Ca2+ homeostasis in the endoplasmic reticulum: coexistence of high and low [Ca2+] subcompartments in intact HeLa cells

Abstract

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).

Figure A1

Detailed mathematical model mimicking the effect of the addition of Ca²⁺ or Sr²⁺ to either erAEQwt- or erAEQmut-expressing HeLa cells. In this model the ER space is divided into three high [Ca²⁺]_er and three low [Ca²⁺]_er subcompartments in which Ca²⁺ or Sr²⁺ were assumed to increase exponentially. Continuous lines represent theoretical Ca²⁺ and Sr²⁺ values obtained from the model. Dotted lines represent typical calibrated values obtained experimentally (Fig. 2).

Figure 1

Simple version of the theoretical model predicting the rates of aequorin consumption and the calibrated [Ca²⁺] during refilling of the ER, assuming the existence of 1 or 2 compartments. (A) The predicted luminescence results recorded during exponential refilling of a single compartment up to a steady state of 2 mM, with a time constant of 120 s. For simplicity it is assumed that the whole cell population could emit 1,000,000 photons in total, 800,000 from the high Ca²⁺ compartment and 200,000 from the low Ca²⁺ compartment. (B) The predicted luminescence recorded during exponential refilling of a single compartment up to a steady state of 10 μM, with a time constant of 5 s. (C) The result of adding the two luminescence records of A and B. (D) The calibrated [Ca²⁺] levels calculated from the data in C, assuming all aequorin to be in the same compartment.

Figure 2

Apparent kinetics of [cation²⁺]_er during store refilling with Ca²⁺ and Sr²⁺ as measured in cells expressing erAEQmut and erAEQwt. (A and B) Continuous lines represent cells expressing erAEQwt. Dotted lines represent cells expressing erAEQmut. Where indicated, the perfusion medium contained 1 mM CaCl₂. A shows the calibrated values of [Ca²⁺]_er with both aequorins, with the inset illustrating the first seconds after Ca²⁺ addition at higher magnification. B shows the data of aequorin luminescence recalculated as an accumulative plot, considering the total amount of photons emitted during the whole experiment (normalized to 100%) and the percentage emitted as a function of time. In this plot it is immediately obvious the residual amount of aequorin available at any given time being compared with the calibrated values of [cation²⁺]_er. (C and D) The erAEQmut-expressing HeLa cells (dotted lines) were challenged where indicated with 1mM Ca²⁺, whereas the erAEQwt-expressing cells (continuous lines) were challenged with 1 mM Sr²⁺. (E and F) The erAEQmut HeLa cells were perfused with 1 mM Sr²⁺ where indicated.

Figure 3

Effects of the Ca²⁺-depletion protocol on the morphology and lumenal continuity of the ER as measured with erGFP. HeLa cells were transiently transfected with the erGFP construct and analyzed 2 d later. (A) ER morphology as revealed by erGFP fluorescence analyzed by confocal microscopy in live cells before (left) and 1 h after incubation in Ca²⁺-free, EGTA-containing KRB, and treatment with 30 μM tBuBHQ (right). (B) Photobleaching and recovery. After the depletion protocol was terminated, the image shown in A (control) was taken with the standard illumination protocol. A mask was then introduced in the exciting light path and the second image was taken (B; bleaching spot). The sample was then continuously illuminated with the highest laser power for 3 min before removing the mask and immediately taking the third image (C; 15 s after bleaching) with the standard settings. The last image (D; 3 min after bleaching) was collected under standard conditions, 3 min after the third. The cells were not illuminated during this recovery period. Bar, 9 μm.

Figure 3

Effects of the Ca²⁺-depletion protocol on the morphology and lumenal continuity of the ER as measured with erGFP. HeLa cells were transiently transfected with the erGFP construct and analyzed 2 d later. (A) ER morphology as revealed by erGFP fluorescence analyzed by confocal microscopy in live cells before (left) and 1 h after incubation in Ca²⁺-free, EGTA-containing KRB, and treatment with 30 μM tBuBHQ (right). (B) Photobleaching and recovery. After the depletion protocol was terminated, the image shown in A (control) was taken with the standard illumination protocol. A mask was then introduced in the exciting light path and the second image was taken (B; bleaching spot). The sample was then continuously illuminated with the highest laser power for 3 min before removing the mask and immediately taking the third image (C; 15 s after bleaching) with the standard settings. The last image (D; 3 min after bleaching) was collected under standard conditions, 3 min after the third. The cells were not illuminated during this recovery period. Bar, 9 μm.

Figure 3

Effects of the Ca²⁺-depletion protocol on the morphology and lumenal continuity of the ER as measured with erGFP. HeLa cells were transiently transfected with the erGFP construct and analyzed 2 d later. (A) ER morphology as revealed by erGFP fluorescence analyzed by confocal microscopy in live cells before (left) and 1 h after incubation in Ca²⁺-free, EGTA-containing KRB, and treatment with 30 μM tBuBHQ (right). (B) Photobleaching and recovery. After the depletion protocol was terminated, the image shown in A (control) was taken with the standard illumination protocol. A mask was then introduced in the exciting light path and the second image was taken (B; bleaching spot). The sample was then continuously illuminated with the highest laser power for 3 min before removing the mask and immediately taking the third image (C; 15 s after bleaching) with the standard settings. The last image (D; 3 min after bleaching) was collected under standard conditions, 3 min after the third. The cells were not illuminated during this recovery period. Bar, 9 μm.

Figure 4

Effects of histamine on [Sr²⁺]_er in erAEQmut-expressing HeLa cells refilled with Sr²⁺ for different periods of time. Cells were exposed to KRB containing 1 mM Sr²⁺ for 0.5 (A), 1 (B), 2 (C), or 5 (D) min before shifting to KRB also containing 100 μM histamine. Other conditions are as in Fig. 2.

Figure 5

Relationship between the drop in aequorin luminescence induced by histamine and the level of aequorin consumption at the moment of histamine addition. Data were obtained from experiments such as those shown in Fig. 4.

Figure 6

Effect of histamine, EGTA, and tBuBHQ on [Sr²⁺]_er. (A) Cells expressing erAEQmut were treated as in Fig. 2 E; i.e., they were allowed to refill the ER with Sr²⁺ for 4 min. Where indicated, the perfusion medium contained 1 mM Sr²⁺ and, as indicated, either 100 μM histamine, 10 μM tBuBHQ, or both. (B) Where indicated, KRB contained 1 mM Sr²⁺ (control), 100 μM EGTA, or 100 μM EGTA + 100 μM histamine. The control shows the artefactual decrease of [Sr²⁺]_er in the absence of additions due to aequorin consumption, as shown in Fig. 2 E. Maximum [Sr²⁺]_er levels were normalized to 100% to facilitate comparison.

Figure 7

Effects of histamine on erAEQmut- (A) and erAEQwt- (B) expressing HeLa cells after refilling with Ca²⁺. Ca²⁺-depleted cells were first incubated with 1 mM Ca²⁺, and then 100 μM histamine was perfused in the same medium for the times indicated in the figure. The inset in A shows the effect of the addition of histamine to erAEQmut-expressing cells at higher resolution.