title: Membrane translocation of TRPC6 channels and endothelial migration are regulated by calmodulin and PI3 kinase activation.
abstract: Lipid oxidation products, including lysophosphatidylcholine (lysoPC), activate canonical transient receptor potential 6 (TRPC6) channels leading to inhibition of endothelial cell (EC) migration in vitro and delayed EC healing of arterial injuries in vivo. The precise mechanism through which lysoPC activates TRPC6 channels is not known, but calmodulin (CaM) contributes to the regulation of TRPC channels. Using site-directed mutagenesis, cDNAs were generated in which Tyr(99) or Tyr(138) of CaM was replaced with Phe, generating mutant CaM, Phe(99)-CaM, or Phe(138)-CaM, respectively. In ECs transiently transfected with pcDNA3.1-myc-His-Phe(99)-CaM, but not in ECs transfected with pcDNA3.1-myc-His-Phe(138)-CaM, the lysoPC-induced TRPC6-CaM dissociation and TRPC6 externalization was disrupted. Also, the lysoPC-induced increase in intracellular calcium concentration was inhibited in ECs transiently transfected with pcDNA3.1-myc-His-Phe(99)-CaM. Blocking phosphorylation of CaM at Tyr(99) also reduced CaM association with the p85 subunit and subsequent activation of phosphatidylinositol 3-kinase (PI3K). This prevented the increase in phosphatidylinositol (3,4,5)-trisphosphate (PIP3) and the translocation of TRPC6 to the cell membrane and reduced the inhibition of EC migration by lysoPC. These findings suggest that lysoPC induces CaM phosphorylation at Tyr(99) by a Src family kinase and that phosphorylated CaM activates PI3K to produce PIP3, which promotes TRPC6 translocation to the cell membrane.
authors: Department of Biomedical Engineering, Cleveland Clinic, Cleveland, OH 44195;
authors: Pinaki
authors: P
authors: Chaudhuri
authors: Surgical Service, Louis Stokes Cleveland Veterans Affairs Medical Center, Cleveland, OH 44106;
authors: Michael A
authors: MA
authors: Rosenbaum
authors: Department of Biological Sciences, Kent State University, Kent, OH 44240;
authors: Pritam
authors: P
authors: Sinharoy
authors: Department of Biological Sciences, Kent State University, Kent, OH 44240;
authors: Derek S
authors: DS
authors: Damron
authors: Neurobiology Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709; Birnbau1@gmail.com grahamL@ccf.org.
authors: Lutz
authors: L
authors: Birnbaumer
authors: Department of Biomedical Engineering, Cleveland Clinic, Cleveland, OH 44195; Department of Vascular Surgery, Cleveland Clinic, Cleveland, OH 44195 Birnbau1@gmail.com grahamL@ccf.org.
authors: Linda M
authors: LM
authors: Graham
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000502:physiology*; D002147:Calmodulin / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D002465:Cell Movement / Q000502:physiology*; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D004789:Enzyme Activation / Q000502:physiology; D006801:Humans; D008244:Lysophosphatidylcholines / Q000235:genetics / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport / Q000502:physiology; D050052:TRPC Cation Channels / Q000235:genetics / Q000378:metabolism*; D000074287:TRPC6 Cation Channel
chemical_list: D002147:Calmodulin; D008244:Lysophosphatidylcholines; D050052:TRPC Cation Channels; D000074287:TRPC6 Cation Channel; C117032:TRPC6 protein, human; D019869:Phosphatidylinositol 3-Kinases; D002118:Calcium
keywords: PI3 kinase; TRPC6; calmodulin; endothelial