Myosin light chain kinase modulates hypotonicity-induced Ca²⁺ entry and Cl^– channel activity in human cervical cancer cells

Abstract.

Hypotonicity-induced Ca²⁺ entry is a critical signal for the normal regulatory volume decrease in human cervical cancer cells. The aim of this study was to explore the role of myosin light chain kinase (MLCK) in the regulation of hypotonicity-induced Ca²⁺ signalling and Cl^– channel activity. Blockade of MLCK activity by MLCK_(11–19) amide, a substrate-specific peptide inhibitor, markedly attenuated hypotonicity-induced Ca²⁺ entry. A similar result was obtained with ML-7, a synthetic naphthalenesulphonyl derivative that inhibits the binding of ATP to MLCK. More than 85% of the activity of the volume-regulated Cl^– channel was suppressed when intracellular Ca²⁺ was buffered to near zero in the absence of extracellular Ca²⁺, suggesting that hypotonicity-induced Ca²⁺ signalling is important for the activation of the volume-regulated Cl^– channel. Intracellular dialysis with MLCK_(11–19) amide or ML-7 concentration-dependently reduced the amplitude and rate of activation of the volume-regulated Cl^– channel. Swelling-activated taurine transport was also inhibited concentration dependently by ML-7 and MLCK_(11–19) amide with IC₅₀ values of 6.4 and 2.0 µM, respectively. Hypotonicity induced MLC phosphorylation which was mediated totally by MLCK and depended on Ca²⁺ entry. However, phosphorylated MLC per se was not involved critically in the regulation of Ca²⁺ entry and activation of volume-sensitive organic osmolyte/anion channels (VSOAC). We propose that MLCK has a novel function in regulating the activation of VSOAC by mediating Ca²⁺ entry in response to hypotonicity. This function of MLCK on Ca²⁺ signalling does not correlate with MLC phosphorylation.