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

Aims The ClC-3 chloride channel (and current, I Cl,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 I Cl,ClC-3 by intercellular signalling is not fully understood. Although it has been suggested that tyrosine phosphorylation is required for I Cl,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 I Cl,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 I Cl,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.