title: Evidence that Orai1 does not contribute to store-operated TRPC1 channels in vascular smooth muscle cells.
abstract: Ca2+-permeable store-operated channels (SOCs) mediate Ca2+entry pathways which are involved in many cellular functions such as contraction, growth, and proliferation. Prototypical SOCs are formed of Orai1 proteins and are activated by the endo/sarcoplasmic reticulum Ca2+sensor stromal interaction molecule 1 (STIM1). There is considerable debate about whether canonical transient receptor potential 1 (TRPC1) proteins also form store-operated channels (SOCs), and if they do, is Orai1 involved. We recently showed that stimulation of TRPC1-based SOCs involves store depletion inducing STIM1-evoked Gαq/PLCβ1 activity in contractile vascular smooth muscle cells (VSMCs). Therefore the present work investigates the role of Orai1 in activation of TRPC1-based SOCs in freshly isolated mesenteric artery VSMCs from wild-type (WT) and Orai1-/-mice. Store-operated whole-cell and single channel currents recorded from WT and Orai1-/-VSMCs had similar properties, with relatively linear current-voltage relationships, reversal potentials of about +20mV, unitary conductances of about 2pS, and inhibition by anti-TRPC1 and anti-STIM1 antibodies. In Orai1-/-VSMCs, store depletion induced PLCβ1 activity measured with the fluorescent phosphatidylinositol 4,5-bisphosphate/inositol 1,4,5-trisphosphate biosensor GFP-PLCδ1-PH, which was prevented by knockdown of STIM1. In addition, in Orai1-/-VSMCs, store depletion induced translocation of STIM1 from within the cell to the plasma membrane where it formed STIM1-TRPC1 interactions at discrete puncta-like sites. These findings indicate that activation of TRPC1-based SOCs through a STIM1-activated PLCβ1 pathway are likely to occur independently of Orai1 proteins, providing evidence that TRPC1 channels form genuine SOCs in VSMCs with a contractile phenotype.
authors.affiliation: a Institute of Cardiovascular & Metabolic Medicine, School of Medicine , University of Leeds , Leeds , UK.
authors.forename: Jian
authors.initials: J
authors.lastname: Shi
authors.affiliation: b Vascular Biology Research Centre, Institute of Molecular & Clinical Sciences Research Institute , St. George's, University of London , Cranmer Terrace, London , UK.|c Institute of Medical & Biomedical Education, St. George's , University of London , Cranmer Terrace, London , UK.
authors.forename: Francesc
authors.initials: F
authors.lastname: Miralles
authors.affiliation: d Laboratory of Allergy and Immunology, Department of Pathology, Beth Israel Deaconess Medical Center , Harvard Medical School , Boston , MA , USA.
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Kinet
authors.affiliation: e Laboratory of Neurobiology , National Institute of Environmental Health Sciences , Research Triangle Park, NC , USA.|f Institute of Biomedical Research (BIOMED) , Catholic University of Argentina , Buenos Aires , Argentina.
authors.forename: Lutz
authors.initials: L
authors.lastname: Birnbaumer
authors.affiliation: b Vascular Biology Research Centre, Institute of Molecular & Clinical Sciences Research Institute , St. George's, University of London , Cranmer Terrace, London , UK.
authors.forename: William A
authors.initials: WA
authors.lastname: Large
authors.affiliation: b Vascular Biology Research Centre, Institute of Molecular & Clinical Sciences Research Institute , St. George's, University of London , Cranmer Terrace, London , UK.
authors.forename: Anthony P
authors.identifier: http://orcid.org/0000-0002-3596-9634
authors.initials: AP
authors.lastname: Albert
mesh_terms: D000818:Animals; D015220:Calcium Channels / Q000378:metabolism*; D020013:Calcium Signaling; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D051379:Mice; D032389:Myocytes, Smooth Muscle / Q000378:metabolism*; D000071740:ORAI1 Protein / Q000235:genetics / Q000378:metabolism*; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D054799:Phospholipase C beta / Q000378:metabolism; D050052:TRPC Cation Channels / Q000378:metabolism*
chemical_list: D015220:Calcium Channels; D000071740:ORAI1 Protein; D019269:Phosphatidylinositol 4,5-Diphosphate; D050052:TRPC Cation Channels; D015544:Inositol 1,4,5-Trisphosphate; D054799:Phospholipase C beta
keywords: Orai1; PLC; STIM1; TRPC1; store-operated; vascular smooth muscle