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Functional roles and regulation of ClC-3B chloride channel in T llymphocytes

T淋巴细胞中ClC-3B氯通道的功能作用和调节

基本信息

批准号：
15590221
负责人：
OGURA Takehiko
金额：
$ 2.3万
依托单位：
Chiba University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15590221/
关键词：
ClC-3B tyrosine kinase T cell proliferation apoptosis chloride channel リンパ球チロシンリン酸化 ClC-3

项目摘要

1.Expression of mRNAs for outwardly rectifying ClC channels (ClC-3,-4,-5 ) was examined in human peripheral lymphocytes by RT-PCR. ClC-3 mRNA was most abundantly expressed in both resting and activated T cells. ClC-3 mRNA was also expressed in both resting and activated B cells. ClC-4 mRNA was detected in both resting and activated B cells, and ClC-5 mRNA was detected in resting T cells and B cells.2.When ClC-3B was co-expressed with tyrosine kinase p56^<lck> in COS1 cells, the ClC-3B protein was tyrosine-phosphorylated by p56^<lck>. Experiments using mutated ClC-3B clones revealed that the 859^<th> tyrosine residue (Y859) in the C-terminal intracellular region was specifically phosphorylated by p56^<lck>. The splicing variant ClC-3A, which dose not carry the corresponding tyrosine residue, was not phosphorylated by p56^<lck>.3.To verify whether tyrosine phosphorylation of the ClC-3B protein has some impact on its subcellular localization, immunocytochemical experiments were conducted. In HT-1080 cells, expression of the ClC-3B protein at surface plasma membrane was enhanced when it was co-expressed with p56^<lck>.4.Number of thymocytes was smaller in ClC-3 knockout mice(ClC-3KO)in comparison with that in wild type mice(WT). Number of spleen T cells was also smaller in ClC-3KO. The rate of proliferation triggered by T cell receptor(TCR) stimulation using anti-CD3 antibody (clone 2C11) was decelerated in T cells of ClC-3KO. Apoptotic cell death induced by TCR stimulation using anti-CD3 antibody (clone 2C11) was normal in activated T cells of ClC-3KO. Apoptotic cell death was also normally induced by fas stimulation using anti-fas antibody (clone Jo-2) in activated T cells of ClC-3KO.

1.用RT-PCR检测人外周血淋巴细胞外向整流型ClC通道（ClC-3、ClC-4、ClC-5）的mRNA表达。ClC-3 mRNA在静息和活化的T细胞中表达最丰富。ClC-3 mRNA在静息和活化的B细胞中均有表达。ClC-4 mRNA在静息和活化的B细胞中均有表达，ClC-5 mRNA在静息T细胞和B细胞中均有表达。2.当ClC-3B与酪氨酸激酶p56^共表达<lck>时，CLC-3B蛋白被p56^酪氨酸磷酸化<lck>。使用突变的ClC-3B克隆的实验显示，<th>C-末端细胞内区域的859^酪氨酸残基（Y859）被p56^特异性磷酸化<lck>。剪接变异体ClC-3A不携带相应的酪氨酸残基，不被p56磷酸化。<lck>3.为了验证ClC-3B蛋白的酪氨酸磷酸化是否对其亚细胞定位有影响，我们进行了免疫细胞化学实验。在HT-1080细胞中，当ClC-3B蛋白与p56蛋白共表达时，ClC-3B蛋白在细胞膜表面的表达增强。<lck>4.与野生型小鼠（WT）相比，ClC-3敲除小鼠（ClC-3 KO）的胸腺细胞数量较少。C1 C-3 KO中脾T细胞的数量也较小。在ClC-3 KO的T细胞中，由使用抗CD 3抗体（克隆2C 11）的T细胞受体（TCR）刺激触发的增殖速率减慢。使用抗CD 3抗体（克隆2C 11）通过TCR刺激诱导的凋亡性细胞死亡在ClC-3 KO的活化T细胞中是正常的。在ClC-3 KO的活化T细胞中，使用抗Fas抗体（克隆Jo-2）通过Fas刺激也正常诱导凋亡细胞死亡。