CA2-RETICULUM - COEXISTENCE OF HIGH AND LOW [CA2+] SUBCOMPARTMENTS ININTACT HELA-CELLS( HOMEOSTASIS IN THE ENDOPLASMIC)

Two recombinant aequorin isoforms with different Ca2+ affinities, spec ifically targeted to the endoplasmic reticulum (ER), were used in para llel to investigate free Ca2+ homeostasis in the lumen of this organel le. Here we show that, although identically and homogeneously distribu ted in the ER system, as revealed by both immunocytochemical and funct ional evidence, the two aequorins measured apparently very different c oncentrations of divalent cations ([Ca2+](er) or [Sr2+](er)). Our data demonstrate that this contradiction is due to the heterogeneity of th e [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, b ut those of one or more subcompartments with lower [Ca2+]. These subco mpartments are not generated artefactually during the experiments, as revealed by the dynamic analysis of the ER structure in living cells c arried out by means of an ER-targeted green fluorescent protein. When the problem of ER heterogeneity was taken into account (and when Sr2was used as a Ca2+ surrogate), the bulk of the organelle was shown to accumulate free [cation(2+)](er) up to a steady state in the millimola r range. A theoretical model, based on the existence of multiple ER su bcompartments of high and low [Ca2+], that closely mimics the experime ntal 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 conclusion s: (a) the changes induced in the ER subcompartments by receptor gener ation of InsP(3) vary depending on their initial [Ca2+]. In the bulk o f the system there is a rapid release whereas in the small subcompartm ents with low [Ca2+] the cation is simultaneously accumulated; (b) sti mulation of Ca2+ release by receptor-generated InsP(3) is inhibited wh en the lumenal level is below a threshold, suggesting a regulation by [cation(2+)](er) of the InsP(3) receptor activity (such a phenomenon h ad already been reported, however, but only in subcellular fractions a nalyzed in vitro); and (c) the maintenance of a relatively constant le vel 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 th ereby regulated (capacitative influx).