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Cardiovasc Res. 2013 Jun 1;98(3):469-78. doi: 10.1093/cvr/cvt063. Epub 2013 Mar 27.

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

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  • 1Department of Pharmacology, Cardiac and Cerebral Vascular Research Center, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou 510080, People's Republic of China.

Abstract

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.

KEYWORDS:

Apoptosis; ClC-3 chloride channel; Phosphorylation; Tyrosine; Vascular smooth muscle cell

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