Two distinct calcium pools in the endoplasmic reticulum of HEK-293T cells

Author:

Aulestia Francisco J.1,Redondo Pedro C.2,Rodríguez-García Arancha1,Rosado Juan A.2,Salido Ginés M.2,Alonso Maria Teresa1,García-Sancho Javier1

Affiliation:

1. Instituto de Biología y Genética Molecular (IBFM), Universidad de Valladolid y Consejo Superior de Investigaciones Científicas (CSIC), c/Sanz y Foré 3, 47003 Valladolid, Spain

2. Cell Physiology Research Group, Department of Physiology, Universidad de Extremadura, Av. de la Universidad s/n, 10071 Cáceres, Spain

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.

Publisher

Portland Press Ltd.

Subject

Cell Biology,Molecular Biology,Biochemistry

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