title: Tyrosine 284 phosphorylation is required for ClC-3 chloride channel activation in vascular smooth muscle cells.
abstract: OBJECTIVE
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  Tyrosine 284 phosphorylation in the ClC-3 channel targeted by Src kinase is an important molecular mechanism for ClC-3 channel activation.
authors.affiliation: Department of Pharmacology, Cardiac and Cerebral Vascular Research Center, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou 510080, People's Republic of China.
authors.forename: Xiao-Guang
authors.initials: XG
authors.lastname: Wang
authors.forename: Jing
authors.initials: J
authors.lastname: Tao
authors.forename: Ming-Ming
authors.initials: MM
authors.lastname: Ma
authors.forename: Yong-Bo
authors.initials: YB
authors.lastname: Tang
authors.forename: Jia-Guo
authors.initials: JG
authors.lastname: Zhou
authors.forename: Yong-Yuan
authors.initials: YY
authors.lastname: Guan
mesh_terms: D000818:Animals; D017209:Apoptosis; D002460:Cell Line; D018118:Chloride Channels / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism; D008564:Membrane Potentials; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism*; D009154:Mutation; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000378:metabolism*; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051381:Rats; D013997:Time Factors; D014162:Transfection; D014443:Tyrosine; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D018118:Chloride Channels; D002712:Chlorides; C115376:ClC-3 channel; D047428:Protein Kinase Inhibitors; D014443:Tyrosine; D019061:src-Family Kinases
keywords: Apoptosis; ClC-3 chloride channel; Phosphorylation; Tyrosine; Vascular smooth muscle cell