Regulation of CLC chloride channels by their beta-subunits

我们研究了细胞内分选机制的肾CLC氯通道的β亚基barttin。为了解决这一问题，我们重点研究了barttin自身的排序机制以及CLC-K与barttin之间的相互作用. CLC-K和barttin相互作用结构域的鉴定通过构建CLC-K和barttin的截短突变体并进行免疫共沉淀分析，我们研究了CLC-K和barttin如何形成异源寡聚体。我们发现barttin在其结构域B和C以及结构域J和K上与CLC-K结合。barttin的两个跨膜区是其与CLC-K结合所必需的。用药物拯救致病突变体巴丁的试验。巴丁将CLC-K募集到质膜上。R8 L barttin是一种致病突变体，保留在内质网（ER）中。但是，它可以与CLC-K结合。因此，CLC-K也保留在ER中。这是Barttin突变引起Bartter综合征的主要机制。为了从ER释放R8 L并使其转运至质膜，使用稳定表达R8 L的MDCK细胞测试了包括化学伴侣和姜黄素在内的多种药物。发现姜黄素（10 μ M）、甘油（1 M）和DMSO（2%）有效地从ER释放R8 L。为了在体内测试这种潜在的治疗策略，我们制备了用于制造R8 L基因敲入小鼠的靶向构建体。barttin基底外侧分选信号的鉴定通过在MDCK细胞中表达barttin的几个截短突变体来确定barttin的基底外侧分选信号。barttin的羧基胞质区有一段15-20个氨基酸的序列是其基底外侧分选所必需的，这可能是一种新的基底外侧分选信号。

We investigated intracellular sorting mechanisms of renal CLC chloride channels by their beta-subunit barttin. To solve this issue, we focused on the sorting mechanisms of barttin itself and how CLC-K and barttin were interacted.1. Identification of CLC-K and barttin inter-acting domains.We investigated how CLC-K and barttin form hetero-oligomer by generating several truncated mutants of CLC-K and barttin and performing co-immunoprecipitation assay. We found that barttin binds to CLC-K on its domain B and C, and domain J and K. Two membrane spanning regions of barttin are necessary for its binding to CLC-K.2. Trial to rescue a disease-causing mutant barttin by drugs.Barttin recruits CLC-K to the plasma membranes. R8L barttin, a disease-causing mutant, is retained in endoplasmic reticulum (ER). However, it can bind to CLC-K. Accordingly, CLC-K is also retained in ER. This is a major mechanism of Bartter syndrome caused by barttin mutaions. In order to release R8L from ER and make it translocate to plasma membranes, several drugs including chemical chaperones and curcumin were tested using R8L stably expressing MDCK cells. Curcumin (10uM), glycerol (1M), and DMSO (2%) were found to be effective in releasing R8L from ER. To test this potential therapeutic strategy in vivo, we prepared a targeting construct for making R8L knock-in mouse.3. Identification of basolateral sorting signal of barttin.Basolateral sorting signal of barttin was determined by expressing several truncated mutants of barttin in MDCK cells. A 15-20 amino acid stretch in the carboxy cytoplasmic region of barttin was necessary for its basolateral sorting, which may be a new type of basolateral sorting signal.