A Direct Mass-action Mechanism Explains Capacitative Calcium Entry in Jurkat and Skeletal L6 Muscle Cells*

We examined capacitative calcium entry (CCE) in Jurkat and in L6 skeletal muscle cells. We found that extracellular Ca2+ can enter the endoplasmic reticulum (ER) of both cell types even in the presence of thapsigargin, which blocks entry into the ER from the cytosol through the CaATPase. Moreover, extracellular Ca2+ entry into the ER was evident even when intracellular flow of Ca2+ was in the direction of ER to cytosol due to the presence of caffeine. ER Ca2+ content was assessed by two separate means. First, we used the Mag-Fura fluorescent dye, which is sensitive only to the relatively high concentrations of Ca2+ found in the ER. Second, we transiently expressed an ER-targeted derivative of aequorin, which reports Ca2+ by luminescence. In both cases, the Ca2+ concentration in the ER increased in response to extracellular Ca2+ after the ER had been previously depleted despite blockade by thapsigargin. We found two differences between the Jurkat and L6 cells. L6, but not Jurkat cells, inhibited Ca2+ uptake at very high Ca2+ concentrations. Second, ryanodine receptor blockers inhibited the appearance of cytosolic Ca2+ during CCE if added before Ca2+ in both cases, but the L6 cells were much more sensitive to ryanodine. Both of these can be explained by the known difference in ryanodine receptors between these cell types. These findings imply that the origin of cytosolic Ca2+ during CCE is the ER. Furthermore, kinetic data demonstrated that Ca2+ filled the ER before the cytosol during CCE. Our results suggest a plasma membrane Ca2+ channel and an ER Ca2+ channel joined in tandem, allowing Ca2+ to flow directly from the extracellular space to the ER. This explains CCE; any decrease in ER [Ca2+] relative to extracellular [Ca2+] would provide the gradient for refilling the ER through a mass-action mechanism.


Summary
We examined capacitative calcium entry (CCE) in Jurkat and in L6 skeletal muscle cells. We found that extracellular Ca 2+ can enter the endoplasmic reticulum (ER) of both cell types even in the presence of thapsigargin, which blocks entry into the ER from the cytosol through the CaATPase.
Moreover, extracellular Ca 2+ entry into the ER was evident even when intracellular flow of Ca 2+ was in the direction of ER to cytosol, due to the presence of caffeine. ER Ca 2+ content was assessed by two separate means. First, we used the Mag-Fura fluorescent dye, which is sensitive only to the relatively high concentrations of Ca 2+ found in the ER. Secondly, we transiently expressed an ERtargeted derivative of aequorin, which reports Ca 2+ by luminescence. In both cases, the Ca 2+ concentration in the ER increased in response to extracellular Ca 2+ after the ER had been previously depleted, despite blockade by thapsigargin. We found two differences between the Jurkat and L6 cells.
L6, but not Jurkat cells inhibited of Ca 2+ uptake at very high Ca 2+ concentrations. Secondly, ryanodine receptor blockers inhibited the appearance of cytosolic Ca 2+ during CCE if added prior to Ca 2+ in both cases, but the L6 cells were much more sensitive to ryanodine. Both of these can be explained by the known difference in ryanodine receptors between these cell types. These findings imply that the origin of cytosolic Ca 2+ during CCE is the ER. Furthermore, kinetic data demonstrated that Ca 2+ filled the ER prior to the cytosol during CCE. Our results suggest a plasma membrane Ca 2+

Introduction
Ca 2+ is a critical regulator for a large number of cells, and it is known that for many, the key signaling event is the release of this ion from the ER (1;2). After release, most of the Ca 2+ is resequestered to the ER through the CaATPase, although some is lost through the plasma membrane to the cell exterior. Maintaining the ER pool of Ca 2+ requires re-entry from outside the cell. Casteels and Droogmans (3) first reported a correlation between depletion of ER [Ca 2+ ] and entry of Ca 2+ from extracellular sources into the cell. This phenomenon was then extensively studied by Putney and coworkers and named "store-operated" or capacitative calcium entry (CCE) (4).
The mechanism for CCE remains unknown, although two general models have been proposed.
One, borrowed from the known association of the skeletal muscle plasma membrane potential sensor (L channel) and ER protein (ryanodine receptor) posits a direct connection in which Ca 2+ depletion within the ER is sensed by an ER protein, transmitted by protein-protein interaction to the plasma membrane protein which then allows entry of Ca 2+ into the cytosol. A second, based on second messenger signaling systems such as that for cyclic AMP, posits a messenger generated within the ER subsequent to Ca 2+ depletion that diffuses to the plasma membrane and allows entry of Ca 2+ into the cytosol (5;6).
When Ca 2+ uptake was originally studied, it appeared that Ca 2+ did not change in concentration in the cytosol (7), and a direct entry from the extracellular space into the ER was postulated.
The discovery of selective inhibitors of the CaATPase (e.g. thapsigargin) changed that view: in the presence of such agents, adding exogenous Ca 2+ to Ca 2+ -free cells leads to an accumulation of cytosolic Ca 2+ (8)(9)(10). This has been interpreted as proving that Ca 2+ enters cells through the cytosol, is blocked from entry into the ER, and accumulates in the cytosol. Almost all of the CCE studies rely on monitoring only changes in cytosolic Ca 2+ levels. While measuring cytosolic Ca 2+ levels afford valuable insights into the phenomenon of CCE, this, by design, remains an indirect way of assessing ER Ca 2+ changes.
Most CCE studies were conducted with non-excitable cells; however, there now exists evidence that this restriction need not apply. We had previously shown that CCE occurs in L6 skeletal muscle cells (11), and others demonstrated subsequently that CCE occurs in skeletal muscle (12;13). In our prior studies of L6 cells, we determined Ca 2+ in both cytosol and ER using fluorescent dyes, finding that Ca 2+ enters the ER even in the presence of thapsigargin (14). In the present study, we explored this hypothesis for Jurkat cells, a well-studied non-excitable cell line. Our previous work used direct measurement of

Materials
Jurkat cells (E6-1 clone from humans) were purchased from American Type Culture Collection (Redding CA). All cell culture media and reagents were obtained from Life Technologies (New York).
The other chemicals and reagents used in this study were obtained from Sigma (St. Louis, MO).

Methods
Cell Culture: Jurkat cells were grown on RPMI-1640 medium supplemented by 10% fetal calf serum.
Media was changed every 48 hours and cells were used when they attained a concentration of 5 x 10 5 cells/ml. L6 cells were grown as described previously (11). Fluorescent Measurements: Measurement of cytosolic Ca 2+ was performed as reported previously, using Indo PE3 (AM) dye (14). For measurement of the ER Ca 2+ of Jurkat cells, the cells were loaded with 8-10 ¼M of the dye Mag-Fura in Hanks Balanced Salt Solution (HBSS). The cells were incubated 1 h at 37 o C at 5% CO 2 tension. Following this, the mixture was centrifuged at 1500 rpm for 2 min and the supernatant was discarded. The cells were resuspended in fresh HBSS for 1.5 h to allow de-esterification of the dye. After dye loading, L6 cells were placed in a quartz cuvette at a 45° angle to both the excitation and emission beams of a Hitachi F2000 Fluorometer in 1.5 ml of HEPES buffer containing, in mM, 20 HEPES, 118 NaCl, 12 NaHCO 3 , 2.6 KCl, 1.2 KH 2 PO 4 , 10 Glucose, pH 7.4. The fluorescence measurements used excitation wavelengths of 347 nm and 373 nm and emission at 507 nm (14). The ratio of the bound to unbound dye with a dissociation constant of 53 µM for Mag-Fura was used to calculate the free ER [Ca 2+ ] as described by Grynkiewicz (22). Maximum and minimum fluorescence was measured at the end of each experiment by adding 0.1% Triton X-100 and 5mM EGTA respectively. Microscopy: L6 cells, grown on glass slides, were loaded as above with Mag-Fura, and a drop of 10% glycerol in 0.9% saline was added, and a cover slip placed on top. Epifluorescence was measured with an Olympus microscope with fluorescent attachment and a blue excitation filter. Exposures were made from an attached Nikon camera.

Statistical Analysis:
The experimental data were analyzed by Student's paired "t" test, ANOVA, and Tukey's test for post-hoc analysis, using SPSS version 9.0. Data are represented as means ± S.E.M and the level of significance was set at p < 0.05.

Results
Since the phenomenon of capacitative calcium entry (CCE), by definition, involves replenishing the ER Ca 2+ stores, we measured ER as well as cytosolic Ca 2+ levels in the Jurkat cells. Figure 1 shows that thapsigargin (Tg) increased cytosolic Ca 2+ in Jurkat cells suspended in a Ca 2+free medium. Subsequent addition of Ca 2+ caused a greater increase in cytosolic Ca 2+ , as expected.
However, as shown in Fig. 1a, this increase was sensitive to inhibition by ryanodine (Ry). As shown in Fig. 1b, ryanodine did not cause inhibition when exogenous Ca 2+ was added prior to Ry. Thus, as we observed previously with L6 muscle cells (14) ryanodine blocked an increase in cytosolic [Ca 2+ ] -the current hallmark of CCE -but only if added prior to exogenous Ca 2+ . Since ryanodine blocks the ER Ca 2+ release channels (the ryanodine receptor, or RyR) the results imply that the origin of cytosolic Ca 2+ during CCE may be the ER rather than the extracellular space, which is usually assumed. DHBP (diheptylbipyridium dibromide), another blocker of the RyR (25;26) was able to inhibit the Tg induced rise in cytosolic Ca 2+ (Fig 2). DHBP also blunted CCE, as measured by the cytosolic Ca 2+ increase subsequent to exogenous Ca 2+ addition. Unlike the effect of Ry (which appeared to act as a competitive inhibitor), DHBP action was unaltered when added prior to or after the addition of Ca 2+ to the media. The blockade of CCE by two kinetically distinct compounds that both target the RyR firmly supports the notion that the origin of the cytosolic Ca 2+ under these conditions is the ER. We next conducted more direct measurements of ER [Ca 2+ ] levels. Ca 2+ concentrations is shown in Fig. 4. It is apparent that ER Ca 2+ concentrations increased over the physiological range of exogenous Ca 2+ and was saturated at about 2 mM. We also found that exogenous Ca 2+ increased the ER Ca 2+ content in the presence of both caffeine and Tg, as shown in

Analysis of ER Ca 2+ by transfection of a modified aequorin
In order to confirm the observations made with Mag-Fura, we sought to measure ER Ca 2+ by an alternative method. We used the technique of transfection of the photo-protein aequorin that has been modified to achieve selectivity for ER Ca 2+ in two ways (19;20) . First, it has a leader sequence that targets the expressed protein into the ER. Second, the Ca 2+ binding site of the protein has been modified so that its affinity for Ca 2+ is much less than aequorin itself, and is suitable for binding Ca 2+ in the ER space. The cofactor coelenterazine is also modified to provide a system for reporting Ca 2+ specific to the ER, with a dissociation constant (about 15 ¼M) that would render it insensitive to Ca 2+ concentration of other cellular pools. Fig. 6 shows the luminescence of cells that have transiently expressed aequorin compared with controls. It is clear that transfected cells had considerable baseline Ca 2+ , and that more was apparent when Ca 2+ was added. This indicates the technique can measure intracellular Ca 2+ pools that have a low Ca 2+ affinity, comparable to the Mag-Fura dye. Fig. 7 shows that the increase in Ca 2+ , which we attribute to ER based upon the two considerations above, occurred over the same range of externally added Ca 2+ as that indicated by the Mag-Fura dye (cf. Fig 4). While the calculated values of ER [Ca 2+ ] were somewhat greater (30%) with the aequorin method, it is not a large difference, considering uncertainties in the binding constants required for calculation of the free [Ca 2+ ].

Lanthanum blocked the increase in ER [Ca 2+ ] measured by both methods
As a further control, we studied the effect of lanthanum, a known inhibitor of Ca 2+ uptake by virtually any pathway into the cell, on the appearance of ER Ca 2+ by both the Mag-Fura (Fig. 8a) as well as the aequorin (Fig. 8b) methods. In both cases, the increase in ER [Ca 2+ ] that occurred in the presence of Tg was completely suppressed by lanthanum. This ruled out the trivial possibility that either dye or protein had leaked to any appreciable extent into the medium, indicating the measurements reflect an intracellular Ca 2+ pool at concentrations of about two orders of magnitude greater than cytosolic or mitochondrial pools.

Complementary results with L6 cells
We have recently shown that Ca 2+ in the SR of the L6 muscle cells increases with increasing exogenous Ca 2+ , but the titration appeared qualitatively different from the one we observed in the present study with Jurkat cells: there was considerable inhibition of Ca 2+ content as Ca 2+ was added at concentrations greater than 3 mM (14). We therefore transfected L6 cells with aequorin using methods similar to those used for Jurkat cells, and titrated the cells with exogenous Ca 2+ . The results are shown in Fig. 9. As with the previous findings with Mag-Fura (14), cells transiently expressing the aequorin protein in the ER showed substantial inhibition of Ca 2+ uptake to the ER when exogenous Ca 2+ was added in concentrations greater than 3 mM. Thus, the similarity of results with the two methods extends to the L6 cells as well.

Kinetic evidence
If Ca 2+ enters the ER prior to its entry into the cytosol, it should be possible to observe a more rapid entry into the ER than the cytosol when exogenous Ca 2+ is added to Ca 2+ -deprived cells. In order to readily resolve the kinetics of Ca 2+ appearance, it was necessary to lower the incubation temperature. Representative traces at 4 o C for the appearance of Ca 2+ are shown for the cytosol (Fig.   10a) and the ER (Fig. 10b). It is evident that the slope of the cytosolic Ca 2+ curve was more shallow and that the time to reach steady state considerably longer than the ER Ca 2+ curve, indicating a slower Ca 2+ entry into the cytosol than the ER. Fig. 11 shows results over a broad range of temperatures, plotting the time required to reach steady state for both compartments. At 37 o C, it was no longer possible to resolve any kinetic differences between these compartments, but at all lower values it is apparent that the ER filling was more rapid than the cytosol, suggesting that Ca 2+ enters the ER prior to the cytosol.

Microsopy
We conducted fluorescence microscopy studies of the L6 cells, as these cells (unlike Jurkat cells) are firmly attached to their support in culture. When L6 cells were incubated with Mag-Fura in the presence of Ca 2+ , we observed virtually all the cells in the field contained spots of discrete fluorescence intensity apparently surrounding the nucleus, typified by the cell shown in Fig. 12. The cells were strikingly similar in appearance to Mag-Fura loaded cells observed with confocal microscopy (27). In experiments not shown, we treated such cells with caffeine, and observed that virtually the entire field was devoid of apparent fluorescence.

Discussion
Our model explaining CCE is diagrammed as Fig. 13. The close linkage of the plasma membrane channel with the ER Ca 2+ channel (1 and 2 in Fig 13) is based on the known proximity of these proteins in the skeletal muscle (28). We suggest that Ca 2+ directly enters the ER, from the extracellular space, traversing both channels (1 and 2) without an intermediate appearance in the cytosol. This suggests the "capacitative" or "store-operated" entry may operate directly by massaction. As more Ca 2+ is depleted from the ER by any means, the gradient for refilling is increased.
The model also accounts for thapsigargin-insensitive ER filling, as the CaATPase is not involved in this step. Moreover, the model explains ER Ca 2+ filling in the presence of submaximal caffeine, when the net flow of Ca 2+ is most likely in the direction of ER to cytosol. The fact that Ca 2+ entry into the ER upon addition of extracellular Ca 2+ was evident even in the presence of caffeine is significant because it provides independent evidence for ER filling from a source other than the cytosol. In the presence of caffeine, it is well established that the ER releases Ca 2+ to the cytosol. It is impossible for the source of this same ER Ca 2+ to be the cytosol, because we have just noted that the net flow is towards the cytosol. A simple solution to this paradox is that Ca 2+ filling proceeds instead through a direct connection between the extracellular space and the ER compartment.
While it is not possible to unambiguously identify the channel proteins 1, 2, and 3, abundant evidence points one of the trp family of proteins for channel protein 1 in Jurkat cells (6). We propose that channel protein 2 may be the RyR3, which currently has no clearly known function despite its location in a wide number of cells that already have other Ca 2+ release channels (29). For skeletal muscles, modeled by the L6 cells, channel protein 1 would be the dihydropyridine receptor (DHPR) and channel protein 2 would be the RyR1. Recent structural studies have indicated that the subunits comprising the Ca 2+ mouth of the DHPR align with those of the RyR1 (30).
Ca 2+ can appear in the cytosol through transport proteins represented by channel protein 3 in Fig. 13; in the case of Jurkat cells, these may be either RyR3 or IP 3 R. Under conditions in which IP 3 is not present, such as those commonly used to demonstrate CCE with Tg, we suggest that channel protein 3 may be the RyR3, as indicated by the inhibition at relatively high concentrations of ryanodine in this study.
Currently, investigators favor two other models to explain the phenomenon of CCE (5). The first of these, a protein-protein interaction, or protein-contact model, was inspired by analogy to skeletal muscle excitation-contraction coupling. The second, a diffusible-messenger model, is based upon second messenger systems such as those involving cAMP or IP 3 . Jurkat cells are known to use IP 3 signaling, so it is not surprising that many investigators use this system as an example of the diffusible-messenger model of CCE.

Complications of the existing CCE Models
The protein-contact model proposes that a lower [Ca 2+ ] in the ER alters the conformational state of the ER-membrane bound Ca 2+ channel. Since this channel is in direct contact with the plasma membrane Ca 2+ channel, the latter is stimulated to release Ca 2+ into the cytosol. This model thus requires that Ca 2+ enter the cytosol from both channel proteins, and yet the channels must also contact each other. The resulting structural requirements are variously depicted in drawings showing protein-protein contact that is separate from the mouths of the channels, from which Ca 2+ must exit (6;31).
While these constructs satisfy the idea that Ca 2+ is elevated in the presence of thapsigargin, they are contrary to what is known about the structures of established ion channels. Indeed, there are no known ion channels with structures that satisfy the sketches. The data for the L-channel:RyR interaction, on which this model is based, indicates that the mouths of the two channels are in direct physical contact with each other, making it difficult for them to also serve as Ca 2+ exit routes (28;32).
Beyond this complication, our observation of an increase in Ca 2+ in the ER in the presence of thapsigargin is inconsistent with currently favored models.
Hofer et al (33) provided simultaneous measurements of ER Ca 2+ , cytosolic Ca 2+ , and Ca 2+ current (I CRAC ) in a fibroblast cell line. They found that the rate of Ca 2+ entry was inversely correlated to the Ca 2+ content of the ER, and that there was no threshold for uptake. This finding, as well as the relatively slow stimulation of uptake after rapidly depleting ER [Ca 2+ ] with an intracellular chelator appears to make the protein-contact model unlikely. They concluded that this makes the diffusible-messenger model more likely, by default. However, other findings in the study of Hofer et al (28) make the soluble messenger model difficult to accept. They demonstrated that the kinetics of Ca 2+ appearance in the cytosol during CCE is independent of the duration of the preceding Ca 2+ free period. An enzyme generating a soluble messenger would be expected to produce more of the signal molecule with longer activation periods.
Taken on its own merits, the diffusible-messenger model proposes a factor, often called the "calcium influx factor" (CIF) that is produced in the ER in response to a depleted ER [Ca 2+ ] (5).
Following this, the CIF diffuses to the plasma membrane, simulating a plasma membrane Ca 2+ channel and subsequently, Ca 2+ entry to the cytosol. Despite elaborate studies of the CIF, its molecular identity remains elusive.
In a recent review, Putney et al. (6) has suggested that the mechanism of CCE may vary with cell type. This is consistent with our findings that Ca 2+ inhibited its own uptake in L6 cells, but not in Jurkat cells. Still, existing models of CCE all require Ca 2+ to flow from the external space into the cytosol, and subsequently enter the ER through the CaATPase. As we provide evidence for the direct entry of Ca 2+ without first entering the cytosol, this is a major point of distinction. This fundamental result, as shown here, holds for cell types as diverse as L6 and Jurkat cells.

Ryanodine Receptor inhibition
The observation that ryanodine and DHBP inhibited Ca 2+ accumulation in the cytosol implicates a RyR (channel 3 in Fig. 13) during CCE. Ryanodine also inhibits Ca 2+ accumulation in the cytosol of the L6 cell (14), with about an order of magnitude greater sensitivity. This corresponds to the known difference of sensitivity between the RyR3 present in Jurkat cells (and most non-muscle type cells) and the RyR1 present in L6 cells (and skeletal muscle in general) (34)(35)(36). It is also possible that ryanodine nonspecifically inhibits another exit channel, perhaps the IP 3 receptor. However, there is no evidence to support this from previous literature. Ryanodine, as has been mentioned in a previous study (37), does not affect the non-specific inward movement of the external Ca 2+ in skeletal muscle unless the cells were preincubated with ryanodine for at least 15 min, and at high concentration (100 ¼M). We showed in a subsequent study that much lower concentrations (10 ¼M), without preincubation, were able to prevent the appearance of cytosolic Ca 2+ during CCE, which had no influence on ER [Ca 2+ ] (14). The lack of effect of Ry on ER [Ca 2+ ] is likely due to the fact that the cytosolic Ca 2+ pool is much smaller than the ER Ca 2+ pool.
The finding that high concentrations of Ca 2+ inhibit L6 cell uptake (this study and (14)) but not Jurkat cell Ca 2+ uptake is also consistent with properties of the ryanodine receptor. The skeletal muscle ryanodine receptor is known to be Ca 2+ sensitive at high concentrations, but the RyR3 is not (36).
These facts are thus consistent with the ryanodine receptor serving as the other half of the dualchannel uptake for Ca 2+ into the ER as proposed here.
The inhibition of cytosolic Ca 2+ filling in response to DHBP, independent of ambient Ca 2+ , further argues that exogenous Ca 2+ does not enter the cytosol first. Additional support for this notion comes from a study of Tg-induced Ca 2+ toxicity (26). In that work, it was concluded that Ca 2+ blockers -including DHBP, but not Ry -abrogated Ca 2+ toxicity. These investigators also suggested a block of cytosolic Ca 2+ , although it was supported only by prevention of toxicity. The inability of Ry itself to suppress the toxicity was not explained. We can propose an explanation based on the observations in this and our prior work (14) : Ca 2+ competes with Ry, but DHBP is unaffected by Ca 2+ levels during CCE (i.e., noncompetitive).

Excitable vs Nonexcitable cells
Most of the work that has been performed on CCE has focused on non-excitable cells (6), presumably to avoid the complication of Ca 2+ entry by voltage-dependent ion channels. However, CCE can occur in excitable cells as well (7). For skeletal muscle cells, there had been some controversy.
The evidence that skeletal muscle CCE was mediated by a "leak channel" was based on observations of Mn 2+ entry, used as a surrogate of Ca 2+ (12). However, we observed that Mn 2+ entry is not capacitative (14). Thus, Mn 2+ is an inappropriate surrogate ion for Ca 2+ in CCE.
Two other misconceptions must be addressed concerning skeletal muscle cells. First, it may be recalled that the Ca 2+ needed for contraction does not require Ca 2+ entry into the cell (38), and this may appear to contradict the notion that the L-channel is involved in Ca 2+ entry in muscle.
However, Ca 2+ entry from the outside is required to replenish the slow but ongoing loss of Ca 2+ from the cell. Just as the NaATPase is required to slowly replenish intracellular Na + lost after firing many volleys of action potentials, the Ca 2+ uptake path -the L-channel -is is required to sustain intracellular Ca 2+ . Secondly, it may be reasoned that another method for distinguishing Ca 2+ pathways into cells would be determining the current carried by Ca 2+ directly. While this can be done, it cannot, as we pointed out previously (14), distinguish between entry into the cytosol and entry into the ER and then into the cytosol, since there is no electrical potential across the ER.
For the non-excitable cells, it is easy to accept the concept that the plasma membrane entry channel must be some protein other than the L-channel. As mentioned above, channel protein 1 in our model may correspond to the trp, or transient receptor potential family of proteins in non-excitable cells (39;40). It has been assumed that trp interacts with the IP 3 receptor, although there is also evidence to suggest that it does not: in particular, when the IP 3 R is genetically ablated, there is still CCE (41). In those cells, it has been argued, CCE is mediated by direct-contact between the trp3 and a RyR. While our findings do imply a close interaction between a plasma membrane channel (perhaps a trp protein) and an ER protein in the Jurkat cell, our evidence suggests the ER protein may be the RyR3 1 . Such an interaction has been reported previously (41). ________________________ 1 This is by implication. This study does not identify the ER protein responsible, but the lack of Ca 2+ product inhibition makes the IP 3 receptor unlikely. For the same reason, the RyR1 would be ruled out, if it in fact existed in the Jurkat cell.
Thus, the RyR3, which is not product inhibited by Ca 2+ is the default choice of the other half of the channel pair for importing Ca 2+ into the ER from the exterior. There are no other known Ca 2+ channels in the ER of Jurkat cells.

Concluding Remarks
Our findings suggest a new and simple mechanism for CCE: mass-action. With the extracellular Ca 2+ supplying an essentially constant pool, any decrease in the ER Ca 2+ will allow an increased uptake. This can easily explain a uniform response to such disparate experimental manipulations as IP 3 -induced ER Ca 2+ release, Tg inhibition of CaATPase, or cytosolic Ca 2+ buffering.
The experimental setup for measuring CCE -removing extracellular Ca 2+ , then observing the increase in cytosolic Ca 2+ after readdition of exogenous Ca 2+ in the presence of thapsigargin -is commonly interpreted as demonstrating that the pathway of Ca 2+ must be from exterior to cytosol directly, with thapsigargin preventing its uptake into the ER. Our evidence suggests an alternative possibility that may at first seem more circuitous: direct entry into the ER through two combined Ca 2+ channels, and subsequent Ca 2+ entry into the cytosol. However, physiologically extracellular Ca 2+ is always present in millimolar concentration. The changes in the extracellular-ER gradient are probably not as dramatic as those used for experimental purposes; the latter serve only to magnify the phenomenon. Thus, it is possible that the increase in the cytosolic Ca 2+ never takes place in vivo. In fact, this would be a physiological advantage to our mechanism: bypassing the cytosol during uptake would ensure that cytosolic signaling is unaffected during ER refilling.
It may be difficult to accept the concept that Ca 2+ does not enter the cytosol first, based on the very large gradient between the extracellular medium and the cytosol. However, this gradient was also the reason for the earlier belief that extracellular rather than ER Ca 2+ is the principal pool of mediator Ca 2+ (42).
It is always possible that despite the fact that our model fits the known data that it is itself incorrect. For example, one feature observed for the Ca 2+ entry current in fibroblasts is a slow inactivation (33), which would require a gating of the entry channel on top of its ability to respond by mass-action. Whether this is controlled by divalent ions such as Ca 2+ itself or Mg 2+ , as is the case for certain K + channels (43) will require future investigation. However, it is significant that direct determination of ER Ca 2+ contents makes the existing hypothesis untenable, and we propose a new model which provides a starting point to clearly explain CCE and determine the pathways for Ca 2+ fluxes between cell spaces.  ER Ca 2+ was measured as in Fig. 3. Tg (used throughout as in Fig. 1   Cells transfected as in Fig. 6 were titrated with exogenous Ca 2+ in a manner parallel to the dye method of Fig. 4. As in the previous figure, 2 mM Ca 2+ saturated the ER Ca 2+ pool. Data are shown as means, SEM are indicated as error bars, n = 4.   added subsequently. a) Cytosolic Ca 2+ record; b) ER Ca 2+ record. It is apparent that the rate of Ca 2+ appearance was slower, and the time to steady state was longer for the cytosol than the ER.   In this scheme, Ca 2+ enters channel protein 1 which is in close apposition to channel protein 2, so that together they form a combined channel for direct entry of Ca 2+ from the cell exterior to the ER.

Figure Legends
This would account for the direct entrance of Ca 2+ into the cell that is both insensitive to thapsigargin (Tg) and accounts for its capacitative nature because it can respond by mass-action, that is, the concentration difference between the cell exterior and the ER. Also shown is the exit route represented by channel protein 3 that allows Ca 2+ to enter the cytosol. Since Ry can inhibit this in resting cells, one candidate for channel protein 3 is RyR3 (see Discussion).