Rapid Turnover of Calcium in the Endoplasmic Reticulum during Signaling

HEK293 cells expressing the thyrotropin-releasing hormone (TRH) receptor were transfected with cameleon Ca2+ indicators designed to measure the free Ca2+ concentration in the cytoplasm, [Ca2+]cyt, and the endoplasmic reticulum (ER), [Ca2+]er. Basal [Ca2+]cyt was about 50 nm; thyrotropin-releasing hormone (TRH) or other agonists increased [Ca2+]cyt to 1 μm or higher. Basal [Ca2+]er averaged 500 μmand fell to 50–100 μm over 10 min in the presence of thapsigargin. TRH consistently decreased [Ca2+]er to 100 μm, independent of extracellular Ca2+, whereas agonists for endogenous receptors generally caused a smaller decline. When added with thapsigargin, all agonists rapidly decreased [Ca2+]er to 5–10 μm, indicating that there is substantial store refilling during signaling. TRH increased [Ca2+]cyt and decreased [Ca2+]er if applied after other agonists, whereas other agonists did not alter [Ca2+]cyt or [Ca2+]er if added after TRH. When Ca2+ was added back to cells that had been incubated with TRH in Ca2+-free medium, [Ca2+]cyt and [Ca2+]er increased rapidly. The increase in [Ca2+]er was only partially blocked by thapsigargin but was completely blocked if cells were loaded with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid. In conclusion, these new Ca2+ indicators showed that basal [Ca2+]er is ∼500 μm, that [Ca2+]er has to be >100 μm to support an increase in [Ca2+]cyt by agonists, and that during signaling, intracellular Ca2+ stores are continuously refilled with cytoplasmic Ca2+ by the sarcoendoplasmic reticulum Ca2+-ATPase pump.

The cytosolic free Ca 2ϩ concentration ([Ca 2ϩ ] cyt ) 1 is a dynamic signal regulating a variety of important cellular functions such as secretion, contraction, metabolism, and gene transcription (1). Ca 2ϩ -mobilizing hormones release inositol 1,4,5-trisphosphate (IP 3 ) which acts on the IP 3 receptor to release Ca 2ϩ from intracellular sites in the endoplasmic reticulum (ER). It is not certain whether the entire ER or specialized compartments within the ER form the IP 3 -releasable Ca 2ϩ store (2)(3)(4)(5). In some tissues, IP 3 receptors are concentrated in a small region of the ER (6). IP 3 receptors are Ca 2ϩ channels that open as the IP 3 concentration increases sufficiently. The cytoplasmic and possibly ER Ca 2ϩ concentrations also regulate the sensitivity of the IP 3 receptor to IP 3 (7)(8)(9). The Ca 2ϩ store is refilled by the sarcoendoplasmic reticulum Ca 2ϩ -ATPase (SERCA), a Ca 2ϩ pump. Like Ca 2ϩ in the cytosol, Ca 2ϩ in intracellular stores is largely bound to Ca 2ϩ -binding proteins; only free Ca 2ϩ is believed to participate directly in Ca 2ϩ signaling (7,10). Acute depletion of Ca 2ϩ stores results in impaired signaling by receptors that activate phospholipase C. Adequate luminal Ca 2ϩ concentrations are also necessary for nuclear transport (11) and protein processing in the ER (3). Chronic depletion of the store by inhibitors of the SERCA pump leads to apoptosis (12).
Although the concentration of free Ca 2ϩ in the cytoplasm, [Ca 2ϩ ] cyt , has been measured routinely for many years, there have been no entirely satisfactory methods for measuring the free Ca 2ϩ concentration in the ER (13)(14)(15)(16). Free Ca 2ϩ in the ER, [Ca 2ϩ ] er , has been estimated using magfura-2, but it is difficult or impossible to load the dye exclusively into the ER in intact cells (16). ER-targeted aequorin has also been used to follow [Ca 2ϩ ] er , but the properties of aequorin limit its ability to report Ca 2ϩ concentrations over time (17). Recently, two groups have developed novel fluorescent Ca 2ϩ indicators based on fluorescence resonance energy transfer between two modified green fluorescent proteins contained in a protein with calmodulin and a calmodulin-binding peptide (14, 18 -20). Cameleons, the Ca 2ϩ indicators from the Tsien group (20), have been targeted to ER and used to monitor [Ca 2ϩ ] er in HeLa cells (14). Total Ca 2ϩ in the ER is usually estimated indirectly by adding a Ca 2ϩ ionophore to cells loaded with a cytoplasmic Ca 2ϩ reporter under conditions where ER Ca 2ϩ is preferentially released. The resultant increase in Ca 2ϩ in the cytoplasm provides an estimate of the amount of Ca 2ϩ released from the ER.
Using Fura-2, we previously characterized the Ca 2ϩ responses of a HEK 293 cell line expressing receptors for thyrotropin-releasing hormone (TRH) and found that the refilling of the total intracellular Ca 2ϩ store, measured indirectly, appeared to be the limiting step in TRH regulation of [Ca 2ϩ ] cyt (21,22). In the present study, we have used the cameleons to measure directly the free Ca 2ϩ concentration in the ER and have followed the depletion and refilling of the intracellular Ca 2ϩ store in response to agonists, SERCA pump inhibitors, and ionomycin. We show that free Ca 2ϩ in the ER is around 500 M and that during signaling, ER Ca 2ϩ is continually replen-ished by the SERCA pump with cytoplasmic Ca 2ϩ that has entered the cell from the extracellular space.
Cell Culture and Transfection-A HEK293 cell line stably expressing the wild type mouse TRH receptor (301 cells) has been described previously (23). Cells were grown in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum as monolayer cultures at 37°C in a humidified 95% air, 5% CO 2 environment. 301 cells were transfected with the plasmids at 5 g/ml using LipofectAMINE. On the second day, cells were plated on ECL-coated coverslips. Cells were used 2 to 5 days after transfection.
Single Cell Ca 2ϩ Imaging-All Ca 2ϩ measurements were performed at room temperature. Cells were bathed in HBSS buffered to pH 7.4 with 15 mM HEPES. The coverslip was washed and put into a Sykes-Moore chamber on a Nikon inverted microscope. Data were collected from 6 -14 cells in a field, generally omitting cells with extremely bright or dim fluorescence. Cells expresssing YC2 were excited at a wavelength of 440 Ϯ 20 nm and emitted fluorescence was collected alternately at 535 Ϯ 15 and 485 Ϯ 20 nm every 3 s. With the YC2 cameleon, baseline fluorescence did not decline much using this protocol. In our preliminary experiments using the ER-targeted YC3er and YC4er cameleons, we observed a steady fall in the 535/485 ratio when fluorescence values were acquired every 3 s. To reduce the apparent photobleaching or photochromism of YC3er and YC4er, we made two acquisitions of fluorescence at 535 and 485 nm (3 s later) every 30 s. This approach largely abolished the baseline drop. The fluorescence images were captured with a Micromax camera (Princeton Instruments, Trenton, NJ) which was controlled by Metafluor software (Universal Imaging, Media, PA). The length of each acquisition was 80 ms for indicator YC2 and 200 ms for indicators YC3er and YC4er. Emission filters were switched manually.
Data Analysis and Presentation-The temporal resolution presented no problems for the indicators YC3er and YC4er since Ca 2ϩ changes inside the ER were slow, but the system may have underestimated peak Ca 2ϩ values in the cytosol where changes were fast. For YC2er, each trace was from a typical individual cell among 8 to 50 cells examined in two to eight independent experiments. Points represent the ratio of emitted fluorescence at 535 nm over that at 485 nm 3 s later. The absolute fluorescence of individual cells differed, but the relative changes in the 535/485 ratio were very consistent. For YC3er and YC4er, each trace was the average of the values of 535/485 ratio over the baseline from 4 to 10 cells in an experiment. All experiments were replicated at least four times with comparable results.
Calibration  (14,20). In attempts to calibrate the ER cameleons, we were unable to raise ER Ca 2ϩ high enough to saturate YC4er, which has a K d of 700 M, by adding ionomycin and high extracellular CaCl 2 as described previously (14). For this reason, we took advantage of the different affinities of the two ER cameleons for Ca 2ϩ in order to calibrate them. These cameleons differ only in that each contains a point mutation in the Ca 2ϩ -binding domain of the calmodulin moiety that alters its Ca 2ϩ binding affinity. We assumed that the maximal and minimal 535/485 ratios were the same for the two cameleons, and that YC3er, which has a K d of 4.4 M, was initially saturated. The minimal ratio was estimated following addition of ionomycin and EGTA, which always reduced the fluorescence ratio to an apparent minimum where it could not be lowered further by thapsigargin or other drugs. [Ca 2ϩ ] er was calculated as described previously, using values for YC3er and YC4er reported in Ref.

Properties of Cameleons in HEK293
Cells-We expressed three of the cameleons described by Miyawaki et al. (14) in HEK293 cells stably expressing the TRH receptor (301 cells): YC2, a cytosolic protein, and YC3er and YC4er, which are targeted to the ER with a calreticulin signal sequence and a C-terminal KDEL. The cameleons were introduced by transient transfection for the studies reported here. YC2 has dual affinity for Ca 2ϩ with reported K d values of 4.3 M and 100 nM. YC3er has a single affinity for Ca 2ϩ with a K d of 4.4 M and YC4er has dual affinity with K d values of 700 M and 83 nM. When transfected into the 301 cells, YC2 appeared to be restricted to the cytoplasm and YC3er and YC4er were both localized in a reticular pattern characteristic of ER (Fig. 1). YC3er and YC4er were distributed throughout the cell, suggesting that these two cameleons would report the average free Ca 2ϩ concentration of the entire endoplasmic reticulum. The localizations of the cameleons in HEK293 cells agrees with those described previously for HeLa cells (14).
When the cameleons are excited at 440 nm, their 535/485 fluorescence emission ratios increase with a rise in local free Ca 2ϩ concentration. The baseline fluorescence ratio of the cameleons was quite stable and was not altered over several minutes by sequential addition of HBSS, 0.2% Me 2 SO, or by 2 mM EGTA, which reduced extracellular free Ca 2ϩ from 1.3 mM to the low nM range (data not shown), indicating that the concentration of free Ca 2ϩ in the cytosol and ER does not fall measurably when extracellular Ca 2ϩ is chelated.
Effects of SERCA Inhibitors-When the SERCA pump is inhibited, Ca 2ϩ moves from the ER into the cytoplasm and capacitative Ca 2ϩ influx is activated as intracellular stores become depleted (24 -26). We used the cameleons to measure free Ca 2ϩ in the ER following addition of SERCA inhibitors (Fig. 2). As expected, the cytoplasmic YC2 reported a gradual, large, sustained increase in [Ca 2ϩ ] cyt when cells were treated with thapsigargin in buffers with normal Ca 2ϩ (Fig. 2). When EGTA was added to chelate extracellular Ca 2ϩ , thapsigargin caused only a small and transient increase in [Ca 2ϩ ] cyt , suggesting that influx of extracellular Ca 2ϩ normally contributes importantly to the sustained increase in [Ca 2ϩ ] cyt induced by SERCA inhibitors (Fig. 2).
Thapsigargin reduced [Ca 2ϩ ] er from ϳ500 M to 50 -100 M over 10 min, as measured with the dual affinity YC4er, whether extracellular Ca 2ϩ was chelated or not (Fig. 2). The decrease in [Ca 2ϩ ] er appeared to be gradual. Not surprisingly, thapsigargin did not decrease the fluorescence ratio of YC3er, the high-affinity ER Ca 2ϩ indicator, because the dye is still nearly saturated at 50 -100 M. Similar results were obtained with another SERCA pump inhibitor, 20 M cyclopiazonic acid (data not shown). Subsequent addition of ionomycin together with or following EGTA caused [Ca 2ϩ ] er to fall rapidly to low nM levels. Thapsigargin treatment prevented agonists such as TRH from increasing [Ca 2ϩ ] cyt .
Effects of Agonists-In a previous report, we estimated total ER pool sizes from the [Ca 2ϩ ] cyt peak obtained in response to ionomycin, added after chelation of extracellular Ca 2ϩ , and estimated that TRH causes more than 95% depletion of total intracellular Ca 2ϩ pools (21). Here, we used the cameleons to determine the changes in free Ca 2ϩ in the ER following TRH treatment. TRH alone, TRH with EGTA, and TRH with thapsigargin stimulated increases in [Ca 2ϩ ] cyt as measured by YC2.
[Ca 2ϩ ] cyt increased to approximately 1 M, in good agreement with data obtained using fura-2 (Fig. 3). TRH causes a biphasic increase in [Ca 2ϩ ] cyt , first releasing Ca 2ϩ from the ER and then stimulating the influx of extracellular Ca 2ϩ , presumably through capacitative Ca 2ϩ channels. With the cameleons, the sustained increase in [Ca 2ϩ ] cyt is particularly evident because of the high sensitivity of the reporter in the low nM range. The [Ca 2ϩ ] cyt spike was short-lived when extracellular Ca 2ϩ was chelated. TRH reduced [Ca 2ϩ ] er from ϳ500 to 50 -100 M over 10 min, as measured with the dual affinity YC4er and single affinity YC3er, whether extracellular Ca 2ϩ was chelated or not (Fig. 3). The decrease was largely complete in the first minute after stimulation, consistent with our previous report using ionomycin to estimate the ER Ca 2ϩ pool (21). The decline in ER Ca 2ϩ occurs rapidly enough to explain the increase in cytoplasmic Ca 2ϩ and the increase in Ca 2ϩ influx, assuming that the signal for influx through store-operated channels is depletion of ER Ca 2ϩ pools. When thapsigargin was added to inhibit the SERCA pump, TRH caused a more severe drop in [Ca 2ϩ ] er , an effect that was particularly evident with the high affinity reporter YC3er. In the presence of thapsigargin, TRH caused [Ca 2ϩ ] er to fall from 500 to below 10 M in the first 30 s.
Using fura2, we previously found that a mixture of agonists working at endogenous G q -coupled receptors increased [Ca 2ϩ ] cyt to the same peak height as TRH but depleted total intracellular Ca 2ϩ stores less completely, ϳ75%, again based on the ionomycin method to measure pool size. Like TRH, the combined agonists caused a robust increase in IP 3 , but for a shorter time (21). Using the cameleons, we found that the agonist mixture increased [Ca 2ϩ ] cyt , measured with YC2, to about the same peak height as TRH but they caused a lower sustained rise (Fig. 4). The combined agonists caused a variable decrease in [Ca 2ϩ ] er , ranging from a decline similar to that caused by TRH to a smaller decline (Figs. 4 and 5), whether extracellular Ca 2ϩ was chelated or not. When added together with thapsigargin, the combined agonists caused [Ca 2ϩ ] er to fall to around 10 M, but as evidenced by traces with YC3er, this drop was not as great as that caused by TRH and thapsigargin.
We also measured the effect of sequential addition of TRH and agonist mixture on free Ca 2ϩ in the ER. The agonist mixture and TRH increased [Ca 2ϩ ] cyt to similar peak values, as reported by YC2 (Fig. 5), with TRH again causing the more sustained increase. The combined agonists were unable to elicit any change in [Ca 2ϩ ] cyt after TRH, whereas TRH was always able to increase [Ca 2ϩ ] cyt effectively when added after the other hormones. This pattern was found with fura2 and has been very consistent in experiments with the cameleons. At the same time, the agonist mixture never produced a sizable decrease in [Ca 2ϩ ] er when added after TRH, but TRH always caused [Ca 2ϩ ] er to decline when added after other agonists. Effects of Ca 2ϩ Readdition-As described above, agonists caused a precipitous fall in [Ca 2ϩ ] er if Ca 2ϩ reuptake into the ER was inhibited. These results suggested that Ca 2ϩ was continually sequestered into the ER during signaling. When cells were incubated with TRH and EGTA, readdition of Ca 2ϩ caused a rapid influx of Ca 2ϩ that could be detected by the cytoplasmic YC2, whether the SERCA pump was inhibited or not (Fig. 6). Removal of extracellular Ca 2ϩ did not, by itself, cause this rapid Ca 2ϩ influx upon readdition of extracellular Ca 2ϩ (Fig. 6, bottom trace in upper left panel). Readdition of extracellular Ca 2ϩ to TRH-treated cells led to a rapid increase in [Ca 2ϩ ] er to about 10 M that was detected by YC3er (Fig. 6). These responses presumably represent capacitative Ca 2ϩ influx. Surprisingly, we found that an increase in [Ca 2ϩ ] er oc- curred when external Ca 2ϩ was re-added to cells following blockade of SERCA pumps with either thapsigargin (Fig. 6) or cyclopiazonic acid (data not shown). A similar increase was observed when the thapsigargin concentration was increased 10-fold. The experiments described in Fig. 6 were repeated after readdition of 2 instead of 20 mM extracellular Ca 2ϩ with very similar results in terms of both the absolute and relative changes in [Ca 2ϩ ] cyt and [Ca 2ϩ ] er . We tested whether an increase in [Ca 2ϩ ] cyt is required for refilling of [Ca 2ϩ ] er by loading cells with BAPTA-AM. Intracellular BAPTA quickly repressed the increase in [Ca 2ϩ ] cyt resulting from Ca 2ϩ readdition and effectively prevented any increase in [Ca 2ϩ ] er (Fig. 6). DISCUSSION We have used the novel cameleon Ca 2ϩ indicators (14) to obtain new information about the concentration of free Ca 2ϩ in the cytoplasm and endoplasmic reticulum during hormonal signaling in a well characterized model system (21,23). The cytoplasmic cameleon YC2 offers several advantages. With K d values of 4.3 M and 100 nM, it is able to detect changes in cytoplasmic Ca 2ϩ in both the micromolar and low nanomolar ranges, where conventional Ca 2ϩ indicators are of limited usefulness. The dual affinity cameleons report changes in free Ca 2ϩ over a broad range, but the absolute changes in fluorescence ratio are small and their sensitivity is fairly low, and calibration is problematic. YC2, YC3er, and YC4er are also sensitive to pH (14,20). This should not have been a problem with TRH, which has almost no effect on cytoplasmic pH unless cells are artificially acidified (27), but was a concern with ionomycin, which is a Ca 2ϩ /proton ionophore. Several lines of evidence, however, suggest that the changes in fluorescence ratio reported here were primarily due to changes in Ca 2ϩ . First, moderate changes in pH affect the 535 nm but not the 485 nm fluorescence emission of the cameleons; in our experiments, fluorescence changed in the expected directions at both wavelengths. Second, Ca 2ϩ changes were in opposite directions for the cytoplasmic YC2 and the ER-targeted dyes. There is reportedly no pH gradient between the cytoplasm and the ER (28). Third, ionomycin did not affect the fluorescence of YC3er or YC4er when added to cells with already depleted intracellular pools. For these reasons, pH effects must have been minor contributors to the overall changes in fluorescence ratio. A newer version of the cytoplasmic reporter has greatly reduced pH sensitivity (20).
It is necessary to postulate that much of the Ca 2ϩ released by both the agonist mixture and TRH was re-sequestered to account for the dramatically greater decline in [Ca 2ϩ ] er seen when SERCA inhibitors were added with hormones. In fact, refilling is expected to accelerate as [Ca 2ϩ ] er drops, because the activity of the SERCA pump is inhibited by high Ca 2ϩ in the ER lumen (9,29), and the rate of Ca 2ϩ transport increases as [Ca 2ϩ ] cyt rises (30,31). An apparent equilibrium is reached when [Ca 2ϩ ] er is around 100 M, at which point the rates of Ca 2ϩ release through the IP 3 receptor and Ca 2ϩ refilling by the SERCA pump become similar. It may be that [Ca 2ϩ ] er is not normally permitted to fall much below 100 M, even when agonists are present, to preserve other functions of the ER. Our results differ from the situation in HeLa cells, where SERCA inhibitors do not potentiate the agonist-promoted decline in [Ca 2ϩ ] er (32). In the HEK293 cells studied here, the balance between Ca 2ϩ reuptake and Ca 2ϩ efflux appears to preserve Ca 2ϩ in the ER better when cells are treated with a mixture of agonists for endogenous receptors than when cells are treated with TRH. It is not clear if this difference is the result of intrinsic differences in signaling through different receptors, or the high level of expression of the transfected TRH receptor. Overall, the results point to a very rapid turnover of Ca 2ϩ following activation of Ca 2ϩ mobilizing, G protein-coupled receptors. In contrast, the rate of turnover of ER Ca 2ϩ appeared to be low in resting cells, since [Ca 2ϩ ] er was quite stable following simple removal of extracellular Ca 2ϩ .
Our conclusions regarding the rapid turnover of Ca 2ϩ in HEK293 cells are in agreement with those of Hofer et al. (33) who used magfura to monitor [Ca 2ϩ ] er in BHK cells and found that uptake of Ca 2ϩ into the ER takes place during agonist stimulation, even under conditions where cytoplasmic Ca 2ϩ is low. Our results, however, differ from those reached by Barrero et al. (32) studying HeLa cells, where agonists seem to release the minimal amount of ER Ca 2ϩ sufficient to produce a cytoplasmic Ca 2ϩ increase. In HeLa cells, the release of ER Ca 2ϩ in response to agonists is halted by the inhibitory effects of high cytoplasmic Ca 2ϩ and low luminal Ca 2ϩ on the IP 3 receptor. In HEK293 cells, agonists still cause a strong decline in [Ca 2ϩ ] er in the face of high cytoplasmic and low luminal Ca 2ϩ and refilling continues during signaling. Differences in IP 3 generation and metabolism, IP 3 receptor subtypes and abundance, and SERCA activity could all contribute to these observed differences between cell types.
Ca 2ϩ Store Refilling-Our data show that extracellular Ca 2ϩ is not critical for the maintenance of the ER Ca 2ϩ store over at least 10 min if no agonists or SERCA inhibitors are present. In the presence of IP 3 , however, the intracellular Ca 2ϩ store is continuously refilled. If stores were allowed to become depleted, then increasing cytoplasmic Ca 2ϩ resulted in increased ER Ca 2ϩ ; this was evident, for example, in the Ca 2ϩ re-addition paradigm. This increase was expected, since K m values for the SERCA pumps are high enough, 0.2-0.4 M (30, 31), that an increase in cytoplasmic Ca 2ϩ would be expected to lead to an increase in the amount of Ca 2ϩ pumped. We found that refilling was completely eliminated with BAPTA-AM, indicating that the Ca 2ϩ for pool refilling entered from the cytoplasm. These results are consistent with recent data showing that even though ER-plasma membrane communication and the IP 3 receptor are critical for store operated Ca 2ϩ influx, stores are replenished with Ca 2ϩ from the cytoplasm (33,34).
Our results suggest that the intracellular Ca 2ϩ store is refilled with Ca 2ϩ from the cytosol by the SERCA pump and perhaps by an additional, thapsigargin-insensitive mechanism.
[Ca 2ϩ ] er increased significantly when extracellular Ca 2ϩ was reintroduced to cells that had been treated with thapsigargin or cyclopiazonic acid in Ca 2ϩ -free medium, although the increase in [Ca 2ϩ ] er was always much greater when SERCA pumps were active. A thapsigargin-insensitive Ca 2ϩ pump has been postulated previously (35,36). Alternatively, residual SERCA activity in thapsigargin-and cyclopiazonic acid-treated cells may have been enough to increase [Ca 2ϩ ] er in the range detected by the high affinity YC3er, or in this paradigm Ca 2ϩ may have been moving down a gradient into the ER.
By using the cameleons to monitor changes in free Ca 2ϩ in the ER and cytoplasm directly, we have shown that there is a very high rate of turnover of ER Ca 2ϩ during hormonally stimulated Ca 2ϩ signaling. During signaling, the intracellular Ca 2ϩ store is continuously refilled by the SERCA pump using Ca 2ϩ from the cytoplasm. Future studies will be directed toward identifying the subcellular localization of the sites in the ER involved in this rapid release and reuptake of Ca 2ϩ .