Myosin light chain kinase modulates hypotonicity-induced Ca2+ entry and Cl- channel activity in human cervical cancer cells.
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 IC(50) values of 6.4 and 2.0 microM, 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.
Shen
Meng-Ru
MR
Department of Obstetrics and Gynaecology, College of Medicine, National Cheng Kung University, Tainan 704, Taiwan.
Furla
Paola
P
Chou
Cheng-Yang
CY
Ellory
J Clive
JC
D000465:Algorithms; D000911:Antibodies, Monoclonal; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology; D018118:Chloride Channels / Q000378:metabolism*; D004558:Electric Stimulation; D004594:Electrophysiology; D005260:Female; D005456:Fluorescent Dyes; D016257:Fura-2; D006801:Humans; D007038:Hypotonic Solutions / Q000494:pharmacology*; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism*; D009997:Osmotic Pressure; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D013654:Taurine / Q000378:metabolism; D014407:Tumor Cells, Cultured; D002583:Uterine Cervical Neoplasms / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D018118:Chloride Channels; D005456:Fluorescent Dyes; D007038:Hypotonic Solutions; D013654:Taurine; D009219:Myosin-Light-Chain Kinase; D002118:Calcium; D016257:Fura-2