramatically induce the expression of CLC-K current. However, it remains unknown how barttin interacts with CLC-K channels in mammalian cells and how the mutations of barttin lead to Bartter syndrome. In an attempt to clarify the effect of barttin expression on CLC-K2 cellular localization, we examined the expression of the CLC-K2 chloride channel and barttin, solely and in combination, in transient and stable expression systems in mammalian cells. In addition, we generated several stable cell lines expressing mutant barttins to clarify the consequence of the previously reported barttin mutations in Bartter syndrome. In immunocytochemistry, CLC-K2 was retained in the Golgi in the absence of barttin expression, but delivered to the plasma membrane when barttin was present. Barttin was coprecipitated with CLC-K2, suggesting a protein-protein interaction. Disease-causing mutant barttins, especially R8L, were retained intracellularly, but their binding ability to CLC-K2 was preserved. This led to a retention of CLC-K2 in intracellular organelles with barttin, and a loss of plasma membrane localization. The stability of the CLC-K2 protein was also markedly increased by coexpression with barttin. These results clarified that barttin determined cellular localization of CLC-K2 by protein-protein interaction. Thus, the mislocalization of CLC-K2 was identified as the molecular pathogenesis of Bartter syndrome by mutant barttins.