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J. Cell Biol.,
Volume 139, Number 3, November 3, 1997 601-611
* 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; 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).
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
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