Tyrosine 284 phosphorylation is required for ClC-3 chloride channel activation in vascular smooth muscle cells.
AIMS
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.


METHODS AND 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.


CONCLUSION
Tyrosine 284 phosphorylation in the ClC-3 channel targeted by Src kinase is an important molecular mechanism for ClC-3 channel activation.
['Wang|Xiao-Guang|XG|', 'Tao|Jing|J|', 'Ma|Ming-Ming|MM|', 'Tang|Yong-Bo|YB|', 'Zhou|Jia-Guo|JG|', 'Guan|Yong-Yuan|YY|']
D000818:Animals D017209:Apoptosis D002460:Cell Line D018118:Chloride Channels D002712:Chlorides D008564:Membrane Potentials D009131:Muscle, Smooth, Vascular D009154:Mutation D032389:Myocytes, Smooth Muscle D010766:Phosphorylation D047428:Protein Kinase Inhibitors D051381:Rats D013997:Time Factors D014162:Transfection D014443:Tyrosine D019061:src-Family Kinases
Tyrosine Phosphorylation ClC-3 chloride channel Vascular smooth muscle cell Apoptosis