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

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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.