PDF(Pubmed)
Abstract：
Early/late endosomes, recycling endosomes, and lysosomes together form the endo-lysosomal recycling pathway. This system plays a crucial role in cell differentiation and survival, and dysregulation of the endo-lysosomal system appears to be important in the pathogenesis of neurodevelopmental and neurodegenerative diseases. Each endo-lysosomal compartment fulfils a specific function, which is supported by ion transporters and channels that modify ion concentrations and electrical gradients across endo-lysosomal membranes. CLC-type Cl-/H+ exchangers are a group of endo-lysosomal transporters that are assumed to regulate luminal acidification and chloride concentration in multiple endosomal compartments. Heterodimers of ClC-3 and ClC-4 localize to various internal membranes, from the endoplasmic reticulum and Golgi to recycling endosomes and late endosomes/lysosomes. The importance of ClC-4-mediated ion transport is illustrated by the association of naturally occurring CLCN4 mutations with epileptic encephalopathy, intellectual disability, and behavioral disorders in human patients. However, how these mutations affect the expression, subcellular localization, and function of ClC-4 is insufficiently understood. We here studied 12 CLCN4 variants that were identified in patients with X-linked intellectual disability and epilepsy and were already characterized to some extent in earlier work. We analyzed the consequences of these mutations on ClC-4 ion transport, subcellular trafficking, and heterodimerization with ClC-3 using heterologous expression in mammalian cells, biochemistry, confocal imaging, and whole-cell patch-clamp recordings. The mutations led to a variety of changes in ClC-4 function, ranging from gain/loss of function and impaired heterodimerization with ClC-3 to subtle impairments in transport functions. Our results suggest that even slight functional changes to the endosomal Cl-/H+ exchangers can cause serious neurological symptoms.
摘要：
早期/晚期内体，回收内体，和溶酶体一起形成内-溶酶体再循环途径。该系统在细胞分化和存活中起着至关重要的作用，和内溶酶体系统的失调似乎在神经发育和神经退行性疾病的发病机理中很重要。每个内溶酶体区室履行特定的功能，它由离子转运蛋白和通道支持，这些离子转运蛋白和通道可以改变离子浓度和跨溶酶体膜的电梯度。CLC型Cl-/H交换剂是一组内溶酶体转运蛋白，被认为可以调节多个内体区室中的管腔酸化和氯化物浓度。ClC-3和ClC-4的异二聚体定位于各种内膜，从内质网和高尔基体到再循环内体和晚期内体/溶酶体。通过自然发生的CLCN4突变与癫痫性脑病的关联，说明了ClC-4介导的离子转运的重要性。智力残疾,和人类患者的行为障碍。然而,这些突变如何影响表达，亚细胞定位，和ClC-4的功能没有被充分理解。我们在这里研究了在X连锁智力障碍和癫痫患者中鉴定出的12种CLCN4变体，并且在早期工作中已经在一定程度上进行了表征。我们分析了这些突变对ClC-4离子转运的影响，亚细胞贩运，和异二聚化与ClC-3使用异源表达在哺乳动物细胞，生物化学,共焦成像，和全细胞膜片钳记录。这些突变导致了ClC-4功能的各种变化，从功能的获得/丧失和与ClC-3的异二聚体化受损到运输功能的细微损害。我们的结果表明，即使内体Cl-/H交换体的轻微功能变化也会引起严重的神经系统症状。
