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J. Biol. Chem., Vol. 278, Issue 45, 44188-44196, November 7, 2003
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¶
From the
Departments of Pharmaceutical Sciences and Biological Sciences, St. John's University, Jamaica, New York 11439
Received for publication, June 19, 2003 , and in revised form, August 26, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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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 Ca2+ 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 Ca2+ 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 Ca2+ depletion that diffuses to the plasma membrane and allows entry of Ca2+ into the cytosol (5, 6).
When Ca2+ uptake was originally studied it appeared that Ca2+ 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 Ca2+ to Ca2+-free cells leads to an accumulation of cytosolic Ca2+ (8–10). This has been interpreted as proving that Ca2+ 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 Ca2+ levels. Although measuring cytosolic Ca2+ levels afford valuable insights into the phenomenon of CCE, this by design remains an indirect way of assessing ER Ca2+ 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 Ca2+ in both cytosol and ER using fluorescent dyes, finding that Ca2+ 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 the ER space of the L6 myotube using the calcium indicator Mag-Fura. This compound has been used for this purpose in the past, and our findings were consistent with measurement of the ER pool (15). However, it is known that, once Mag-Fura is added to cells (as the acetoxy ester) it appears in all three significant Ca2+ pools; that is, the cytosol, the mitochondria, and the ER. Given that the Mag-Fura dissociation constant (53 µM) is far above the Ca2+ concentrations in the first two compartments, it is unlikely that they contribute significantly to the signal. Still, earlier investigators suggested cytosolic Ca2+ and Mg2+ signals may be sensed by Mag-Fura (16, 17).
Therefore, we sought to investigate the question of whether Mag-Fura faithfully reports the ER space by an entirely separate measurement system, expression of an aequorin protein. This photo-protein selectively binds to Ca2+ and can be synthesized with a leader sequence targeted to the ER exclusively and modified so that its binding constant is much higher than cytosol or mitochondrial Ca2+ concentrations (18–20). We also examined Mag-Fura-loaded cells by fluorescence microscopy to determine whether Ca2+ accumulated in vesicles (presumably ER) or was diffuse (presumably cytosolic). Finally, we conducted additional experiments to probe the model for direct Ca2+ entry into the ER. Our findings support the idea that Mag-Fura specifically reports ER Ca2+ and also support the model suggesting that Ca2+ entry to the ER does not involve the cytosol.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Methods
Cell Culture—Jurkat cells were grown on RPMI 1640 medium supplemented by 10% fetal calf serum. Media was changed every 48 h, and cells were used when they attained a concentration of 5 x 105 cells/ml. L6 cells were grown as described previously (11).
Fluorescent Measurements—Measurement of cytosolic Ca2+ was performed as reported previously using Indo PE3 (AM) dye (14). For measurement of the ER Ca2+ of Jurkat cells, the cells were loaded with 8–10 µM concentrations of the dye Mag-Fura in Hanks' balanced salt solution. The cells were incubated for 1 h at 37 °C at 5% CO2 tension. After this, the mixture was centrifuged at 1500 rpm for 2 min, and the supernatant was discarded. The cells were resuspended in fresh Hanks' balanced salt solution for 1.5 h to allow de-esterification of the dye.
L6 cells were loaded with 8–10 µM concentrations of the dye Mag-Fura in phosphate-buffered saline. The loading time was 1 h at 37 °C in an atmosphere of 5% CO2, after which cells were washed with Hank's balanced salt solution. The cells were incubated further for 1.5 h to allow de-esterification of the dye. For both L6 and Jurkat cells, saponin (1 mg/ml) did not show an increase in the fluorescence ratio (347/373), but subsequent treatment with Triton X-100 (0.1%) did show an increase in this ratio, indicating ER localization of the dye (21).
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 20 mM HEPES, 118 mM NaCl, 12 mM NaHCO3, 2.6 mM KCl, 1.2 mM KH2PO4, 10 mM glucose, pH 7.4. The fluorescence measurements used excitation wavelengths of 347 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 [Ca2+] 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 5 mM EGTA, respectively.
Microscopy—L6 cells, grown on glass slides, were loaded as above with Mag-Fura, a drop of 10% glycerol in 0.9% saline was added, and a coverslip was 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.
Aequorin Plasmid Amplification and Transfection of Jurkat Cells— Competent Escherichia coli cells were transformed with 25 ng/µl aequorin plasmid pSVAEQERK and amplified, and the presence of plasmid was confirmed with the restriction enzymes SalI and EcoRI. Jurkat cells were grown in RPMI 1640 medium supplemented with 10% fetal calf serum in 75-cm2 Falcon flasks. Approximately 5 x 106 cells were transferred into each well in a 6-well plate and transfected with FuGENE 6, as described in the manufacturer's protocol. Briefly, 3 µl of FuGENE 6 was diluted to 100 µl in serum-free Dulbecco's modified Eagle's medium, and 2 µg of DNA was added. After 30 min of incubation at room temperature, this mixture was added to the cells. After 24 h, the cells were used for calcium measurement.
Reconstitution of Aequorin—To obtain an initially low [Ca2+] in the lumen of the ER, cells were treated for 2 min in Ca2+-free medium (125 mM NaCl, 5 mM KCl, 1 mM MgSO4, 5.5 mM glucose, 20 mM HEPES, 0.1 mM EGTA, pH 7.4) containing in addition 10 mM caffeine, 3 mM EGTA and 30 µM tBuBHQ (2,5-di(terbutyl)-1,4-benzohydroquinone) (17, 18). It is important to lower the [Ca2+] in the ER before reconstitution since the presence of Ca2+ will cause the reconstituted aequorin to decompose to apoaequorin, coelenteramide, and CO2. After washing with the Ca2+-free medium, 5 µM coelenterazine-n was added, and incubation was continued for 2 h at 37 °C (18). Subsequently, cells were centrifuged at 1000 rpm for 3 min and resuspended in Ca2+-free media. The aequorin light emission was measured with a Zylox luminometer. At the end of each experiment, the total light units from the unconsumed aequorin was estimated by permeabilizing the cells with 0.5% Triton X-100 in the presence of 10 mM CaCl2 (19, 23, 24). The [Ca2+] was calculated from the value of –log(L/Lmax), where L is the rate of luminescence (in arbitrary units), and Lmax is the rate of luminescence remaining in the sample. This value is proportional to the pCa (18).
Statistical Analysis—The experimental data were analyzed by Student's paired t test, analysis of variance, and Tukey's test for post hoc analysis using SPSS, Version 9.0. Data are represented as the means ± S.E., and the level of significance was set at p < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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DHBP, another blocker of the RyR (25, 26), was able to inhibit the Tg-induced rise in cytosolic Ca2+ (Fig. 2). DHBP also blunted CCE, as measured by the cytosolic Ca2+ increase subsequent to exogenous Ca2+ addition. Unlike the effect of Ry (which appeared to act as a competitive inhibitor), DHBP action was unaltered when added before or after the addition of Ca2+ 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 Ca2+ under these conditions is the ER. We next conducted more direct measurements of ER [Ca2+] levels.
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Direct Analysis of ER Ca2+ by Fluorescent Chelator—Fig. 3, a tracing of raw data, shows that after Tg addition, both 0.5 and 1 mM exogenous Ca2+ increased ER [Ca2+]. Subsequent addition of caffeine, known to release Ca2+ from the ER, decreased the ER [Ca2+]. A titration of ER [Ca2+] response to a wide range of exogenously added Ca2+ concentrations is shown in Fig. 4. It is apparent that ER Ca2+ concentrations increased over the physiological range of exogenous Ca2+ and was saturated at about 2 mM. We also found that exogenous Ca2+ increased the ER Ca2+ content in the presence of both caffeine and Tg, as shown in Fig. 5. Because ER [Ca2+] was lowered in the presence of caffeine and net flux was very likely to be in the direction of ER to cytosol, this result indicates ER [Ca2+] filling occurred directly from the extracellular space.
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Analysis of ER Ca2+ by Transfection of a Modified Aequorin—To confirm the observations made with Mag-Fura, we sought to measure ER Ca2+ by an alternative method. We used the technique of transfection of the photo-protein aequorin that has been modified to achieve selectivity for ER Ca2+ in two ways (19, 20). First, it has a leader sequence that targets the expressed protein into the ER. Second, the Ca2+ binding site of the protein has been modified so that its affinity for Ca2+ is much less than aequorin itself and is suitable for binding Ca2+ in the ER space. The cofactor coelenterazine is also modified to provide a system for reporting Ca2+ specific to the ER, with a dissociation constant (about 15 µM) that would render it insensitive to Ca2+ 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 base-line Ca2+ and that more was apparent when Ca2+ was added. This indicates the technique can measure intracellular Ca2+ pools that have a low Ca2+ affinity, comparable with the Mag-Fura dye. Fig. 7 shows that the increase in Ca2+, which we attribute to ER based upon the two considerations above, occurred over the same range of externally added Ca2+ as that indicated by the Mag-Fura dye (cf. Fig. 4). Although the calculated values of ER [Ca2+] 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 [Ca2+].
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Lanthanum Blocked the Increase in ER [Ca2+] Measured by Both Methods—As a further control, we studied the effect of lanthanum, a known inhibitor of Ca2+ uptake by virtually any pathway into the cell, on the appearance of ER Ca2+ by both the Mag-Fura (Fig. 8a) as well as the aequorin (Fig. 8b) methods. In both cases the increase in ER [Ca2+] 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 Ca2+ pool at concentrations of about 2 orders of magnitude greater than cytosolic or mitochondrial pools.
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Complementary Results with L6 Cells—We have recently shown that Ca2+ in the SR of the L6 muscle cells increases with increasing exogenous Ca2+, but the titration appeared qualitatively different from the one we observed in the present study with Jurkat cells; there was considerable inhibition of Ca2+ content as Ca2+ 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 Ca2+. 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 Ca2+ uptake to the ER when exogenous Ca2+ was added in concentrations greater than 3 mM. Thus, the similarity of results with the two methods extends to the L6 cells as well.
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Kinetic Evidence—If Ca2+ enters the ER before its entry into the cytosol, it should be possible to observe a more rapid entry into the ER than the cytosol when exogenous Ca2+ is added to Ca2+-deprived cells. To readily resolve the kinetics of Ca2+ appearance, it was necessary to lower the incubation temperature. Representative traces at 4 °C for the appearance of Ca2+ are shown for the cytosol (Fig. 10A) and the ER (Fig. 10B). It is evident that the slope of the cytosolic Ca2+ curve was more shallow and that the time to reach steady state was considerably longer than the ER Ca2+ curve, indicating a slower Ca2+ 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 °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 Ca2+ enters the ER before the cytosol.
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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 Ca2+, we observed virtually that 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.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Although 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 Ca2+ release channels (29). For skeletal muscles, modeled by the L6 cells, channel protein 1 would be the dihydropyridine receptor, and channel protein 2 would be the RyR1. Recent structural studies indicate that the subunits comprising the Ca2+ mouth of the dihydropyridine receptor align with those of the RyR1 (30).
Ca2+ 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 IP3 receptor. Under conditions in which IP3 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 IP3. Jurkat cells are known to use IP3 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 [Ca2+] in the ER alters the conformational state of the ER membrane-bound Ca2+ channel. Because this channel is in direct contact with the plasma membrane Ca2+ channel, the latter is stimulated to release Ca2+ into the cytosol. This model, thus, requires that Ca2+ 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 Ca2+ must exit (6, 31). Although these constructs satisfy the idea that Ca2+ 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 Ca2+ exit routes (28, 32). Beyond this complication our observation of an increase in Ca2+ in the ER in the presence of thapsigargin is inconsistent with currently favored models.
Hofer et al. (33) provide simultaneous measurements of ER Ca2+, cytosolic Ca2+, and Ca2+ current (ICRAC) in a fibroblast cell line. They found that the rate of Ca2+ entry was inversely correlated to the Ca2+ 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 [Ca2+] 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 (28) make the soluble messenger model difficult to accept. They demonstrated that the kinetics of Ca2+ appearance in the cytosol during CCE are independent of the duration of the preceding Ca2+-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" that is produced in the ER in response to a depleted ER [Ca2+] (5). After this, the calcium influx factor diffuses to the plasma membrane, simulating a plasma membrane Ca2+ channel and, subsequently, Ca2+ entry to the cytosol. Despite elaborate studies of the calcium influx factor, its molecular identity remains elusive.
In a recent review, Putney et al. (6) suggest that the mechanism of CCE may vary with cell type. This is consistent with our findings that Ca2+ inhibited its own uptake in L6 cells but not in Jurkat cells. Still, existing models of CCE all require Ca2+ to flow from the external space into the cytosol and subsequently enter the ER through the CaATPase. Since we provide evidence for the direct entry of Ca2+ 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 Ca2+ accumulation in the cytosol implicates a RyR (channel 3 in Fig. 13) during CCE. Ryanodine also inhibits Ca2+ accumulation in the cytosol of the L6 cell (14) with about one 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) (34–36). It is also possible that ryanodine nonspecifically inhibits another exit channel, perhaps the IP3 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 nonspecific inward movement of the external Ca2+ in skeletal muscle unless the cells were preincubated with ryanodine for at least 15 min and at high concentration (100 µM). We showed in a subsequent study that much lower concentrations (10 µM) without preincubation were able to prevent the appearance of cytosolic Ca2+ during CCE, which had no influence on ER [Ca2+] (14). The lack of effect of Ry on ER [Ca2+] is likely due to the fact that the cytosolic Ca2+ pool is much smaller than the ER Ca2+ pool.
The finding that high concentrations of Ca2+ inhibit L6 cell uptake (this study and Ref. 14) but not Jurkat cell Ca2+ uptake is also consistent with properties of the ryanodine receptor. The skeletal muscle ryanodine receptor is known to be Ca2+-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 dual-channel uptake for Ca2+ into the ER as proposed here.
The inhibition of cytosolic Ca2+ filling in response to DHBP, independent of ambient Ca2+, further argues that exogenous Ca2+ does not enter the cytosol first. Additional support for this notion comes from a study of Tg-induced Ca2+ toxicity (26). In that work it was concluded that Ca2+ blockers, including DHBP but not Ry, abrogated Ca2+ toxicity. These investigators also suggest a block of cytosolic Ca2+, 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); Ca2+ competes with Ry, but DHBP is unaffected by Ca2+ levels during CCE (i.e. noncompetitive).
Excitable Versus 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 Ca2+ 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 Mn2+ entry, used as a surrogate of Ca2+ (12). However, we observed that Mn2+ entry is not capacitative (14). Thus, Mn2+ is an inappropriate surrogate ion for Ca2+ in CCE.
Two other misconceptions must be addressed concerning skeletal muscle cells. First, it may be recalled that the Ca2+ needed for contraction does not require Ca2+ entry into the cell (38), and this may appear to contradict the notion that the L-channel is involved in Ca2+ entry in muscle. However, Ca2+ entry from the outside is required to replenish the slow but ongoing loss of Ca2+ from the cell. Just as the NaATPase is required to slowly replenish intracellular Na+ lost after firing many volleys of action potentials, the Ca2+ uptake path, the L-channel, is required to sustain intracellular Ca2+. Second, it may be reasoned that another method for distinguishing Ca2+ pathways into cells would be determining the current carried by Ca2+ directly. Although 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 IP3 receptor, although there is also evidence to suggest that it does not; in particular, when the IP3 receptor 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. Although 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 RyR3.2 Such an interaction has been reported previously (41).
Concluding Remarks—Our findings suggest a new and simple mechanism for CCE: mass-action. With the extracellular Ca2+ supplying an essentially constant pool, any decrease in the ER Ca2+ will allow an increased uptake. This can easily explain a uniform response to such disparate experimental manipulations as IP3-induced ER Ca2+ release, Tg inhibition of CaATPase, or cytosolic Ca2+ buffering.
The experimental setup for measuring CCE, removing extracellular Ca2+ and then observing the increase in cytosolic Ca2+ after readdition of exogenous Ca2+ in the presence of thapsigargin, is commonly interpreted as demonstrating that the pathway of Ca2+ 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 Ca2+ channels and subsequent Ca2+ entry into the cytosol. However, physiologically extracellular Ca2+ is always present in millimolar concentrations. 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 Ca2+ 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 Ca2+ 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 Ca2+ is the principal pool of mediator Ca2+ (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 Ca2+ 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 Ca2+ itself or Mg2+, as is the case for certain K+ channels (43), will require future investigation. However, it is significant that direct determination of ER Ca2+ contents makes the existing hypothesis untenable, and we propose a new model that provides a starting point to clearly explain CCE and determine the pathways for Ca2+ fluxes between cell spaces.
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FOOTNOTES |
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¶ To whom correspondence should be addressed: Dept. of Pharmaceutical Sciences, School of Pharmacy, St. John's University, 8000 Utopia Pkwy., Jamaica, NY 11439. Tel.: 718-990-1678; Fax: 718-990-1936; E-mail: ochsr{at}stjohns.edu.
1 The abbreviations used are: ER, endoplasmic reticulum (also used in this work for muscle endoplasmic reticulum, often called sarcoplasmic reticulum); CCE, capacitative calcium entry; Tg, thapsigargin; Ry, ryanodine; RyR, Ry receptor (Ca2+ channel) and subtypes RyR1 and RyR3; Mag-Fura, MagFura 2-AM (acetoxymethyl); DHBP, 1,1-diheptyl-4,4-bipyridiniumdibromide; IP3, inositol 1,4,5-trisphosphate.
2 This is by implication. This study does not identify the ER protein responsible, but the lack of Ca2+ product inhibition makes the IP3 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 Ca2+, is the default choice of the other half of the channel pair for importing Ca2+ into the ER from the exterior. There are no other known Ca2+ channels in the ER of Jurkat cells.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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, p < 0.05 versus Tg + Ca2+; n = 4.
