title: Threonine532 phosphorylation in ClC-3 channels is required for angiotensin II-induced Cl(-) current and migration in cultured vascular smooth muscle cells.
abstract: OBJECTIVE
  Angiotensin 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.
  
  
  METHODS
  The 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,
  
  
  RESULTS
  In 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
  ClC-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.
authors.affiliation: Department of Pharmacology, Cardiac and Cerebral Vascular Research Center, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, China.
authors.forename: Ming-Ming
authors.initials: MM
authors.lastname: Ma
authors.affiliation: Department of Pharmacology, Cardiac and Cerebral Vascular Research Center, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, China.
authors.forename: Cai-Xia
authors.initials: CX
authors.lastname: Lin
authors.affiliation: Department of Pharmacology, Cardiac and Cerebral Vascular Research Center, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, China.
authors.forename: Can-Zhao
authors.initials: CZ
authors.lastname: Liu
authors.affiliation: Department of Pharmacology, Cardiac and Cerebral Vascular Research Center, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, China.
authors.forename: Min
authors.initials: M
authors.lastname: Gao
authors.affiliation: Department of Pharmacology, Cardiac and Cerebral Vascular Research Center, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, China.
authors.forename: Lu
authors.initials: L
authors.lastname: Sun
authors.affiliation: Department of Pharmacology, Cardiac and Cerebral Vascular Research Center, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, China.
authors.forename: Yong-Bo
authors.initials: YB
authors.lastname: Tang
authors.affiliation: Department of Pharmacology, Cardiac and Cerebral Vascular Research Center, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, China.
authors.forename: Jia-Guo
authors.initials: JG
authors.lastname: Zhou
authors.affiliation: Department of Pharmacology, Cardiac and Cerebral Vascular Research Center, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, China.
authors.forename: Guan-Lei
authors.initials: GL
authors.lastname: Wang
authors.affiliation: Department of Pharmacology, Cardiac and Cerebral Vascular Research Center, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, China.
authors.forename: Yong-Yuan
authors.initials: YY
authors.lastname: Guan
mesh_terms: D000804:Angiotensin II / Q000502:physiology*; D000818:Animals; D001488:Basilar Artery / Q000166:cytology; D002460:Cell Line; D002465:Cell Movement; D002478:Cells, Cultured; D018118:Chloride Channels / Q000235:genetics / Q000502:physiology*; D008297:Male; D018345:Mice, Knockout; D009131:Muscle, Smooth, Vascular / Q000166:cytology; D032389:Myocytes, Smooth Muscle / Q000502:physiology*; D010766:Phosphorylation; D051381:Rats; D013912:Threonine / Q000378:metabolism*; D054460:rho-Associated Kinases / Q000502:physiology*
chemical_list: D018118:Chloride Channels; C115376:ClC-3 channel; D000804:Angiotensin II; D013912:Threonine; C515855:Rock2 protein, mouse; D054460:rho-Associated Kinases