Threonine532 phosphorylation in ClC‐3 channels is required for angiotensin II‐induced Cl⁻ current and migration in cultured vascular smooth muscle cells

Abstract

BACKGROUND AND PURPOSEAngiotensin 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 À current, but the underlying mechanism is not clear. EXPERIMENTAL APPROACHThe 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, KEY RESULTSIn VSMC, AngII induced Cl À 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 À 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 À 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 À 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 AND IMPLICATIONSClC-3 protein phosphorylation at Thr 532 by ROCK2 is required for AngII-induced Cl À current and VSMC migration that are involved in AngII-induced vascular remodelling in hypertension.Abbreviations ΔCT, C-terminal truncated ClC-3; ΔNT, N-terminal truncated ClC-3; BASMC, basilar artery smooth muscle cells; caROCK2, constitutively active ROCK2; dnROCK2, dominant negative ROCK2; N1, EGFP-N1 plasmid; VRCC, volume-regulated chloride channel; VSMC, vascular smooth muscle cells BJP British Journal of Pharmacology

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