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Received September 5, 2005
Revised September 22, 2005
Accepted after revision November 9, 2005
1 Department of Pathophysiology, School of Pharmaceutical Sciences, Showa University
2 Center for Integrative Bioscience, National Institute for Physiological Sciences
3 Department of Information Physiology, National Institute for Physiological Sciences
4 Mitsubishi Kagaku Institute of Life Sciences
5 Institute of Life Science Research, Asahi Kasei Corporation
* To whom correspondence should be addressed. E-mail: shun{at}pharm.showa-u.ac.jp.
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 TRPC5 channels. Here we investigated the role of calmodulin, an important sensor of intracellular Ca2+ change, and its downstream cascades in the activation of recombinant TRPC5 channels in human embryonic kidney (HEK) 293 cells. Ca2+ entry through TRPC5 induced upon stimulation of the G-protein- coupled ATP receptor was abolished by the treatment with W-13, an inhibitor of calmodulin. ML-9 and wortmannin, the inhibitors of Ca2+/calmodulin-dependent myosin light chain kinase (MLCK), and the expression of a dominant-negative mutant of MLCK inhibited the TRPC5 activity, revealing an essential role of MLCK in maintaining TRPC5 channel activity. Importantly, ML-9 impaired the plasma membrane localization of TRPC5. Furthermore, the TRPC5 activity measured using the whole- cell mode of the patch-clamp technique was inhibited by ML-9, whereas the TRPC5 activity observed in the cell- excised, inside-out patch was unaffected by ML-9. The antibody which 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 distribution under the control of phosphorylation/dephosphorylation equilibrium of MLC.
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