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Elementary Calcium-Release Units Induced by Inositol Trisphosphate

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Science  13 Jun 1997:
Vol. 276, Issue 5319, pp. 1690-1693
DOI: 10.1126/science.276.5319.1690

Abstract

The extent to which inositol 1,4,5-trisphosphate (InsP3)–induced calcium signals are localized is a critical parameter for understanding the mechanism of effector activation. The spatial characteristics of InsP3-mediated calcium signals were determined by targeting a dextran-based calcium indicator to intracellular membranes through the in situ addition of a geranylgeranyl lipid group. Elementary calcium-release events observed with this indicator typically lasted less than 33 milliseconds, had diameters less than 2 micrometers, and were uncoupled from each other by the calcium buffer EGTA. Cellwide calcium transients are likely to result from synchronized triggering of such local release events, suggesting that calcium-dependent effector proteins could be selectively activated by localization near sites of local calcium release.

Inositol 1,4,5-trisphosphate–induced increases in cytosolic Ca2+ concentration are essential for a large number of receptor-initiated signaling pathways in nearly all cell types (1). Although InsP3-mediated Ca2+ transients are often largely uniform across the cytosol, it has been proposed that they result from the synchronized triggering of highly localized Ca2+-release events (2), much like those of ryanodine receptor channels (3). Such localized events could target specific Ca2+-sensitive proteins, thus increasing the precision by which InsP3-mediated signal transduction activates downstream targets.

Resolving the spatial aspects of Ca2+ signaling is problematic when the Ca2+ indicator itself is readily diffusable, and could be more accurately resolved with an immobilized Ca2+ indicator. To design such an indicator, we relied on the endogenous posttranslational protein-modification machinery to add a geranylgeranyl lipid group to a peptide containing a COOH-terminal CAAX sequence. This CAAX peptide was coupled to dextran–calcium green with a homobifunctional amine cross-linker (4). The resulting indicator (CAAX green) was soluble and uniformly distributed in the cytosol immediately after electroporation into rat basophilic leukemia (RBL) cells (5) and was then processed by addition of a geranylgeranyl lipid group within 3 hours (Fig. 1) (6). This processing led to the nonspecific localization of CAAX green to internal membranes, as indicated by its localization to the plasma membrane, nuclear membrane, and various reticular structures in the cytosol (Fig. 1).

Figure 1

Localization of CAAX green to internal membranes in RBL cells. Differential interference contrast (DIC) and confocal fluorescence images with fluorescein optics are shown for cells at 0, 3, and 6 hours after electroporation (7). Bar, 10 μm for all images. The dissociation constant (K d) of membrane-bound CAAX green is 650 nM (4).

The time course of membrane attachment of CAAX green was more accurately determined by two additional approaches: (i) The diffusion coefficient, as determined by photobleaching experiments (7), was 6.55 ± 1.1 μm2/s (n = 9) immediately after electroporation and decreased to 0.64 ± 0.11 μm2/s (n = 4) at 1 hour. (ii) Maximal retention of CAAX green fluorescence in digitonin-permeabilized cells was reached within 45 min (95 ± 12%, n = 5) (8). These results suggest that CAAX green is lipid-modified in less than 1 hour after introduction into the cytosol, resulting in membrane attachment and immobilization.

A rapid increase in cytosolic Ca2+ concentration (<66 ms) (Fig. 2A) was triggered when a saturating amount of InsP3 was uniformly released throughout the cell by ultraviolet (UV)-mediated photorelease of caged InsP3(9). Subtracted images were used to determine the spatial aspects of this rapid increase (Fig. 2B) (10). In response to a maximal dose, InsP3-mediated Ca2+ release was largely uniform across the cell, similar to the spatial characteristics observed for receptor-mediated Ca2+transients observed in these and other cells (1,11). Similar results were observed in 10 cells analyzed.

Figure 2

Increase in Ca2+concentration after UV laser–triggered photorelease of saturating amounts of InsP3 in the absence and presence of EGTA. (A) The change in average relative fluorescence intensity of CAAX green in an individual cell as a function of time. Images were recorded every 33 ms and corrected for background fluorescence before the average fluorescence intensity was determined. The arrow indicates a 20-ms UV-laser pulse illuminating the entire cell. The peak free Ca2+ concentration was estimated to be 400 nM with the in situ K d calibration of the indicator (4). (B) Subtracted images show a near uniform increase in local Ca2+ concentration. A 10% increase in CAAX green fluorescence corresponds to a ∼50 nM increase in the free Ca2+ concentration (4). Consecutive images were recorded at the times indicated (relative to the UV pulse). Decreases in Ca2+ concentration are shown in black. (C) Same intensity trace as in (A) but measured in the presence of EGTA. An estimated intracellular concentration of 0.5 to 1 mM EGTA was introduced by addition of 20 mM extracellular EGTA during the electroporation of caged InsP3. (D) Subtracted images in the presence of EGTA show localized Ca2+-release events.

Local Ca2+-release events have been observed inXenopus leavis oocytes and pancreatic acinar cells in response to minimal increases in InsP3 concentration (12). These events have thus far been observed only in specialized regions of cells and may result from nonuniform distributions of InsP3 receptors or a locally increased sensitivity to InsP3 (13). Nevertheless, it has been proposed that receptor-mediated Ca2+ transients in all cell types are the result of rapid coupling of individual, all-or-none Ca2+-release units (2). The coupling between such local release units would be mediated by Ca2+diffusion and triggered by Ca2+ activation of InsP3-dependent Ca2+ release (14). We reasoned that if such elementary Ca2+-release units were the building blocks of cellwide Ca2+ transients, individual units could be resolved by blocking the Ca2+-mediated synchronization with the slow Ca2+ buffer EGTA.

In the presence of 0.5 to 1 mM intracellular EGTA, the rise time of the global Ca2+ increase induced by a saturating dose of InsP3 was 15 times longer than in the absence of EGTA (Fig.2C). This suggests that Ca2+ diffusion is required to generate the rapid, global release event observed in Fig. 2, A and B. Strikingly, underlying this slowed release were rapid, localized, and stochastic release events that often changed location in subsequent frames (Fig. 2D). Similar results were observed in six cells analyzed. Under these conditions the basal Ca2+ concentration was the same with EGTA [51 ± 12 nM (n = 10)] and without EGTA [51 ± 17 nM (n = 10)]. These images reveal the existence of elementary Ca2+-release units and suggest that these units were resolved only because EGTA reduced the probability of triggering individual release events. Lower EGTA concentrations (100 to 250 μM) were not sufficient to uncouple these elementary Ca2+-release units. One can therefore calculate that the time required for coupling of release events is ∼1 ms (15).

It is difficult to determine the exact dimensions of individual release events at the resolution of light microscopy. We determined an upper limit to the size of localized Ca2+-release events by marking locations with a 6% or higher increase in intensity (Fig.3A). The size distribution of these areas was represented as a histogram, with the number of events plotted as a function of the maximal diameter of the area (Fig. 3B). Ca2+-release events with diameters from 0 to 2 μm were most frequently observed. A plot of the local frequency of Ca2+-release events showed that they are triggered in all regions of the cytosol, with slightly higher frequencies in the perinuclear and plasma membrane regions (16). Comparison of the distribution of release events with the distribution of indicator showed no correlation, indicating that the localization of release events was not an artifact resulting from uneven indicator distribution.

Figure 3

Analysis of elementary Ca2+-release units. Subtracted images the same as those shown in Fig. 2D were used to analyze the spatial distribution of local Ca2+-release events. (A) A threshold of 6% ΔF/F was imposed on the subtracted image, and the resulting binary image shows areas of the cell above this threshold in white and areas below in black. (B) Histogram of the number of release events as a function of the size of elementary release units (largest diameter). A series of 20 consecutive images during the rising phase (Fig. 3) were processed as in (A) and analyzed with Image-1 object analysis. (C) The time course of fluorescence increases from several regions of the cell analyzed in two ways. (Top) Relative local fluorescence increase; (bottom) subtractive plot showing the increase in fluorescence relative to the previous frame. The global increase across the cell is shown in the top left panel and is also plotted in each of the local release traces (solid line). (D) Comparison of the fluctuations in local fluorescence intensity in unstimulated cells (top), after addition of thapsigargin (middle), and after uncaging of InsP3(bottom). Local fluctuations were plotted by counting the number of times a given change in local fluorescence intensity was measured (in every 33 ms, at different 1-μm2 areas). Gaussian fits to each histogram are shown as solid lines.

We analyzed the time course of Ca2+-release events by comparing the Ca2+- release kinetics measured from the whole cell to that measured at several 1-μm2 boxes in various regions of the cell (Fig. 3C). This comparison showed that different regions of the cell experience different release kinetics. The local transient can coincide with, lag behind, or precede the global increase, and rapid local increases were often observed. The duration of these shorter events was typically less than 100 ms, with many events below the resolution limit of 33 ms. Therefore, although the global increase in Ca2+ elicited by InsP3is slowed in the presence of EGTA, the localized Ca2+-release events remained rapid. The effect of EGTA appears to be to prevent synchronization by lowering the probability of triggering local Ca2+-release events.

The noise contribution to the observed local increases in fluorescence intensity was determined by histogram analysis of the amplitude of fluorescence intensity fluctuations (Fig. 3D). No significant difference was observed between the fluctuations at basal Ca2+ concentration (top panel) and those of cells with thapsigargin-depleted Ca2+ stores (middle panel), suggesting that the small basal fluctuations are not spontaneous release events. In contrast, the average local intensity fluctuations increased by 260% after an increase in InsP3concentration, demonstrating that the observed Ca2+-release events are significantly above the photon noise (bottom panel).

An estimate of the Ca2+ current responsible for individual release events can be made from the local change in fluorescence intensity. At least 30 pA of current is required to generate a 10% increase in CAAX green fluorescence, assuming that a release volume of 1 μm3 contains more than 50 μM of Ca2+buffer. This is similar to the current responsible for Ca2+“puffs” in Xenopus oocytes (10 to 20 pA) (12). The conductance of InsP3-receptor channels incorporated into lipid bilayers is ∼2 pA (14), suggesting that the observed localized events are likely to result from the simultaneous opening of several InsP3-gated Ca2+ channels.

Our study shows that InsP3-induced Ca2+transients are likely the result of synchronized triggering of elementary Ca2+-release units. Because the amplitudes of localized release events were similar at increasing Ca2+concentrations, it is likely that Ca2+ does not control the size of Ca2+-release events but instead regulates the stochastic probability at which elementary Ca2+-release units are triggered. The existence of localized all-or-none Ca2+-release events suggests that Ca2+-dependent enzymes can be selectively activated by their localization with, or translocation to, such local sites of Ca2+ release.

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