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MOLECULAR AND GENOMIC |
1 Department of Pathophysiology, School of Pharmaceutical Sciences, Showa University, Tokyo 142-8555, Japan
2 Center for Integrative Bioscience
3 Department of Information Physiology, National Institute for Physiological Sciences, Okazaki, Aichi 444-8585, Japan
4 Department of Physiology, Kagoshima University Faculty of Medicine, Kagoshima, Kagoshima 890-8520, Japan
5 Mitsubishi Kagaku Institute of Life Sciences, Machida, Tokyo 194-8511, Japan
6 Institute for Life Science Research, Asahi Kasei Corporation, Fuji, Shizuoka 416-0934, Japan
Mammalian homologues of Drosophila transient receptor potential (TRP) proteins are responsible for receptor-activated Ca2+ influx in vertebrate cells. We previously reported the involvement of intracellular Ca2+ in the receptor-mediated activation of mammalian canonical transient receptor potential 5 (TRPC5) channels. Here we investigated the role of calmodulin, an important sensor of changes in intracellular Ca2+, and its downstream cascades in the activation of recombinant TRPC5 channels in human embryonic kidney (HEK) 293 cells. Ca2+ entry through TRPC5 channels, induced upon stimulation of the G-protein-coupled ATP receptor, was abolished by treatment with W-13, an inhibitor of calmodulin. ML-9 and wortmannin, inhibitors of Ca2+–calmodulin-dependent myosin light chain kinase (MLCK), and the expression of a dominant-negative mutant of MLCK inhibited the TRPC5 channel activity, revealing an essential role of MLCK in maintaining TRPC5 channel activity. It is important to note that ML-9 impaired the plasma membrane localization of TRPC5 channels. Furthermore, TRPC5 channel activity measured using the whole-cell patch-clamp technique was inhibited by ML-9, whereas TRPC5 channel activity observed in the cell-excised, inside-out patch was unaffected by ML-9. An antibody that recognizes phosphorylated myosin light chain (MLC) revealed that the basal level of phosphorylated MLC under unstimulated conditions was reduced by ML-9 in HEK293 cells. These findings strongly suggest that intracellular Ca2+–calmodulin constitutively activates MLCK, thereby maintaining TRPC5 channel activity through the promotion of plasma membrane TRPC5 channel distribution under the control of phosphorylation/dephosphorylation equilibrium of MLC.
(Received 5 September 2005;
accepted after revision 9 November 2005;
first published online 10 November 2005)
Corresponding author S. Shimizu: Department of Pathophysiology, School of Pharmaceutical Sciences, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan. Email: shun{at}pharm.showa-u.ac.jp
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