title: Ca2+-calmodulin-dependent myosin light chain kinase is essential for activation of TRPC5 channels expressed in HEK293 cells.
abstract: Mammalian homologues of Drosophila transient receptor potential (TRP) proteins are responsible for receptor-activated Ca(2+) influx in vertebrate cells. We previously reported the involvement of intracellular Ca(2+) 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 Ca(2+), and its downstream cascades in the activation of recombinant TRPC5 channels in human embryonic kidney (HEK) 293 cells. Ca(2+) 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 Ca(2+)-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 Ca(2+)-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.
authors.affiliation: Department of Pathophysiology, School of Pharmaceutical Sciences, Showa University, Shinagawa-ku, Tokyo, Japan. shun@pharm.showa-u.ac.jp
authors.forename: Shunichi
authors.initials: S
authors.lastname: Shimizu
authors.forename: Takashi
authors.initials: T
authors.lastname: Yoshida
authors.forename: Minoru
authors.initials: M
authors.lastname: Wakamori
authors.forename: Masakazu
authors.initials: M
authors.lastname: Ishii
authors.forename: Takaharu
authors.initials: T
authors.lastname: Okada
authors.forename: Masami
authors.initials: M
authors.lastname: Takahashi
authors.forename: Minoru
authors.initials: M
authors.lastname: Seto
authors.forename: Katsuhiko
authors.initials: K
authors.lastname: Sakurada
authors.forename: Yuji
authors.initials: Y
authors.lastname: Kiuchi
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Mori
mesh_terms: D000595:Amino Acid Sequence; D000730:Androstadienes / Q000494:pharmacology; D001381:Azepines / Q000494:pharmacology; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000502:physiology*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D018994:Myosin Light Chains / Q000502:physiology; D009219:Myosin-Light-Chain Kinase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013449:Sulfonamides / Q000494:pharmacology; D050052:TRPC Cation Channels / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D000077191:Wortmannin
chemical_list: D000730:Androstadienes; D001381:Azepines; D002147:Calmodulin; D018994:Myosin Light Chains; D012333:RNA, Messenger; D013449:Sulfonamides; D050052:TRPC Cation Channels; C491780:TRPC5 protein, human; C056218:ML 9; C033772:N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide; D009219:Myosin-Light-Chain Kinase; D002118:Calcium; D000077191:Wortmannin