The role of the carboxyl terminus in ClC chloride channel function
The role of the carboxyl terminus in ClC chloride channel function
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DOI：
10.1074/jbc.m312649200
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发表时间：
2004-03-26
影响因子：
4.8
通讯作者：
Fahlke, C
中科院分区：
文献类型：
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作者：
Hebeisen, S;Biela, A;Fahlke, C
人肌肉氯离子通道ClC-1具有位于细胞质中的398-氨基酸羧基末端结构域，并含有两个CBS（胱硫醚-β-合酶）结构域。为了研究这个区域的作用，我们研究了各种羧基末端截断在哺乳动物细胞中的异源表达，全细胞膜片钳记录，共聚焦成像。通道结构缺乏远端CBS结构域，CBS 2的部分，不产生功能性通道，而删除CBS 1是可以容忍的。ClC通道是具有两个离子传导途径（原孔）的二聚体蛋白质。在由一个野生型亚基和一个羧基末端完全缺失的亚基组成的异二聚体通道中，只有野生型原孔是功能性的，表明羧基末端支持原孔的功能。所有羧基末端截短的突变体通道融合到黄色荧光蛋白被翻译，大多数插入到质膜显示通过共聚焦显微镜。连接到羧基末端的各种片段的青色荧光蛋白的融合蛋白形成可溶性蛋白，其可以通过结合到某些截短的通道亚基而重新分布到表面膜。稳定的结合仅发生在单个亚基的羧基末端片段之间，而不发生在不同亚基的羧基末端之间，也不发生在羧基末端和跨膜结构域之间。然而，与跨膜结构域的相互作用可以改变特定羧基末端蛋白质的结合特性。我们的研究结果表明，CLC-1的羧基末端是不必要的细胞内运输，但通道功能的关键。羧基末端独立折叠并修饰双管通道的单个原孔。
The human muscle chloride channel ClC-1 has a 398-mino acid carboxyl-terminal domain that resides in the cytoplasm and contains two CBS (cystathionine-beta-synthase) domains. To examine the role of this region, we studied various carboxyl-terminal truncations by heterologous expression in mammalian cells, whole-cell patch clamp recording, and confocal imaging. Channel constructs lacking parts of the distal CBS domain, CBS2, did not produce functional channels, whereas deletion of CBS1 was tolerated. ClC channels are dimeric proteins with two ion conduction pathways (protopores). In heterodimeric channels consisting of one wild type subunit and one subunit in which the carboxyl terminus was completely deleted, only the wild type protopore was functional, indicating that the carboxyl terminus supports the function of the protopore. All carboxyl-terminal-truncated mutant channels fused to yellow fluorescent protein were translated and the majority inserted into the plasma membrane as revealed by confocal microscopy. Fusion proteins of cyan fluorescent protein linked to various fragments of the carboxyl terminus formed soluble proteins that could be redistributed to the surface membrane through binding to certain truncated channel subunits. Stable binding only occurs between carboxyl-terminal fragments of a single subunit, not between carboxyl termini of different subunits and not between carboxyl-terminal and transmembrane domains. However, an interaction with transmembrane domains can modify the binding properties of particular carboxyl-terminal proteins. Our results demonstrate that the carboxyl terminus of ClC-1 is not necessary for intracellular trafficking but is critical for channel function. Carboxyl termini fold independently and modify individual protopores of the double-barreled channel.