Department of Pharmacology, Cardiac and Cerebral Vascular Research Center, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou 510080, People's Republic of China.
Cardiovasc Res. 2013 Jun 1;98(3):469-78. doi: 10.1093/cvr/cvt063. Epub 2013 Mar 27.
The ClC-3 chloride channel (and current, ICl,ClC-3) plays an important role in cell volume regulation, proliferation, and apoptosis in vascular smooth muscle cells, and is a potential target for prevention of vascular remodelling and stroke. However, modulation of ICl,ClC-3 by intercellular signalling is not fully understood. Although it has been suggested that tyrosine phosphorylation is required for ICl,ClC-3 activation, the potential tyrosine residues in the ClC-3 protein are not clear. In the present study, the critical tyrosine residues in ClC-3 protein were investigated.
Site-specific mutagenesis, immunoprecipitation, patch clamp, and Cl(-) transport imaging techniques were employed. We found that activation of ICl,ClC-3 was associated with tyrosine phosphorylation of the ClC-3 protein. Three potential tyrosine residues, Y284, Y572, and Y631, were mutated to phenylalanine, and only mutation, at Y284 within a consensus Src-phosphorylation site, completely blocked ICl,ClC-3. Phosphomimetic mutation Y284D increased the Cl(-) current and Cl(-) efflux mediated by ClC-3. The Y284F mutation completely abolished the protective effect of ClC-3 on apoptosis, whereas the Y284D mutation potentiated it. There was an interaction between Src kinase and ClC-3 protein, and the Y284D mutation abrogated the inhibitory effect of SU6656, a Src family kinase inhibitor, on ClC-3 Cl(-) current.
Tyrosine 284 phosphorylation in the ClC-3 channel targeted by Src kinase is an important molecular mechanism for ClC-3 channel activation.
氯离子通道 3（ClC-3 氯离子通道和电流，ICl，ClC-3）在血管平滑肌细胞的细胞体积调节、增殖和凋亡中发挥重要作用，是预防血管重塑和中风的潜在靶点。然而，细胞间信号对 ICl，ClC-3 的调节作用尚未完全阐明。尽管已经提出酪氨酸磷酸化是 ICl，ClC-3 激活所必需的，但 ClC-3 蛋白中的潜在酪氨酸残基尚不清楚。本研究探讨了 ClC-3 蛋白中的关键酪氨酸残基。
采用定点突变、免疫沉淀、膜片钳和 Cl(-)转运成像技术。我们发现 ICl，ClC-3 的激活与 ClC-3 蛋白的酪氨酸磷酸化有关。三个潜在的酪氨酸残基 Y284、Y572 和 Y631 突变为苯丙氨酸，只有位于 Src 磷酸化位点保守序列内的 Y284 突变完全阻断了 ICl，ClC-3。磷酸模拟突变 Y284D 增加了 ClC-3 介导的 Cl(-)电流和 Cl(-)外排。Y284F 突变完全消除了 ClC-3 对细胞凋亡的保护作用，而 Y284D 突变增强了这种作用。Src 激酶与 ClC-3 蛋白之间存在相互作用，Y284D 突变消除了 Src 家族激酶抑制剂 SU6656 对 ClC-3 Cl(-)电流的抑制作用。
Src 激酶靶向的 ClC-3 通道中酪氨酸 284 的磷酸化是 ClC-3 通道激活的一个重要分子机制。
