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J Biol Chem, Vol. 274, Issue 50, 35318-35324, December 10, 1999

Ca²⁺ Entry Activated by S-Nitrosylation
RELATIONSHIP TO STORE-OPERATED Ca²⁺ ENTRY^*

Hong-Tao Ma, Cécile J. Favre, Randen L. Patterson, Michele R. Stone, and Donald L. Gill^§

From the Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

The coupling between Ca²⁺ pools and store-operated Ca²⁺ entry channels (SOCs) remains an unresolved question. Recently, we revealed that Ca²⁺ entry could be activated in response to S-nitrosylation and that this process was stimulated by Ca²⁺ pool emptying (Favre, C. J., Ufret-Vincenty, C. A., Stone, M. R., Ma, H-T., and Gill, D. L. (1998) J. Biol. Chem. 273, 30855-30858). In DDT₁MF-2 smooth muscle cells and DC-3F fibroblasts, Ca²⁺ entry activated by the lipophilic NO donor, GEA3162 (5-amino-3-(3,4-dichlorophenyl)1,2,3,4-oxatriazolium), or the alkylator, N-ethylmaleimide, was observed to be strongly activated by transient external Ca²⁺ removal, closely resembling activation of SOC activity in the same cells. The nonadditivity of SOC and NO donor-activated Ca²⁺ entry suggested a single entry mechanism. Calyculin A-induced reorganization of the actin cytoskeleton prevented SOC but had no effect on GEA3162-induced Ca²⁺ entry. However, a single entry mechanism could account for both SOC and NO donor-activated entry if the latter reflected direct modification of the entry channel by S-nitrosylation, bypassing the normal coupling process between channels and pools. Small differences between SOC and GEA3162-activated Ba²⁺ entry and sensitivity to blockade by La³⁺ were observed, and in HEK293 cells SOC activity was observed without a response to thiol modification. It is concluded that in some cells, S-nitrosylation modifies an entry mechanism closely related to SOC and/or part of the regulatory machinery for SOC-mediated Ca²⁺ entry.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Cytosolic Ca²⁺ signals control a vast array of cellular functions ranging from short term responses such as contraction and secretion to longer term regulation of cell growth and proliferation (1). The generation of receptor-induced cytosolic Ca²⁺ signals is complex, involving two closely coupled components: rapid, transient release of Ca²⁺ stored in the endoplasmic reticulum (ER),¹ followed by slowly developing extracellular Ca²⁺ entry (1-5). G protein-coupled receptors and tyrosine kinase receptors, through activation of phospholipase C, generate the second messenger, inositol 1,4,5-trisphosphate. This chemical message diffuses rapidly within the cytosol to interact with inositol 1,4,5-trisphosphate receptors located on the ER, which serve as Ca²⁺ channels to release luminal stored Ca²⁺ and generate the initial Ca²⁺ signal phase (1, 3). The resulting depletion of Ca²⁺ stored within the ER lumen serves as the primary trigger for a message that is returned to the plasma membrane, resulting in the slow activation of "store-operated" Ca²⁺ entry channels (2, 4-6). This second Ca²⁺ entry phase of Ca²⁺ signals serves to mediate longer term cytosolic Ca²⁺ elevations and provides a means to replenish intracellular stores (2, 4). Whereas receptor-induced generation of inositol 1,4,5-trisphosphate and the function of Ca²⁺ release channels to mediate the initial Ca²⁺-signaling phase is well understood, the mechanism for coupling ER Ca²⁺ store depletion with Ca²⁺ entry remains a crucial but unresolved question (4-6).

Recently, several major channels have been shown to be regulated by thiol nitrosylation, a process becoming recognized as an important nitric oxide (NO)-mediated posttranslational modification affecting control over a diverse array of signaling and regulatory proteins (7-12). Such S-nitrosylation-mediated effects are direct and independent of activation of guanylyl cyclase, which is a major target for NO and a frequent mediator of the actions of NO (13, 14). Studies have revealed that nitrosothiol formation underlies the direct modifying action of NO on a number of important plasma membrane and intracellular channels for Ca²⁺ and other ions including the N-methyl-D-aspartate receptor (8), cyclic nucleotide-gated cation channel (15, 16), Ca²⁺-activated K⁺ channel (17), L-type Ca²⁺ channel (18), and the ryanodine receptor Ca²⁺ release channel (19). For several of these channels, NO donor-induced S-nitrosylation results in channel activation, and this activation is mimicked by alkylation of the same thiol groups (15-19). Because of the reactivity of thiols toward NO, the sphere of influence of NO can be highly restricted and, rather than diffusion-dependent, NO (or an equivalent of the nitrosonium ion, NO⁺) may be donated and exchanged between neighboring protein thiols by local transnitrosation events (7, 9-11, 15, 16).

We recently utilized a combination of membrane-permeant NO donors and alkylators to probe the role of S-nitrosylation in the process of Ca²⁺ entry and its relationship to Ca²⁺ pool depletion (20). A novel class of lipophilic NO donors, including the oxatriazole-5-imine derivative, GEA3162, activated Ca²⁺ entry independent of the well defined NO target, guanylyl cyclase. Strikingly similar Ca²⁺ entry induced by cell permeant alkylators indicated that this Ca²⁺ entry process was activated through thiol modification. Significantly, Ca²⁺ entry activated by NO donors or alkylators was stimulated by Ca²⁺ pool depletion, which increased the rate and size of the Ca²⁺ response and the sensitivity to thiol modifiers. These results led us to postulate that S-nitrosylation may underlie activation of an important store-operated Ca²⁺ entry mechanism. Here we have examined the relationship between store-operated Ca²⁺ entry occurring independently of S-nitrosylation and Ca²⁺ entry activated in response to S-nitrosylation.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Culture of Cells-- DDT₁MF-2 smooth muscle cells derived from hamster vas deferens were cultured in Dulbecco's modified Eagle's medium supplemented with 2.5% calf serum as described previously (21, 22); DC-3F Chinese hamster lung fibroblasts were cultured in -modified Eagle's medium supplemented with 5% heat-inactivated fetal bovine serum as described previously (23, 24).

Measurement of Intracellular Calcium-- Cells grown on coverslips for 1 day were transferred to Hepes-buffered Krebs medium (107 mM NaCl, 6 mM KCl, 1.2 mM MgSO₄, 1 mM CaCl₂, 1.2 mM KH₂PO₄, 11.5 mM glucose, 0.1% bovine serum albumin, 20 mM Hepes-KOH, pH 7.4) and loaded with fura-2/AM (2 µM) for 10 min at 20 °C. Cells were washed, and the dye was allowed to deesterify for a minimum of 15 min at 20 °C. Approximately 95% of the dye was confined to the cytoplasm as determined by the signal remaining after saponin permeabilization (25, 26). Fluorescence emission at 505 nm was monitored with excitation at 340 and 380 nm; Ca²⁺ measurements are shown as 340/380-nm ratios obtained from a groups of 10-12 cells. Details of these Ca²⁺ measurements were recently described for DDT₁MF-2 (27) and DC-3F cells (24). Resting Ca²⁺ levels in DDT₁MF-2 cells were approximately 60-90 nM and 25-50 nM in DC-3F cells; maximal activation by GEA3162 resulted in up to 600 nM Ca²⁺. All measurements shown are representative of a minimum of three, and in most cases, a much larger number of independent experiments.

Materials and Miscellaneous Procedures-- GEA3162 was from Alexis Corp., San Diego, CA. 2,5-di-tert-butylhydroquinone and 4-vinylpyridine were from Aldrich. Thapsigargin was from LC Services, Woburn, MA. Fura-2/acetoxymethylester was from Molecular Probes, Eugene, OR. N-Ethylmaleimide (NEM) and all other compounds were from Sigma.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Our previous results revealed that NO donors including nitroprusside, nitrite, and the lipophilic donor, GEA3162, were effective in directly inducing Ca²⁺ entry (20). A highly similar entry of Ca²⁺ was induced with alkylators including NEM and 4-vinylpyridine, indicating that the activation of Ca²⁺ entry resulted from thiol modification, either nitrosylation or alkylation (20). The Ca²⁺ entry observed with NO donors or with alkylators, in both cases, was substantially enhanced by emptying Ca²⁺ pools before administration of the activator. Pool emptying increased three parameters of thiol modifier-induced Ca²⁺ entry: the time-dependence of entry, the size of the Ca²⁺ entry response, and the sensitivity to thiol modifier (20). The most prominent of these effects was the time dependence. Thus, in normal cells with filled pools, there was a pronounced lag in the Ca²⁺ entry response to thiol modifiers of at least 1 min; after pool emptying, the Ca²⁺ entry response was extremely rapid, suggesting that pool emptying had allowed the Ca²⁺ entry channel to alter its configuration to expose a thiol group that was important in modifying channel activity (20). The question of whether this putative entry channel was indeed the store-operated Ca²⁺ channel was important to address.

Store-operated Ca²⁺ entry channels display a further important characteristic. In many cells, the entry of Ca²⁺, activated after pool depletion, becomes deactivated with time, and transient removal and readdition of extracellular Ca²⁺ is a well described means for reactivating the entry mechanism (24, 28-31). The effect is frequently referred to as the Ca²⁺ "overshoot" response, since it results in a transiently high reactivation of Ca²⁺ entry, which then deactivates once again with time (24, 28). The results in Fig. 1 reveal that transient removal of external Ca²⁺ has a dramatic enhancing effect on the operation of Ca²⁺ entry activated by GEA3162. Untreated DDT₁MF-2 cells exposed briefly to nominally Ca²⁺-free medium then returned to medium containing normal Ca²⁺ showed no change in cytosolic Ca²⁺ (Fig. 1A). The addition of GEA3162 at 25 µM, a submaximal concentration under normal conditions (20), in the continued presence of external Ca²⁺ induced a modest rise of Ca²⁺ after a lag of approximately 2 min. If external Ca²⁺ was removed before the addition of 25 µM GEA3162 (Fig. 1B), no significant change in Ca²⁺ occurred for several minutes, confirming the lack of any effect of the NO donor on release of internal Ca²⁺. However, upon readdition of external Ca²⁺, a rapid entry of Ca²⁺ occurred, resulting in a considerably larger peak of Ca²⁺ (Fig. 1B) than that observed in the continuous presence of external Ca²⁺ (Fig. 1A). Thus the removal and readdition of Ca²⁺ considerably enhanced the effectiveness of GEA3162. Control experiments revealed that prolonged (10 min) removal of external Ca²⁺ did not cause any release of Ca²⁺ from pools and that following such prolonged external Ca²⁺ removal, no entry of Ca²⁺ was observed upon the readdition of Ca²⁺ in the absence of GEA3162. The stimulatory effect of transient Ca²⁺ removal on the action of GEA3162 was further characterized as shown in Fig. 2. In this experiment external Ca²⁺ was transiently removed after the addition of different GEA3162 concentrations. After adding GEA3162 at 1 µM, a 3-min period of external Ca²⁺ removal resulted in only a very slight entry of Ca²⁺ (Fig. 2A). However, after the addition of 10 µM GEA3162, the transient removal of external Ca²⁺ triggered a much more significant and rapid increase in Ca²⁺ following Ca²⁺ readdition (Fig. 2B). Under normal conditions of external Ca²⁺, GEA3162 at 10 µM was below its effective threshold and induced almost no Ca²⁺ entry (20). Therefore, the entry of Ca²⁺ observed after the brief removal of external Ca²⁺ represents a real potentiation of the effect of GEA3162. The resultant increase in Ca²⁺ was rapid but transient and began to decline after 1 min. Removal of Ca²⁺ prevented any further entry of Ca²⁺, and the Ca²⁺ level fell rapidly. After a further 3 min, readdition of Ca²⁺ resulted again in a rapid and transient entry of Ca²⁺. The entry of Ca²⁺ could be repeatedly reactivated by transient removal of Ca²⁺ (Fig. 2B). Although the peak size of the response to 10 µM GEA3162 after the initial transient Ca²⁺ depletion was smaller, the peaks following subsequent brief periods of Ca²⁺ removal were larger and approached the maximal size attainable. Thus, external Ca²⁺ removal followed by the readdition in the presence of 25 µM GEA3162 (Fig. 2C) resulted in a rapid and maximal activation of Ca²⁺ entry. Again, the activation rapidly deactivated with time, and cycling of reactivation of Ca²⁺ entry in response to transient Ca²⁺ removal could be repeated several times in succession.

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Ca2+ entry into DDT1MF-2 cells activated by the NO donor GEA3162 is stimulated by removal and readdition of external Ca2+. Cytosolic Ca2+ was measured in fura-2-loaded DDT1MF-2 cells as described under "Experimental Procedures." Standard conditions included 1 mM Ca2+ in the external medium; medium was replaced with nominally Ca2+-free medium (bars labeled no Ca2+) for the times shown. A, after removal and the readdition of Ca2+, 25 µM GEA3162 (GEA) was added at the time indicated by the arrow. B, 25 µM GEA3162 was added (arrow) shortly after replacing the bathing medium with nominally Ca2+-free medium, followed by the addition of standard Ca2+ medium in the continued presence of GEA3162.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Repeated transient Ca2+ depletion induces a large potentiation of Ca2+ entry activated by varying concentrations of the NO donor, GEA3162, in DDT1MF-2 cells. Bars indicate times of replacement of medium with nominally Ca2+-free medium (no Ca2+). GEA3162 (GEA) was added at either 1 µM (A), 10 µM (B), or 25 µM (C) at the times indicated (arrows) and maintained at these levels throughout the remainder of traces.

This pattern of deactivation and reactivation by transient removal of Ca²⁺ is highly similar to the operation of store-operated Ca²⁺ entry channels. As shown in Fig. 3A, after thapsigargin-induced pool emptying in the absence of external Ca²⁺, readdition of Ca²⁺ caused a large increase in cytosolic Ca²⁺, reflecting a high level of store-operated Ca²⁺ entry. The entry of Ca²⁺ rapidly deactivated with time, and subsequent removal of external Ca²⁺ prevented any further Ca²⁺ entry. Upon the readdition of external Ca²⁺, maximal store-operated Ca²⁺ entry was restored. This overshoot response pattern classically reflects the operation of store-operated Ca²⁺ entry and is believed to represent the function of Ca²⁺-binding sites, which negatively control store-operated Ca²⁺ entry (24, 28, 29, 31). According to such a model, as Ca²⁺ increases in the cytosol, binding of Ca²⁺ to such regulatory sites inhibits entry; transient external Ca²⁺ removal prevents Ca²⁺ entry, allowing cytosolic Ca²⁺ to fall rapidly as Ca²⁺ is pumped out of the cell. As a result, Ca²⁺ dissociates from the regulatory site, permitting the channel to become fully reactivated; upon readdition of Ca²⁺, a high level of Ca²⁺ entry is again observed. As shown in Fig. 3A, this process could be repeated many times. However, it is important to reiterate that the entry observed was completely dependent on pool depletion. Thus, transient removal of Ca²⁺ at the beginning of the trace before pools were emptied induced no entry of Ca²⁺. Indeed, in experiments with normal, pool-filled cells, repeated transient removal and readdition of Ca²⁺ over a period of 30 min induced no change in cytosolic Ca²⁺ (not shown). The means of activation, the appearance, and the size of the overshoot responses after pool emptying were all remarkably similar to those described above, activated in response to the NO donor. Yet in the case of the NO donor, pools were not emptied. This point is reinforced from the data shown in Fig. 3B. Thus addition of 15 µM GEA3162 before thapsigargin had no effect on the size of the Ca²⁺ pool released by subsequent addition of thapsigargin. Moreover, in this experiment the size of overshoots induced after application of both GEA3162 and thapsigargin was not measurably different from that induced by each agent alone. Also, addition of 15 µM GEA3162 to cells after pool depletion with thapsigargin resulted in little significant change in the size of overshoots induced by repeated transient Ca²⁺ removal (Fig. 3A). Thus, thapsigargin and NO donor induced similar shaped and sized overshoots that did not appear to be additive. This suggested they were activating either the same or a closely coupled entry mechanism.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Thapsigargin-induced pool emptying in DDT1MF-2 cells induces overshoots of store-operated Ca2+ entry, which are similar to and nonadditive with the effects of GEA3162. Bars indicate times of replacement of medium with nominally Ca2+-free medium (no Ca2+). A, 2 µM thapsigargin (TG) and 15 µM GEA3162 (GEA) were added at the times shown. B, as for A except 15 µM GEA3162 was added before 1 µM thapsigargin. In each case, thapsigargin and GEA3162, once added, were maintained throughout the experiment.

As described above and earlier (20), thiol modification by either nitrosylation or alkylation activated a very similar entry of Ca²⁺. We therefore examined whether the stimulatory action of transient Ca²⁺ removal also activated Ca²⁺ entry induced by alkylators. Experiments utilized the DC-3F fibroblast cell line in which responses to NO donors and alkylators were similar to DDT₁MF-2 cells. As shown in Fig. 4A, the addition of the alkylator, NEM, at 10 µM induced only a slight increase in cytosolic Ca²⁺ (Fig. 4A). However, if extracellular Ca²⁺ was transiently removed for just a short (2 min) period, a substantial entry of Ca²⁺ immediately followed the readdition of Ca²⁺ (Fig. 4B). As with DDT₁MF-2 cells, transient Ca²⁺ removal without alkylator or NO donor present had no effect on cytosolic Ca²⁺ in DC-3F cells (24). The shape and time dependence of the transient Ca²⁺ removal-induced entry response seen after NEM treatment (Fig. 4B) and after pool emptying (24) were impressively similar in the DC-3F cells. NEM-induced entry of Ca²⁺ into DDT₁MF-2 cells was similarly potentiated by transient removal of Ca²⁺ (data not shown).

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Transient Ca2+ depletion induces a large potentiation of N-ethylmaleimide-activated Ca2+ entry in DC-3F cells. A, 10 µM NEM was added under standard external conditions. B, 10 µM NEM addition was followed by replacement of medium with nominally Ca2+-free medium for 2 min as shown by the bar (no Ca2+) followed by return of standard external Ca2+ medium. NEM was maintained after the addition.

Taken together, the above results and those published previously (20) revealed that Ca²⁺ entry activated by S-nitrosylation was stimulated by both Ca²⁺ pool depletion and transient external Ca²⁺ removal, the two primary conditions for activating store-operated Ca²⁺ entry channels. From this it could be concluded that S-nitrosylation was affecting a process closely linked with store-operated Ca²⁺ entry. However, other approaches to determining the relationship between the Ca²⁺ entry mechanisms have revealed some interesting differences. In recent work, we determined that the operation of store-operated Ca²⁺ entry appears to involve trafficking of the ER toward the plasma membrane (32). One of the approaches to this work was to utilize the phosphatase inhibitor, calyculin A, which in two distinct cell types induced a profound redistribution of actin resulting in the formation of a tight ring of cortical actin filaments subjacent to the plasma membrane. This cortical actin appeared to act as a physical barrier to prevent close interaction between the ER and plasma membrane (32). Under this condition, the activation of store-operated Ca²⁺ entry by pool emptying with thapsigargin was blocked in both cell types. We therefore compared the action of calyculin A on Ca²⁺ entry activated by thapsigargin-induced Ca²⁺ pool depletion with its effects on Ca²⁺ entry activated by NO donor.

As shown in Fig. 5, we were able to directly observe the two means of Ca²⁺ entry activation in a single trace. In normal DDT₁MF-2 cells, typical activation of Ca²⁺ entry via pool depletion and application of GEA3162 is shown in Fig. 5A. After the addition of 2 µM thapsigargin, rapid release of Ca²⁺ from pools was observed. The slower secondary peak was due to entry of Ca²⁺ dependent on pool depletion; this became deactivated with time, and cytosolic Ca²⁺ decreased to a reduced level. The basal Ca²⁺ level reached was slightly higher than normal resting Ca²⁺, reflecting a small level of residual Ca²⁺ entry. After removal of external Ca²⁺, this low level of entry was abolished, and upon subsequent readdition of Ca²⁺, the typical large overshoot of Ca²⁺ entry was observed, consistent with that described above. After the store-operated entry had once again deactivated, the addition of a 100 µM GEA3162 clearly activated a large increase in Ca²⁺ entry, and this entry again deactivated with time. In similar experiments, lower GEA3162 concentrations also induced entry of Ca²⁺, although the entry was less prolonged (not shown). Note that in these experiments, deactivation of store-operated Ca²⁺ entry occurred for a longer period of time compared with that in Fig. 3, and there was no further removal of external Ca²⁺. Thus, if GEA3162 was activating the same Ca²⁺ entry pathway as pool emptying, then this result would suggest that GEA3162 was able to reverse the deactivation process occurring as a result of Ca²⁺ inhibition.

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Calyculin A treatment blocks store-operated Ca2+ entry in response to thapsigargin-induced pool emptying in DDT1MF-2 cells but does not block Ca2+ entry activated by the NO donor, GEA3162. Bars indicate times of replacement of medium with nominally Ca2+-free medium (no Ca2+). A, 2 µM thapsigargin (TG) and 100 µM GEA3162 (GEA) were added at the times shown. B, cells were initially treated with the phosphatase inhibitor calyculin A (CalyA) at 100 nM for 10 min before the addition of 2 µM thapsigargin and 100 µM GEA3162 at the times indicated by the respective arrows. Each of the agents was maintained in medium after addition throughout successive changes of medium with or without Ca2+.

Clearly, the picture was very different in the presence of 100 nM calyculin A. The experiment shown in Fig. 5B included a 10-min pretreatment with the phosphatase inhibitor that was sufficient to rearrange cortical actin into a tight band closely associated with the plasma membrane (32) and prevent store-operated Ca²⁺ entry. As seen in this experiment, whereas the size of the release peak of Ca²⁺ in response to thapsigargin was unchanged, almost no Ca²⁺ entry followed, and hence the second peak was eliminated. A very slight level of Ca²⁺ entry appeared to remain, as seen by the removal of external Ca²⁺; however, subsequent readdition of Ca²⁺ caused only a small reactivation of Ca²⁺ entry (that is, almost no overshoot), conclusively demonstrating that activation of store-operated entry had been blocked. Whereas the overshoot response was blocked, application of 100 µM GEA3162 gave an immediate and large activation of Ca²⁺ entry. The effects of applying lower GEA3162 concentrations was also unaltered by calyculin A treatment (not shown); thus, although the duration and peak sizes of the responses were smaller than with 100 µM GEA3162, they were not different to the responses observed without calyculin A. Therefore, on the basis of sensitivity to modification by calyculin A, it might be concluded that the entry activated by pool emptying was quite distinct from that activated by S-nitrosylation.

However, this interpretation is not necessarily valid. Thus, we concluded from our recent work that the coupling mechanism between pools and store-operated Ca²⁺ entry channels is through a trafficking event involving physical movement of some component of the ER toward the plasma membrane (32). In this model, the channels may be preexisting within the plasma membrane, but activation may be elicited by close approach of the ER membrane. Indeed, recent evidence suggests that for one type of Ca²⁺ entry channel, the TRP3 channel, activation may occur via a reversible interaction between the ER-located inositol 1,4,5-trisphosphate receptor and the TRP3 channel itself or a closely related component within the plasma membrane (33). Although there is some uncertainty about whether pool emptying is necessary for activation of this particular channel (33, 34), there is certainly precedent for believing that the activation of store-operated Ca²⁺ entry channels may involve interactions between the ER and plasma membrane (32). The action of calyculin A on preventing store-operated Ca²⁺ entry is believed to result from physical interruption of the coupling process that occurs between the ER and plasma membrane as a result of reorganization of F-actin into a tight cortical layer beneath the plasma membrane (32). Evidence for this action of calyculin A was based on the morphological redistribution of actin observed. In addition, the inhibitory action of calyculin A on store-operated Ca²⁺ entry by cytochalasin D could be reversed by cytochalasin D. Thus, depolymerization of actin with cytochalasin D after calyculin A caused the cortical actin barrier to be removed and the coupling between ER and the plasma membrane to be reestablished, permitting activation of store-operated Ca²⁺ entry (32). Since this action of calyculin A is not considered to be a direct effect on the channel itself but rather a prevention of the interaction with ER, it appeared to us that the lack of effect of calyculin A on GEA3162-induced Ca²⁺ entry might reflect a direct action of the NO donor on the entry channel, perhaps circumventing or even mimicking the activation that results from interaction with ER.

Therefore, we sought to determine other parameters defining operation of the entry mechanisms to examine any differences between store-operated and GEA3162-activated entry. Our attention turned toward examination of cation sensitivity and specificity of the entry processes. We investigated the passage of different divalent alkaline-earth cations and of the blocking action of La³⁺. The passage of Sr²⁺ and Ba²⁺ ions could be assessed directly by fura-2 ratio-fluorimetry. Experiments revealed that both store-operated entry and entry activated by GEA3162 allowed passage of Sr²⁺ ions (Fig. 6). The addition of 25 µM GEA3162 to DDT₁MF-2 cells in medium containing 1 mM Sr²⁺ in place of 1 mM Ca²⁺ activated entry of Sr²⁺ with kinetics similar to that of Ca²⁺ (Fig. 6A). When using cells in which pools had been pool-depleted in the absence of external Ca²⁺ (Fig. 6B), the addition of Sr²⁺ caused a rapid entry of Sr²⁺ through store-operated Ca²⁺ entry channels. The kinetics of deactivation of Sr²⁺ entry were not dissimilar from those for Ca²⁺ entry (compare with Fig. 5A), although there did not appear to be a residual of Sr²⁺ entry as appeared for Ca²⁺. This might suggest that a second entry channel was activated by store depletion. Application of 25 µM GEA3162 also clearly induced Sr²⁺ entry (Fig. 6B), and clearly, the emptying of pools activated both the rate and extent of entry, consistent with the results on Ca²⁺ entry (20). The slightly slower deactivation of Sr²⁺ entry may result from less efficient pumping of Sr²⁺ out of the cell and/or a difference in the relative ability of Sr²⁺ to effect deactivation of entry channels. We did not observe any significant difference in the Sr²⁺ concentration dependence of entry activated by pool emptying as opposed to GEA3162 (not shown). However, this result was significantly different from results obtained with Ba²⁺ (Fig. 7). In this experiment, the concentration dependence of externally applied Ba²⁺ entry activated by pool emptying was compared with that for Ba²⁺ entry activated by GEA3162. In cells in which pools were emptied, removal followed by the readdition of Ca²⁺ caused the familiar overshoot of Ca²⁺ entry (Fig. 7). Further removal of Ca²⁺ followed by the addition instead of Ba²⁺ resulted in Ba²⁺ entry, which, as for Sr²⁺, was also detectable by fura-2 ratio-fluorimetry. The entry appeared long-lasting, since Ba²⁺ is a poor Ca²⁺ pump substrate (31) and, hence, was not removed from cells by the plasma membrane pump even if the entry mechanism did become activated (compare with Sr²⁺ and Ca²⁺ in Figs. 5A and 6B, respectively). Under these conditions, only very slow entry of Ba²⁺ was observed at 0.1 mM Ba²⁺, although entry was larger and more rapid with higher levels of Ba²⁺ (Fig. 7A). If the experiment was undertaken with pool-filled cells in the absence of Ca²⁺, the addition of 25 µM GEA3162 activated entry when Ba²⁺ was subsequently added (Fig. 7B). However, the Ba²⁺ dependence of entry appeared measurably different. Now, even 0.02 mM Ba²⁺ resulted in significant entry. From analyses from several experiments using ranges of Ba²⁺, it appeared that the rate of store-operated Ba²⁺ entry was half-maximal at approximately 1 mM Ba²⁺, whereas the rate of GEA3162-dependent Ba²⁺ entry was activated half-maximally with approximately 0.1 mM Ba²⁺.

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Ca2+ pool depletion stimulates Sr2+ entry activated by the NO donor GEA3162 in DDT1MF-2 cells. Bars indicate that throughout the experiment medium was nominally Ca2+-free medium (no Ca2+). Medium was changed to include 1 mM Sr2+ (first arrow) followed by the addition of 25 µM GEA3162 (second arrow). A, normal cells with filled pools. B, cells had been pretreated under standard conditions with the Ca2+ pump blocker, 2,5-di-tert-butylhydroquinone (DBHQ), for 10 min before the addition of Sr2+ to empty pools.

View larger version (19K): [in this window] [in a new window]   Fig. 7.   Entry of Ba2+ into DDT1MF-2 cells in response to thapsigargin-induced Ca2+ pool emptying (A) or activated by the NO donor, GEA3162 (B). Bars indicate times of replacement of medium with nominally Ca2+-free medium (no Ca2+). A, Ca2+ pools were emptied by pretreated the cells with Ca2+ pump blocker, thapsigargin (2 µM), for 10 min in the absence of Ca2+ before transient readdition and removal of Ca2+ followed by addition of Ca2+-free medium containing the indicated mM concentrations of Ba2+ (arrow). B, normal pool-filled cells in Ca2+-free medium were treated with 25 µM GEA3162 (GEA; first arrow) followed by the addition of the indicated mM concentrations of Ba2+ (second arrow).

The results with Ba²⁺ indicated a significant difference in the apparent selectivity for passage of cations activated by store emptying as opposed to GEA3162. In other studies on Ca²⁺ entry, criteria for defining differences between putative entry channels have rested on the effectiveness of La³⁺ in blocking passage of Ca²⁺ ions (35). Although we noted that 10 µM La³⁺ was sufficient to block both pool emptying as well as NO donor-activated entry, a more careful analysis of the La³⁺ dependence of such blockade again revealed a significant difference between the two modes of activation of entry (Fig. 8). The experimental approach was similar to that used in Fig. 7, except varying concentrations of La³⁺ were added either after pool emptying and subsequent Ca²⁺ removal (Fig. 8A) or following 25 µM GEA3162 addition in the absence of Ca²⁺ (Fig. 8B). The Ca²⁺ entry activated by subsequent Ca²⁺ addition to the pool-emptied cells was completely blocked by 10 µM La³⁺. Indeed, 1 µM La³⁺ induced a large decrease in the rate of Ca²⁺ entry, and even 0.1 µM La³⁺ had a significant inhibitory action (Fig. 8A). From other experiments, induction of a half-maximal rate of Ca²⁺ entry was observed with approximately 0.5 µM La³⁺. After GEA-dependent activation of entry by Ca²⁺ readdition (Fig. 8B), whereas Ca²⁺ entry was blocked by 10 µM La³⁺, the sensitivity to La³⁺ appeared measurably different from the store-operated entry. Thus, 1 µM La³⁺ had only a small effect, and the entry of Ca²⁺ was blocked half-maximally at approximately 5 µM La³⁺.

View larger version (19K): [in this window] [in a new window]   Fig. 8.   La3+ blockade of Ca2+ entry into DDT1MF-2 cells in response to thapsigargin-induced Ca2+ pool emptying (A) or Ca2+ entry activated by the NO donor, GEA3162. Bars indicate times of replacement of medium with nominally Ca2+-free medium (no Ca2+). A, Ca2+ pools were emptied by pretreating the cells with Ca2+ pump blocker, thapsigargin (2 µM), for 10 min in the presence of Ca2+ before transient removal and readdition of Ca2+; during the absence of Ca2+, La3+ at the indicated µM concentrations was added (arrow) and maintained after the readdition of Ca2+. B, normal pool-filled cells in Ca2+-free medium were treated with 25 µM GEA3162 (GEA; first arrow) followed by the addition of the indicated µM concentrations of La3+ (second arrow), which were maintained after subsequent readdition of Ca2+.

Overall, our conclusions concerning the nature and relationship of the two modes of activating Ca²⁺ entry present an interesting picture. The S-nitrosylation-activated entry is stimulated by both of the two conditions that define the operation of store-activated Ca²⁺ entry, Ca²⁺ pool emptying and transient external Ca²⁺ removal. Moreover, when both mechanisms are simultaneously activated by external Ca²⁺ addition, their effects can appear nonadditive. Whereas the effects of cytoskeletal reorganization by calyculin A indicate that the mode of activation of the entry mechanisms may be quite distinct, we could reconcile such a difference by considering a possible direct action of S-nitrosylation on the channel, which might circumvent the process of pool-emptying. If this were the case, then the action of pool emptying might help to change the conformation of the channel to increase the availability of a reactive thiol toward nitrosylation or alkylation. In addition, transient Ca²⁺ removal may also cause reconfiguration of the channel into a more susceptible conformation. However, careful analysis of divalent cation selectivity and La³⁺ blockade does reveal a significant difference between the two modes of Ca²⁺ entry. A difference in ion conductivity is difficult to reconcile with the premise that both activities result from activation of a single channel, even though recent evidence for the "slip-mode" operation of Na⁺ channels (36) provides some precedent for such changes. At this stage, we might conclude that the two entry mechanisms are distinct but related. This conclusion may derive strength from other observations. Thus, we have observed that in human embryonic kidney HEK293 cells, in which store-operated Ca²⁺ entry can be activated, the entry of Ca²⁺ is not stimulated by GEA3162 or alkylators.² This may underscore an impression that has been previously suggested (2, 4, 6) that store-operated Ca²⁺ entry differs significantly between cells and may represent a family of distinct channel proteins and/or different association with regulatory proteins. Interest in the TRP family of channels as potential members of the store-operated family has revealed at least six different gene products that vary significantly with respect to their ion selectivity and, more importantly, their possible relationship to pool emptying (33, 34, 37, 38). We utilized the HEK293 cell lines stably transfected by Zhu et al. (35) to express the TRP-3 channel protein.² In these cells as well as the parent and control-transfected cells, the lack of action of either GEA3162 or alkylators indicated that the TRP-3 channel was not a likely target for activation of Ca²⁺ entry by S-nitrosylation. These results notwithstanding, it is interesting to consider that very significant differences in both the conductance properties and the ability to couple to store depletion were noted by Xu et al. (39) for operation of Drosophila-derived transient receptor potential (TRP) and TRP-like (TRPL) proteins cotransfected into mammalian cells. Thus, these experiments suggested that the coassembly of channel monomers into multimeric channels could confer quite different properties related to the relative makeup of the distinct subunits within the channel assemblies. Thus, it is possible that differences in physiological operation (for example, pool coupling and desensitization by Ca²⁺), susceptibility to S-nitrosylation, as well as ion selectivity among different cells may all be related to the relative expression of an extended family of related but distinct channel proteins.

	ACKNOWLEDGEMENTS

We greatly thank Dr. Kim Collins for his invaluable assistance in the completion of this work, Dr. Carmen Ufret-Vincenty and Dr. Richard Waldron for help in the early part of these studies, and Dr. Lutz Birnbaumer for providing the transfected HEK293 cells.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant HL55426, a fellowship (to H-T. M.) from the American Heart Association, Maryland affiliate, and a fellowship from the Swiss Federal Research Foundation (to C. J. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Current address: Dept. of Anatomy, University of California at San Francisco, 513 Parnassus Ave., San Francisco, CA 94143.

^§ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Maryland, School of Medicine, 108 North Greene St., Baltimore, MD 21201. Tel.: 410-706-2593; Fax: 410-706-6676; E-mail: dgill@umaryland.edu.

² H-T. Ma and D. L. Gill, unpublished results.

	ABBREVIATIONS

The abbreviations used are: ER, endoplasmic reticulum; NO, nitric oxide; fura-2/AM, fura-2 acetoxymethylester; GEA3162, 5-amino-3-(3,4-dichlorophenyl)1,2,3,4-oxatriazolium; NEM, N-ethylmaleimide.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

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