Evidence that Orai1 does not contribute to store-operated TRPC1 channels in vascular smooth muscle cells |
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Authors: | Jian Shi Francesc Miralles Jean-Pierre Kinet Lutz Birnbaumer William A Large |
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Institution: | 1. Institute of Cardiovascular &2. Metabolic Medicine, School of Medicine, University of Leeds, Leeds, UK;3. Vascular Biology Research Centre, Institute of Molecular &4. Clinical Sciences Research Institute, St. George's, University of London, Cranmer Terrace, London, UK;5. Institute of Medical &6. Biomedical Education, St. George's, University of London, Cranmer Terrace, London, UK;7. Laboratory of Allergy and Immunology, Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA;8. Laboratory of Neurobiology, National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA;9. Institute of Biomedical Research (BIOMED), Catholic University of Argentina, Buenos Aires, Argentina |
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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. |
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Keywords: | Orai1 PLC STIM1 store-operated TRPC1 vascular smooth muscle |
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