Coexpression of complementary fragments of ClC-5 and restoration of chloride channel function in a Dent's disease mutation.
Coexpression of complementary fragments of ClC-5 and restoration of chloride channel function in a Dent's disease mutation.
复制标题
Dent 病突变中 ClC-5 互补片段的共表达和氯离子通道功能的恢复。
DOI：
10.1152/ajpcell.00009.2003
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发表时间：
2004
期刊：
影响因子：
--
通讯作者：
Wills,NK
中科院分区：
文献类型：
--
作者：
Mo,L;Xiong,W;Qian,T;Sun,H;Wills,NK
人类遗传性疾病登特氏病与氯离子通道ClC-5的功能丧失突变有关。这些突变中的许多涉及过早终止密码子的插入，导致蛋白质的截短。我们确定是否可以通过共表达截短的蛋白质（含有NH 2-末端区域）与其互补的“缺失”COOH-末端区域来恢复ClC-5的功能活性。在COOH-末端区域（V655）的任意位点和四个Dent病突变位点（R347、Y 617、R648和R704）处产生用于ClC-5的由互补的N和C蛋白区域组成的分流通道构建体。在V655的分裂通道的互补片段的共表达产生的电流与阴离子和pH敏感性类似的野生型ClC-5。当共表达Dent突变R648的互补分裂通道构建体时，通道活性类似地恢复，但当共表达突变R347、Y 617或R704的分裂通道时，没有发现ClC-5电流。COS-7细胞的免疫印迹和免疫荧光研究表明，N或C蛋白片段可以瞬时表达，并在质膜中检测到，即使在分裂通道，未能显示功能活性。结果表明，C1 C-5通道活性可以通过表达C1 C-5分子的缺失部分来恢复特定的Dent突变。
The human hereditary disorder Dent's disease is linked to loss-of-function mutations of the chloride channel ClC-5. Many of these mutations involve insertion of premature stop codons, resulting in truncation of the protein. We determined whether the functional activity of ClC-5 could be restored by coexpression of the truncated protein (containing the NH2-terminal region) with its complementary “missing” COOH-terminal region. Split channel constructs for ClC-5, consisting of complementary N and C protein regions, were created at an arbitrary site in the COOH-terminal region (V655) and at four Dent's disease mutation sites (R347, Y617, R648, and R704). Coexpression of complementary fragments for the split channel at V655 produced currents with anion and pH sensitivity similar to those of wild-type ClC-5. Channel activity was similarly restored when complementary split channel constructs made for Dent's mutation R648 were coexpressed, but no ClC-5 currents were found when split channels for mutations R347, Y617, or R704 were coexpressed. Immunoblot and immunofluorescence studies of COS-7 cells revealed that N or C protein fragments could be transiently expressed and detected in the plasma membrane, even in split channels that failed to show functional activity. The results suggest that ClC-5 channel activity can be restored for specific Dent's mutations by expression of the missing portion of the ClC-5 molecule.