title: Platelet-derived growth factor maintains stored calcium through a nonclustering Orai1 mechanism but evokes clustering if the endoplasmic reticulum is stressed by store depletion.
abstract: BACKGROUND
  Calcium entry through Orai1 channels drives vascular smooth muscle cell migration and neointimal hyperplasia. The channels are activated by the important growth factor platelet-derived growth factor (PDGF). Channel activation is suggested to depend on store depletion, which redistributes and clusters stromal interaction molecule 1 (STIM1), which then coclusters and activates Orai1.
  
  
  OBJECTIVE
  To determine the relevance of STIM1 and Orai1 redistribution in PDGF responses.
  
  
  RESULTS
  Vascular smooth muscle cells were cultured from human saphenous vein. STIM1 and Orai1 were tagged with green and red fluorescent proteins to track them in live cells. Under basal conditions, the proteins were mobile but mostly independent of each other. Inhibition of sarco-endoplasmic reticulum calcium ATPase led to store depletion and dramatic redistribution of STIM1 and Orai1 into coclusters. PDGF did not evoke redistribution, even though it caused calcium release and Orai1-mediated calcium entry in the same time period. After chemical blockade of Orai1-mediated calcium entry, however, PDGF caused redistribution. Similarly, mutagenic disruption of calcium flux through Orai1 caused PDGF to evoke redistribution, showing that calcium flux through the wild-type channels had been filling the stores. Acidification of the extracellular medium to pH 6.4 caused inhibition of Orai1-mediated calcium entry and conferred capability for PDGF to evoke complete redistribution and coclustering.
  
  
  CONCLUSIONS
  The data suggest that PDGF has a nonclustering mechanism by which to activate Orai1 channels and maintain calcium stores replete. Redistribution and clustering become important, however, when the endoplasmic reticulum stress signal of store depletion arises, for example when acidosis inhibits Orai1 channels.
authors.affiliation: Multidisciplinary Cardiovascular Research Centre, Faculty of Biological Sciences, University of Leeds, United Kingdom.
authors.forename: Lynn
authors.initials: L
authors.lastname: McKeown
authors.forename: Nicholas K
authors.initials: NK
authors.lastname: Moss
authors.forename: Paul
authors.initials: P
authors.lastname: Turner
authors.forename: Jing
authors.initials: J
authors.lastname: Li
authors.forename: Nikki
authors.initials: N
authors.lastname: Heath
authors.forename: Dermot
authors.initials: D
authors.lastname: Burke
authors.forename: David
authors.initials: D
authors.lastname: O'Regan
authors.forename: Mark S
authors.initials: MS
authors.lastname: Gilthorpe
authors.forename: Karen E
authors.initials: KE
authors.lastname: Porter
authors.forename: David J
authors.initials: DJ
authors.lastname: Beech
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism*; D002478:Cells, Cultured; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D018715:Microscopy, Video; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism*; D009154:Mutation; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D000071740:ORAI1 Protein; D010982:Platelet-Derived Growth Factor / Q000378:metabolism*; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012501:Saphenous Vein / Q000378:metabolism; D013312:Stress, Physiological; D000071737:Stromal Interaction Molecule 1; D019284:Thapsigargin / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection; D000097573:Red Fluorescent Protein
chemical_list: D001426:Bacterial Proteins; D002118:Calcium; D015220:Calcium Channels; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D000071740:ORAI1 Protein; D010982:Platelet-Derived Growth Factor; D011993:Recombinant Fusion Proteins; D000071737:Stromal Interaction Molecule 1; D019284:Thapsigargin; D000097573:Red Fluorescent Protein; C510746:ORAI1 protein, human; C103530:STIM1 protein, human; C485184:enhanced green fluorescent protein; C054562:yellow fluorescent protein, Bacteria