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© The Rockefeller University Press, 0021-9525/1997//601 $5.00
The Journal of Cell Biology, Volume 139, Number 3, , 1997 601-611

Article

Ca2+ Homeostasis in the Endoplasmic Reticulum: Coexistence of High and Low [Ca2+] Subcompartments in Intact HeLa Cells



Mayte Montero*, Javier Alvarez*, Wilhelm J.J. Scheenen{ddagger}, Rosario Rizzuto{ddagger}, Jacopo Meldolesi§, and Tullio Pozzan{ddagger}

* Department of Biochemistry and Molecular Biology and Physiology, Insituto de Biologia y Genetica Molecular, University of Valladolid and Consejo Superior de Investigaciones Cientificas, E-47005 Valladolid, Spain; {ddagger} Department of Biomedical Sciences, Consiglio Nazionale delle Ricerche (CNR) Center of Biomembranes, University of Padova, I-35121 Padova, Italy; and § Department of Pharmacology, University of Milan, CNR Center of Molecular and Cellular Pharmacology and Department of Biological and Technological Research, St. Raffaele Science Institute, I-20132 Milan, Italy

Two recombinant aequorin isoforms with different Ca2+ affinities, specifically targeted to the endoplasmic reticulum (ER), were used in parallel to investigate free Ca2+ homeostasis in the lumen of this organelle. Here we show that, although identically and homogeneously distributed in the ER system, as revealed by both immunocytochemical and functional evidence, the two aequorins measured apparently very different concentrations of divalent cations ([Ca2+]er or [Sr2+]er). Our data demonstrate that this contradiction is due to the heterogeneity of the [Ca2+] of the aequorin-enclosing endomembrane system. Because of the characteristics of the calibration procedure used to convert aequorin luminescence into Ca2+ concentration, the [Ca2+]er values obtained at steady state tend, in fact, to reflect not the average ER values, but those of one or more subcompartments with lower [Ca2+]. These subcompartments are not generated artefactually during the experiments, as revealed by the dynamic analysis of the ER structure in living cells carried out by means of an ER-targeted green fluorescent protein. When the problem of ER heterogeneity was taken into account (and when Sr2+ was used as a Ca2+ surrogate), the bulk of the organelle was shown to accumulate free [cation2+]er up to a steady state in the millimolar range. A theoretical model, based on the existence of multiple ER subcompartments of high and low [Ca2+], that closely mimics the experimental data obtained in HeLa cells during accumulation of either Ca2+ or Sr2+, is presented. Moreover, a few other key problems concerning the ER Ca2+ homeostasis have been addressed with the following conclusions: (a) the changes induced in the ER subcompartments by receptor generation of InsP3 vary depending on their initial [Ca2+]. In the bulk of the system there is a rapid release whereas in the small subcompartments with low [Ca2+] the cation is simultaneously accumulated; (b) stimulation of Ca2+ release by receptor-generated InsP3 is inhibited when the lumenal level is below a threshold, suggesting a regulation by [cation2+]er of the InsP3 receptor activity (such a phenomenon had already been reported, however, but only in subcellular fractions analyzed in vitro); and (c) the maintenance of a relatively constant level of cytosolic [Ca2+], observed when the cells are incubated in Ca2+-free medium, depends on the continuous release of the cation from the ER, with ensuing activation in the plasma membrane of the channels thereby regulated (capacitative influx).

Abbreviations used in this paper: [Ca2+]c, cytosolic-free Ca2+; [Ca2+]er, Ca2+ level within the endomembrane system; FCCP, carbonylcyanide p(trifluoromethoxy) phenylhydrazone; KRB, Krebs-Ringer modified buffer; SERCA, sarcoendoplasmic reticulum Ca2+ ATPases; tBuBHQ, 2,5-di(tert-butyl)-1,4-benzohydroquinone.

M. Montero is a recipient of an European Union Training and Mobility of Researchers return grant and W.J.J. Scheenen of a European Union Human Capital and Mobility fellowship. This work was supported by grants from the Fondo de Investigaciones Sanitarias from the Spanish Health Ministry (96/0456) to J. Alvarez, from Telethon, from the EU programs Human Capital and Mobility, Biomed 2, and Copernicus, and from Human Frontier Science Program to T. Pozzan, R. Rizzuto, and J. Meldolesi.

Address all correspondence to Mayte Montero, Department of Biochemistry and Molecular Biology and Physiology, Faculty of Medicine, University of Valladolid, E-47005 Valladolid, Spain. Tel.: 34.83.423085. Fax: 34.83.423588. E-mail: mmontero{at}cpd.uva.es


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