Threonine532 phosphorylation in ClC-3 channels is required for angiotensin II-induced Cl- current and migration in cultured vascular smooth muscle cells
Threonine532 phosphorylation in ClC-3 channels is required for angiotensin II-induced Cl- current and migration in cultured vascular smooth muscle cells
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ClC-3 通道中苏氨酸532 磷酸化是血管紧张素 II 诱导的 Cl(-) 电流和培养血管平滑肌细胞迁移所必需的
DOI：
10.1111/bph.13385
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发表时间：
2016-02-01
影响因子：
7.3
通讯作者：
Guan, Yong-Yuan
中科院分区：
文献类型：
--
作者：
Ma, Ming-Ming;Lin, Cai-Xia;Guan, Yong-Yuan
背景与目的血管紧张素II(AngII)诱导血管平滑肌细胞(VSMC)迁移和生长，参与某些心血管疾病的血管重塑。血管紧张素转换酶II也可激活氯电流，但其机制尚不清楚。实验方法采用A10细胞系和原代培养的VSMC，分别来自对照组、ClC-3通道缺失小鼠和WT小鼠。采用的技术包括全细胞膜片钳、免疫共沉淀、定点突变和Western blotting。在VSMC中，血管紧张素Ⅱ诱导的氯电流是由氯离子通道ClC-3携带的。CLC-3通道缺失小鼠的VSMC不存在该电流。血管紧张素Ⅱ诱导的氯电流涉及ClC-3通道与Rho-kinase2(ROCK2)之间的相互作用，C端或C端截短ClC-3蛋白、ROCK2 siRNA和免疫共沉淀实验显示。ROCK2对Thr(532)处ClC-3通道的磷酸化是血管紧张素转换酶诱导的氯电流和VSMC迁移的关键。CLC-3 T532D突变体(Thr(532)突变为天冬氨酸)模拟磷酸化ClC-3蛋白，显著增强Angii诱导的氯电流和VSMC迁移，而ClC-3 T532A(Thr(532)突变为丙氨酸)具有相反的作用。在对ROCK2抑制剂Y27632不敏感的ClC-3通道缺失小鼠的VSMC中，Angii诱导的细胞迁移显著减少。此外，在ClC-3基因缺失的小鼠中，可能通过ROCK2途径减少了血管紧张素Ⅱ诱导的脑血管重塑。结论和意义在血管紧张素Ⅱ诱导的氯离子电流和VSMC迁移过程中，ROCK2对Thr(532)处的ClC-3蛋白的磷酸化是必需的，这可能参与了血管紧张素Ⅱ诱导的高血压血管重塑。
Background and PurposeAngiotensin II (AngII) induces migration and growth of vascular smooth muscle cell (VSMC), which is responsible for vascular remodelling in some cardiovascular diseases. Ang II also activates a Cl- current, but the underlying mechanism is not clear.Experimental ApproachThe A10 cell line and primary cultures of VSMC from control, ClC-3 channel null mice and WT mice made hypertensive with AngII infusions were used. Techniques employed included whole-cell patch clamp, co-immunoprecipitation, site-specific mutagenesis and Western blotting,Key ResultsIn VSMC, AngII induced Cl- currents was carried by the chloride ion channel ClC-3. This current was absent in VSMC from ClC-3 channel null mice. The AngII-induced Cl- current involved interactions between ClC-3 channels and Rho-kinase 2 (ROCK2), shown by N- or C-terminal truncation of ClC-3 protein, ROCK2 siRNA and co-immunoprecipitation assays. Phosphorylation of ClC-3 channels at Thr(532) by ROCK2 was critical for AngII-induced Cl- current and VSMC migration. The ClC-3 T532D mutant (mutation of Thr(532) to aspartate), mimicking phosphorylated ClC-3 protein, significantly potentiated AngII-induced Cl- current and VSMC migration, while ClC-3 T532A (mutation of Thr(532) to alanine) had the opposite effects. AngII-induced cell migration was markedly decreased in VSMC from ClC-3 channel null mice that was insensitive to Y27632, an inhibitor of ROCK2. In addition, AngII-induced cerebrovascular remodelling was decreased in ClC-3 null mice, possibly by the ROCK2 pathway.Conclusions And ImplicationsClC-3 protein phosphorylation at Thr(532) by ROCK2 is required for AngII-induced Cl- current and VSMC migration that are involved in AngII-induced vascular remodelling in hypertension.