title: Two distinct calcium pools in the endoplasmic reticulum of HEK-293T cells.
abstract: Agonist-sensitive intracellular Ca2+ stores may be heterogeneous and exhibit distinct functional features. We have studied the properties of intracellular Ca2+ stores using targeted aequorins for selective measurements in different subcellular compartments. Both, HEK-293T [HEK (human embryonic kidney)-293 cells expressing the large T-antigen of SV40 (simian virus 40)] and HeLa cells accumulated Ca2+ into the ER (endoplasmic reticulum) to near millimolar concentrations and the IP3-generating agonists, carbachol and ATP, mobilized this Ca2+ pool. We find in HEK-293T, but not in HeLa cells, a distinct agonist-releasable Ca2+ pool insensitive to the SERCA (sarco/endoplasmic reticulum Ca2+ ATPase) inhibitor TBH [2,5-di-(t-butyl)-benzohydroquinone]. TG (thapsigargin) and CPA (cyclopiazonic acid) completely emptied this pool, whereas lysosomal disruption or manoeuvres collapsing endomembrane pH gradients did not. Our results indicate that SERCA3d is important for filling the TBH-resistant store as: (i) SERCA3d is more abundant in HEK-293T than in HeLa cells; (ii) the SERCA 3 ATPase activity of HEK-293T cells is not fully blocked by TBH; and (iii) the expression of SERCA3d in HeLa cells generated a TBH-resistant agonist-mobilizable compartment in the ER. Therefore the distribution of SERCA isoforms may originate the heterogeneity of the ER Ca2+ stores and this may be the basis for store specialization in diverse functions. This adds to recent evidence indicating that SERCA3 isoforms may subserve important physiological and pathophysiological mechanisms.
authors.affiliation: Instituto de Biología y Genética Molecular (IBFM), Universidad de Valladolid y Consejo Superior de Investigaciones Científicas, c/ Sanz y Foré 3, 47003 Valladolid, Spain.
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Aulestia
authors.forename: Pedro C
authors.initials: PC
authors.lastname: Redondo
authors.forename: Arancha
authors.initials: A
authors.lastname: Rodríguez-García
authors.forename: Juan A
authors.initials: JA
authors.lastname: Rosado
authors.forename: Ginés M
authors.initials: GM
authors.lastname: Salido
authors.forename: Maria Teresa
authors.initials: MT
authors.lastname: Alonso
authors.forename: Javier
authors.initials: J
authors.lastname: García-Sancho
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000331:Aequorin / Q000235:genetics / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects; D002217:Carbachol / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D006873:Hydroquinones / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D015544:Inositol 1,4,5-Trisphosphate / Q000819:agonists / Q000378:metabolism; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism; D007700:Kinetics; D049990:Membrane Transport Modulators / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
chemical_list: D006873:Hydroquinones; D007211:Indoles; D007527:Isoenzymes; D049990:Membrane Transport Modulators; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C019359:2,5-di-tert-butylhydroquinone; D000331:Aequorin; D015544:Inositol 1,4,5-Trisphosphate; D000255:Adenosine Triphosphate; D002217:Carbachol; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; C506949:ATP2A3 protein, human; D002118:Calcium; C000543:cyclopiazonic acid