ClC-3 chloride channels facilitate endosomal acidification and chloride accumulation
ClC-3 chloride channels facilitate endosomal acidification and chloride accumulation
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DOI：
10.1074/jbc.m407030200
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发表时间：
2005-01-14
影响因子：
4.8
通讯作者：
Verkman, AS
中科院分区：
文献类型：
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作者：
Hara-Chikuma, M;Yang, BX;Verkman, AS
我们通过测量对照组与缺乏ClC-3的肝细胞以及对照组与转染了ClC-3的中国仓鼠卵巢细胞的内体pH和氯离子浓度[Cl-]，研究了ClC-3氯离子通道在内体酸化中的作用。核内体用pH或[Cl-]感应荧光转铁蛋白（Tf）或α(2)-巨球蛋白（α (2)M）标记，前者靶向早期/循环核内体，后者靶向晚期核内体。在脉冲标记追踪实验中，野生型小鼠肝细胞原代培养中，[Cl-]在α (2) m标记的内体中内化后仅为19 mM，在45分钟内增加到58 mM，而pH从7.1下降到5.4。ClC-3敲除小鼠肝细胞内体酸化和[Cl-]积累明显受损，[Cl-]从16 mM增加到43 mM， pH从7.1降低到6.0。酸化和Cl的积累被巴菲霉素阻断。在tf标记的内体中，内化后15分钟，野生型的[Cl-]为46 mM，而clc -3缺陷肝细胞为35 mM，相应的pH为6.1，而6.5。从野生型小鼠肝细胞中分离的α (2) m标记的内体中发现Cl-电导率比ClC-3缺失小鼠增加了大约4倍。相反，高尔基酸化在clc -3缺失的肝细胞中没有受损。在转染表达ClC-3A的中国仓鼠卵巢细胞中，内体酸化和[Cl-]积累增强。α (2) m标记的内体中[Cl-]在45分钟时为42 mM（对照）和53 mM (ClC-3A)，相应的pH为5.8和5.2；15分钟时，tf标记内体中的[Cl-]为37 mM（对照）和49 mM (ClC-3A)， pH分别为6.3和5.9。我们的研究结果提供了直接证据，证明ClC-3参与了内体酸化，通过液泡H+泵产生的内正膜电位的Cl-分流。
We investigated the involvement of ClC-3 chloride channels in endosomal acidification by measurement of endosomal pH and chloride concentration [Cl-] in control versus ClC-3-deficient hepatocytes and in control versus ClC-3-transfected Chinese hamster ovary cells. Endosomes were labeled with pH or [Cl-]-sensing fluorescent transferrin (Tf), which targets to early/recycling endosomes, or alpha(2)-macroglobulin (alpha(2)M), which targets to late endosomes. In pulse label-chase experiments, [Cl-] was 19 mM just after internalization in alpha(2)M-labeled endosomes in primary cultures of hepatocytes from wildtype mice, increasing to 58 mM over 45 min, whereas pH decreased from 7.1 to 5.4. Endosomal acidification and [Cl-] accumulation were significantly impaired in hepatocytes from ClC-3 knock-out mice, with [Cl-] increasing from 16 to 43 mM and pH decreasing from 7.1 to 6.0. Acidification and Cl- accumulation were blocked by bafilomycin. In Tf-labeled endosomes, [Cl-] was 46 mM in wild-type versus 35 mM in ClC-3-deficient hepatocytes at 15 min after internalization, with corresponding pH of 6.1 versus 6.5. Approximately 4-fold increased Cl- conductance was found in alpha(2)M-labeled endosomes isolated from hepatocytes of wild-type versus ClC-3 null mice. In contrast, Golgi acidification was not impaired in ClC-3-deficient hepatocytes. In transfected Chinese hamster ovary cells expressing ClC-3A, endosomal acidification and [Cl-] accumulation were enhanced. [Cl-] in alpha(2)M-labeled endosomes was 42 mM ( control) versus 53 mM (ClC-3A) at 45 min, with corresponding pH 5.8 versus 5.2; [Cl-] in Tf-labeled endosomes at 15 min was 37 mM (control) versus 49 mM (ClC-3A) with pH 6.3 versus 5.9. Our results provide direct evidence for involvement of ClC-3 in endosomal acidification by Cl- shunting of the interior-positive membrane potential created by the vacuolar H+ pump.