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Molecular mechanism of endocytosis disorder induced by mutation of CIC-5 Chloride Channel gene

CIC-5氯离子通道基因突变引起内吞障碍的分子机制

基本信息

批准号：
12671030
负责人：
SAKAMOTO Hisato
金额：
$ 2.18万
依托单位：
Kitasato University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

项目摘要

A number of mutations in the human CIC-5 chloride (CI-) channel genes might be responsible for Dent's disease. In the present study, we determined whether mutant CIC-5 is involved in the trafficking disorder using stable transfection system in the mammalian cultured cells, CHO-K1. When expressed in CHO-K1 cells, two kinds of mutants CIC-5 (S270R and R280P) with naturally occurring missense mutations in Dent's disease were correctly processed and trafficked to specific destination, early endosome, even if associated with aberrant structure. In contrast, the trafficking of the channel to plasma membrane was only detected in wild type. Because the sites of both mutations belong to the specific loop between the predicted transmembrane domain D5 and D6 of CIC-5, these findings suggest that the structural modification in this region affect on the assembling and subsequent trafficking of the channel in the endosome rather than intracellular sorting of channel. Furthermore, we identified a new … More mutant with flame-shift in C-terminal region might result in retention and rapid degradation in an intracellular compartment. Taken together, these findings suggest that to presume the function of defective gene product at the level of intracellular sorting is of fundamental importance to assess the possibility of therapeutic intervention.We also identified and characterized that CIC-5 channel expressed in osteoclast and gastric parietal cells, in both of witch the channel co-localized with H^+-ATPase. A novel finding is that calcitonin might inhibit the function of osteoclast through the internalization of CIC-5 related channel in an actin-associated manner.The specific anti-ClC-3 monoclonal antibody was successfully established to evaluate the interrelationship between two homologous channels, CIC-3 and CIC-5, in the intercalated cells (IC). CIC-3 was differentially localized in type C IC compared with CIC-5 localized in type A IC. Further experiments are necessary to confirm the role ofCIC-5 and CIC-3 in IC. Less

人类CIC-5氯离子（CI-）通道基因的一些突变可能是邓特病的原因。在本研究中，我们使用稳定转染系统在哺乳动物培养细胞CHO-K1中确定突变体CIC-5是否参与运输障碍。当在CHO-K1细胞中表达时，两种在邓氏病中自然发生错义突变的ic -5 （S270R和R280P）被正确处理并运输到特定的目的地，即早期内体，即使与结构异常有关。相比之下，仅在野生型中检测到通道到质膜的运输。由于这两个突变位点都属于CIC-5预测的跨膜结构域D5和D6之间的特定环，这些发现表明，该区域的结构修饰影响了内体中通道的组装和随后的运输，而不是通道的细胞内分选。此外，我们还发现了一个新的…更多的突变体，其c端区域的火焰位移可能导致细胞内隔室的保留和快速降解。综上所述，这些发现表明，在细胞内分选水平上推测缺陷基因产物的功能对于评估治疗干预的可能性具有重要意义。我们还发现并表征了ic -5通道在破骨细胞和胃壁细胞中表达，在这两种情况下，通道都与H^+- atp酶共定位。一项新的研究发现，降钙素可能通过内化CIC-5相关通道，以肌动蛋白相关的方式抑制破骨细胞的功能。成功地建立了特异性的抗clc -3单克隆抗体，以评价插层细胞（IC）中两个同源通道CIC-3和CIC-5之间的相互关系。CIC-3在C型IC中的定位与CIC-5在A型IC中的定位存在差异，需要进一步的实验来证实CIC-5和CIC-3在IC中的作用