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Originally published In Press as doi:10.1074/jbc.M400005200 on January 8, 2004

J. Biol. Chem., Vol. 279, Issue 12, 11106-11111, March 19, 2004
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Inhibitory Mechanism of Store-operated Ca2+ Channels by Zinc*

Ariel Gore{ddagger}, Arie Moran{ddagger}§, Michal Hershfinkel¶, and Israel Sekler{ddagger}||

From the {ddagger}Physiology and Morphology, Faculty of Health Science and the Zlotowski Center for Neuroscience, Ben Gurion University of the Negev, POB 653, Beer-Sheva, 84105, Israel

Received for publication, January 2, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Capacitative calcium influx plays an important role in shaping the Ca2+ response of various tissues and cell types. Inhibition by heavy metals is a hallmark of store-operated calcium channel (SOCC) activity. Paradoxically, although zinc is the only potentially physiological relevant ion, it is the least investigated in terms of inhibitory mechanism. In the present study, we characterize the inhibitory mechanism of the SOCC by Zn2+ in the human salivary cell line, HSY, and rat salivary submandibular ducts and acini by monitoring SOCC activity using fluorescence imaging. Analysis of Zn2+ inhibition indicated that Zn2+ acts as a competitive inhibitor of Ca2+ influx but does not permeate through the SOCC, suggesting that Zn2+ interacts with an extracellular site of SOCC. Application of the reducing agents, dithiothreitol (DTT) and {beta}-mercaptoethanol, totally eliminated Zn2+ and Cd2+ inhibition of SOCC, suggesting that cysteines are part of the Zn2+ and Cd2+ binding site. Interestingly, reducing conditions failed to eliminate the inhibition of SOCC by La3+ and Gd3+, indicating that the Zn2+ and lanthanides binding sites are distinct. Finally, we show that changes in redox potential and Zn2+ are regulating, via SOCC activity, the agonist-induced Ca2+ response in salivary ducts. The presence of a specific Zn2+ site, responsive to physiological Zn2+ and redox potential, may not only be instrumental for future structural studies of various SOCC candidates but may also reveal novel physiological aspects of the interaction between zinc, redox potential, and cellular Ca2+ homeostasis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The release of Ca2+ from inositol 1,4,5-trisphosphate (InsP3)-sensitive intracellular Ca2+ stores is followed in many cell types by the opening of store-operated Ca2+ channels (SOCC)1 that refill the Ca2+ stores (1–5). The genes linked to SOCC activity have not been clearly identified. However, an accumulating body of evidence indicates that members of the family of membrane proteins, Trp, first discovered in Drosophila but with many mammalian homologues, are mediating the store-operated Ca2+ flux (6). The cation influx mediated by SOCC is highly Ca2+-specific. Other divalent cations such as Ba2+ and Sr2+ are also permeable albeit at a much slower rate (3, 7). In the absence of divalent cations, it has been reported that the SOCC becomes permeable to Na+ (8, 9). However, recent studies have indicated that this mono-valent cation influx is not mediated by the SOCC but rather by MIC channels (10).

Calcium influx mediated by the SOCC participates in refilling of internal Ca2+ stores in addition to modulating the Ca2+ response triggered by various agonists (2). Such regulation of the Ca2+ response has been suggested to control a wide range of processes including secretion, cell growth, and proliferation (11). The opening of the SOCC under metabolic stress, it should be added, results in a prolonged rise of intracellular Ca2+, an important element in the signaling cascade leading to cell death (12, 13).

Trivalent cations such as La3+ and Gd3+ and the divalent cations Cd2+ and Zn2+ have been employed previously as inhibitors of the SOCC and have contributed to understanding of this entry route for cations (14–16). Paradoxically, La3+,Gd3+, and Cd2+, which are not physiologically relevant, have been the most commonly studied cations, whereas zinc, which is highly relevant physiologically, is the least understood with respect to the mechanism by which it affects inhibition (1). Endogenous Zn2+, by virtue of its interactions with specific Zn2+ binding sites, modulates a number of ion channels, including NMDA and GABA receptors, as well as regulating transporters such as the dopamine transporter (17–20). Zinc inhibition of Ca2+ influx, mediated by SOCC, has been shown to modulate intracellular Ca2+ signals, thereby modifying cellular functions (14). The majority of zinc ions are complexed to metalloproteins such as enzymes, metallothioneins, and transcription factors. It is, therefore, unclear whether the minute concentrations of free zinc, found in most organs, are sufficient to block SOCC-mediated Ca2+ influx. It is also not known whether zinc binding sites are allosteric or whether they are associated with the cation permeation pathway. In the present study, we describe the basic characteristics of the Zn2+ inhibitory site on the SOCC. Our results indicate that Zn2+, at physiological concentrations, competitively blocks the Ca2+ flux via SOCC, without permeating through this pathway. We further show that the SOCC inhibition by zinc, but not by lanthanides, is completely eliminated by an increase in redox potential, indicating that cysteines are part of the Zn2+ inhibitory site that is distinct from the lanthanide site. Such interplay between zinc and redox, shown previously with regard to sequestration and release of zinc ions by cysteine-rich metallothioneins (21), may have important physiological implications with respect to the regulation of the Ca2+ response in salivary submandibular acini and duct cells.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell and Tissue Preparation—HSY (human salivary gland cell line) were grown in Dulbecco's modified Eagle's medium as described previously (22). Min-6 cells (a mouse insulinoma cell line) were grown in Dulbecco's modified Eagle's medium with 1% penicillin-streptomycin, 1% L-glutamine, 71.5 µM {beta}-mercaptoethanol, and 15% calf serum bovine donor (23). Rat submandibular ducts and acini were prepared essentially as described previously (24). The preparations were mounted on glass coverslips that were coated with CellTak (BD Biosciences), according to the manufacturer's instructions, 30 min prior to the Fura-2 loading.

Fluorescent Calcium Imaging—Cells grown on coverslips were loaded for 30 min at room temperature with 5 µM Fura-2 AM (1 mM stock in Me2SO, Tef Labs, Austin, TX) in Ringer's solution composed of (in mM): 120 NaCl; 5.4 KCl; 1.8 CaCl2; 0.8 MgCl2; 10 HEPES; 10 glucose; pH 7.4, containing 0.1% bovine serum albumin. Ducts and acini were loaded by 2.5 µM Fura-2 AM in the presence of 0.025% pluronic F-127 (Molecular Probes). Fura-2-loaded cells were washed twice in Ringer's solution containing 0.1% bovine serum albumin and incubated for an additional 15 min at room temperature to facilitate the hydrolysis of the dye. Coverslips were then mounted in a perfusion chamber placed on the microscope stage, and Ca2+ imaging was carried out as described previously (25). The zinc-dependent Ca2+ rise triggered by ZnR (25) was eliminated by pretreating HSY cells with 80 µM zinc for 30 min and then thoroughly washing with zinc-free Ringer's. All the results shown are the average of at least 120 cells, from at least three independent experiments. For the sake of clarity, only every 4–5th data point is shown in the graphs.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Zinc Acts as a Competitive Inhibitor of Ca2+ Permeation through SOCC—The opening of the SOCC following depletion of intracellular stores plays an important role in the physiology of exocrine glands (14, 26). To activate and monitor SOCC in HSY cells, loaded with the Ca2+-sensitive dye Fura-2, we depleted intracellular calcium stores using 200 nM thapsigargin (TG, Alomone Labs) in nominally calcium-free Ringer's solution. Application of TG was followed by a slow rise of [Ca2+]i, leaking from the intracellular pools, and recovery of Ca2+ to resting levels. The subsequent addition of Ca2+ to the perfusing solution was followed by a massive calcium influx (Fig. 1a), which was totally blocked by application of 20 µM Gd3+ or La3+ (Fig. 1b), indicating that it is mediated by SOCC (1). Application of 20 µM Zn2+ had a similar inhibitory effect (Fig. 1b). The free Zn2+ concentration in extracellular fluids is estimated to be in the range of 10–7-10–5 molar (27). Therefore, to assess the physiological significance, it was important to determine the concentration range at which Zn2+ strongly inhibits the SOCC. Varying concentrations of Zn2+ were applied, and the maximal initial calcium influx rate was determined at different Ca2+ concentrations. To quantify the inhibitory effect of Zn2+, d[Ca2+]i/dt was plotted against the Zn2+ concentration in the perfusing solution (Fig. 2a). Fitting the data with a Michaelis-Menten equation yielded apparent Ki for each calcium concentration. The Ki values of Zn2+ inhibition of SOCC suggest that Zn2+ ions have the potential to inhibit SOCC activity at concentrations found not only in exocrine glands but also in many other tissues, including pancreatic islets of Langerhans (28), brain (29), colon (30), and the male reproductive system (27).



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  		FIG. 1.
The experimental paradigm of SOCC opening and its inhibition by heavy metals. In a, SOCC was activated by emptying intracellular Ca2+ pools, in nominally Ca2+-free Ringer's solution in the presence of 0.2 µM thapsigargin followed by the addition of 1 mM CaCl2. Changes in intracellular Ca2+ concentrations were monitored using Fura-2 fluorescence, allowing estimation of SOCC activity. In b, Ca2+ permeation through the SOCC was determined as described in a. Calcium (1 mM) was added in the presence or absence of the indicated metals (20 µM). All three metals strongly inhibited Ca2+ permeation through the SOCC.

 



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  		FIG. 2.
Zinc is a high affinity competitive inhibitor of Ca2+ permeation through SOCC. a, dose-response analysis of Zn2+ inhibition of SOCC, using the same experimental paradigm described in Fig. 1a, at various Ca2+ concentrations (0.45, 0.6, 1, 1.5, 2 mM). The calcium influx rate at the indicated zinc concentrations was expressed as the percentage of maximal influx rate (no zinc added) for each calcium concentration used. The line is a fit to a Michaelis-Menten equation, yielding an apparent Ki for each Ca2+ concentration. The inset shows the full range of Zn2+ concentration that was used. In b, the apparent Ki exhibits linear dependence on Ca2+ concentration. The extrapolated Ki for Zn2+ at the absence of Ca2+ is 0.2 ± 0.1 µM. The linear relationship between the Ki values for Zn2+ and extracellular Ca2+ concentration indicates that Zn2+ competitively inhibits Ca2+ influx.

 
Zinc modulates the activity of channels and transporters such as the dopamine transporter, GABA, and NMDA channels by interacting with allosteric sites (31, 32). On other proteins such as the glycine receptor, CIC chloride channel, and the L-type Ca2+ channel, zinc acts as a competitive inhibitor of ion permeation (33–35). To distinguish between these two modes of inhibition, the Ki for Zn2+ inhibition of Ca2+ influx through the SOCC was plotted versus extracellular Ca2+ concentration (Fig. 2b). The linear dependence indicates that Zn2+ acts as a competitive inhibitor of Ca2+ influx through the SOCC.

Zn2+ Does Not Permeate through the SOCC—The permeation of Zn2+ through several types of cationic channels, most notably glutamate channels, purinergic P2X receptor, and the L-type Ca2+ channels, has important physiological and pathophysiological implications (36, 37). In contrast to these, Zn2+ does not permeate through, but rather modulates channels such as the GABA receptor and the dopamine transporter (31, 38, 39). To determine whether Zn2+ modulation of the SOCC also involves permeation through the SOCC, we used a similar experimental paradigm to that described in Fig. 1a but replaced the Ca2+ applied following the depletion of the stores with 80 µM Zn2+ (Fig. 3a). Because Fura-2 is ~100-fold more sensitive to Zn2+ than to Ca2+, it can be effectively used to determine whether Zn2+ permeates into cells (40, 41). As shown in Fig. 3a, no change in Fura-2 fluorescence was observed following the opening of the SOCC in the presence of zinc, suggesting that the SOCC is impermeable to this ion. To confirm that this result in HSY cells did not stem from insufficient sensitivity, we monitored Zn2+ permeation in Min-6 insulinoma cells that functionally express both SOCC and LTCC (23, 42, 43). As shown in Fig. 3b, depolarization that triggers the opening of LTCC was followed by a robust Zn2+ influx. Using the SOCC opening paradigm, no change in the fluorescent signal was observed when Zn2+ was applied (Fig. 3b), although Ca2+ permeation is monitored. Our results, therefore, indicate that the SOCC is impermeable to Zn2+.



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  		FIG. 3.
SOCC is impermeable to Zn2+. In a, SOCC was activated by depleting the stores with 100 µM UTP and 0.2 µM TG. Cells were then perfused with Ca2+-free Ringer's solution with Zn2+ (80 µM), and finally, Zn2+ was removed, and Ca2+ (1.5 mM) added. A fluorescent rise is monitored following the addition of Ca2+, indicating that SOCC was indeed opened. Although Fura-2 is highly sensitive to zinc, no change in fluorescence was monitored following its addition. Thus, zinc is a reversible blocker of SOCC that does not permeate through this channel. In b, to determine the sensitivity of this assay, permeation of zinc through LTCC was monitored in Min-6 cells, loaded with Fura-2 and treated with UTP and TG as in a. Cells were depolarized by Ca2+-free Ringer's solution containing 50 mM K+, in the presence of 100 µM zinc. The rapid reduction of the signal by N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN, 50 µM), a membrane-permeable heavy metal chelator, indicates that the rise in fluorescence is related to zinc. To determine whether Zn2+ permeates via the SOCC, in Min-6 cells, SOCC was activated by emptying intracellular calcium stores, as above (not shown on graph). Cells were then perfused with Ca2+-free Ringer's solution with Zn2+ (100 µM) or 1.5 mM Ca2+ (ions added at the time indicated by *). No change in fluorescence was monitored following Zn2+ application, whereas Ca2+ permeation via SOCC, not affected by TPEN, is clearly monitored. Taken together, the data indicate that in Min-6 cells, as in HSY cells, zinc does not permeate through this pathway.

 
The Zn2+ Inhibitory Site—The amino acid residues that are involved in the Zn2+ inhibitory sites on the SOCC are unknown. Previous work on several membrane proteins including the purinergic receptors subunits P2X2 and P2X4 (44), glycine receptor (45) NMDA and GABA receptors, and the CIC chloride channel (17, 35), implicate cysteine residues as part of the Zn2+ binding site. The role of cysteines in Zn2+ inhibition of SOCC was assessed, therefore, under reducing and non-reducing conditions. As shown in Fig. 4a, application of the Zn2+ in the presence of the reducing agent {beta}-mercaptoethanol (5 mM) totally eliminated the inhibitory effect of Zn2+. Cadmium is another known inhibitor of the SOCC, although at a much lower affinity than Zn2+, and it also does not permeate cells via this channel (46). Similarly to Zn2+, this ion is known to interact with cysteine residues of membrane proteins. Although application of Cd2+, at a concentration range shown previously to inhibit SOCC (14), completely blocked Ca2+ permeation, the addition of {beta}-mercaptoethanol (5 mM) eliminated the SOCC inhibition by Cd2+ (Fig. 4a). A similar effect was observed in the presence of DTT (not shown). This indicates that cysteines, affected by the change in redox potential, are involved in Zn2+ inhibition of the SOCC and that the inhibitory site may also interact with Cd2+. A small degree of permeation by Cd2+ was monitored by Fura-2 fluorescence, and its rate was similar when Cd2+ was applied following the opening of SOCC (Fig. 4a) or when Cd2+ was applied prior to the opening of SOCC (not shown). This Cd2+ flux is known to be mediated by the maitotoxin-sensitive pathway, which is distinct from the SOCC (46).



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  		FIG. 4.
Reducing agents prevent blocking of SOCC by Zn2+. SOCC activity was monitored following the depletion of intracellular Ca2+ stores using UTP (100 µM) and TG (0.2 µM) in Ca2+-free Ringer's solution. In a, although application of Zn2+ (50 µM) or Cd2+ (500 µM) inhibited the Ca2+ influx (x, •), {beta}-mercaptoethanol ({beta}-Me, 5 mM) treatment together with the heavy metals ({circ}-Zn2+, {blacksquare}-Cd2+) eliminated the inhibition of the SOCC. Control cells were superfused only with Ca2+-containing Ringer's solution. In b, following the opening of SOCC, cells were perfused with Zn2+ ({blacksquare},50 µM), or Cd2+ ({circ}, 500 µM), in the presence of Ca2+ (1.5 mM). Subsequently, DTT (60 µM or 4 mM respectively) was added. The application of the reducing agent totally reversed the block of SOCC. When cells were perfused with DTT and Zn2+ without Ca2+ ({diamondsuit}), no change in fluorescence was monitored, indicating that DTT does not change the permeability of SOCC to Zn2+.

 
We next asked whether an increase in redox potential may reverse the zinc-mediated block of the SOCC. This is of potential physiological importance, considering the changes in redox potential that are monitored in multiple cell types (47). Following the block of SOCC by Zn2+ and Cd2+, application of DTT restored Ca2+ permeation (Fig. 4b). Using this experimental paradigm, we found that as little as 60 µM DTT was sufficient to totally eliminate zinc inhibition of the SOCC.

Although Ca2+ permeates strongly in the presence of DTT, no apparent increase in Fura-2 fluorescence was observed when Ca2+ was replaced with zinc, suggesting that DTT treatment does not render SOCC more permeable to zinc (Fig. 4b). Our results, therefore, suggest that the zinc inhibitory site precedes the cation selectivity region on the SOCC.

To determine whether redox potential will have an effect on SOCC inhibition by lanthanides, we determined their inhibition potency in the presence and absence of reducing agents. In contrast to the striking effects that reducing conditions had on Zn2+ inhibition, both DTT (Fig. 5a) and {beta}-mercaptoethanol (data not shown) failed to eliminate the inhibition of SOCC by 20 µM La3+ or Gd3+. To address the possibility that DTT is acting by chelating the metal ions, thereby affecting SOCC inhibition, we determined the inhibitory effect of 2 µM La3+ or 1 mM zinc in the presence of 4 mM DTT. As shown in Fig. 5b, La3+ at a minute concentration (2 µM) was still effective in inhibiting SOCC, and the elimination of zinc inhibition by DTT persisted even in the presence of 1 mM Zn2+. Thus, our results indicate that it was the change in the redox potential induced by DTT and {beta}-mercaptoethanol and not the chelation of zinc that eliminated the inhibition of SOCC. The effect of DTT on zinc inhibition of SOCC suggests that the Zn2+ inhibitory site may involve cysteines. Furthermore, the lack of effect on lanthanide inhibition of SOCC following the change in redox potential suggests that the inhibitory sites for Zn2+ and lanthanides are distinct.



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  		FIG. 5.
Reducing agents do not prevent the blocking of SOCC by lanthanides. In a, SOCC was opened by UTP (100 µM) followed by application of the SOCC inhibitors La3+ or Gd3+ (20 µM) in the presence of the reducing agent DTT (4 mM) and Ca2+ (1.5 mM). In contrast to its effect on zinc, DTT did not eliminate the inhibition by La3+ or Gd3+, indicating that the lanthanide and zinc inhibitory sites are distinct. In b, SOCC inhibition by La3+ was also not eliminated at a much lower concentration of 2 µM. However, zinc inhibition of SOCC was still eliminated in the presence of high concentration of Zn2+ (1 mM).

 
Changes in Redox Potential Have a Physiological Effect on Agonist-induced Ca2+ Response by Altering the Zn2+ Sensitivity of SOCC—Changes in redox potential play a fundamental physiological and pathophysiological role in salivary secretion (48). We hypothesized that redox potential is involved in shaping the agonist-induced calcium response by modulating the zinc inhibitory effect of the SOCC. To test this, we first monitored the Ca2+ response in Fura-2-loaded rat submandibular salivary ducts attached to glass coverslips (Fig. 6a) following depletion of intracellular stores using TG, in the presence and absence of Zn2+ (50 µM). A similar inhibition of SOCC was apparent when stores were depleted using TG or carbachol in ducts (Fig. 6, a and b). This experimental paradigm was also applied to acini and yielded similar results (not shown). We then determined the Ki for the inhibitory effects of Zn2+ in both acini and ducts, by monitoring Ca2+ influx following the depletion of intracellular Ca2+ stores using TG in the presence of varying zinc concentrations (Fig. 6c). The apparent Ki of both acini and duct was similar. The physiological effect on agonist-induced Ca2+ response in the presence of 50 µM Zn2+, a concentration sufficient to inhibit the SOCC, was next determined. Ducts were superfused with Ca2+ Ringer's solution, and SOCC opening was induced using carbachol (100 µM). As shown in Fig. 6d, application of zinc was followed by a rapid return of Ca2+ to resting levels within 1 min and eliminated the elevated plateau triggered by SOCC. Similar results were obtained using 20 µM La3+ (not shown). In contrast, the addition of zinc in the presence of DTT significantly prolonged the Ca2+ response (to about 3 min) and re-established the elevated plateau, completely reversing the inhibitory effect of zinc. The application of DTT in the absence of zinc did not significantly change the Ca2+ response, indicating that it is not the DTT which modulates the Ca2+ response. These results show that under conditions of increased redox potential, the zinc inhibition of SOCC is totally eliminated, thereby dynamically regulating the profile of the Ca2+ response. Interestingly, the duration of the Ca2+ response in the presence of DTT (in the absence or presence of zinc) was even slightly longer than in the control, suggesting that endogenous zinc is present in a concentration sufficient to partially inhibit the SOCC.



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  		FIG. 6.
Zinc blocking of SOCC regulates agonist-induced Ca2+ response of salivary gland ducts. In a, ducts were treated with TG (0.2 µM) in calcium-free Ringer's solution to deplete the intracellular stores. Subsequently, Ca2+ (1.5 mM) was added in the presence or absence of Zn2+ (50 µM). Calcium permeation through the SOCC was only apparent following TG treatment (not shown). In b, the same experimental paradigm was applied using carbachol (CCh, 100 µM), instead of TG, to deplete the intracellular stores. In c, a dose-response analysis of Zn2+ inhibition of SOCC performed on ducts and acini, using the Michaelis-Menten equation, yielded calculated Ki values of 0.48 ± 0.05 and 0.49 ± 0.05 µM, respectively. In d, ducts were perfused with Ca2+-containing Ringer's solution with carbachol (100 µM). The calcium response was monitored in the presence or absence of 50 µM zinc. In the presence of zinc, the duration of the Ca2+ response was drastically reduced, and the elevated plateau phase was eliminated, indicating that both effects are mediated by zinc inhibition of Ca2+ permeation via the SOCC. The same paradigm was used while applying both Zn2+ (50 µM) and DTT (4 mM). The reducing agent restored the full duration of the Ca2+ response and the elevated plateau phase following carbachol application. As shown, application of DTT did not change the calcium response profile as compared with control, indicating that DTT does not affect SOCC activity.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The main goal of the present study was to characterize the mechanism by which Zn2+ blocks SOCC. Because of the prominent role of SOCC in shaping the Ca2+ response in exocrine cells following stimulation, we focused on the rat salivary ductal and acinar cells as well as the HSY cell line. We found that Zn2+ inhibits Ca2+ influx through SOCC with an apparent Ki of 0.5–2 ± 0.1 µM in salivary gland and HSY cells, which is well within the concentration of extracellular Zn2+ (27). Our results indicate that although Zn2+ acts as a competitive inhibitor of Ca2+ influx, it does not permeate through this pathway. An important finding of this work is that Zn2+ inhibition is eliminated by reducing conditions, whereas the inhibition by the lanthanides is not affected, indicating that the SOCC harbors a redox-sensitive Zn2+ inhibitory site that is distinct from the lanthanides site.

Zinc and cadmium share similar physico-chemical characteristics, the most notable of which is the interaction with thiolate groups. Indeed, Cd2+ and Zn2+ interact strongly with cysteines of numerous proteins, e.g. metallothioneins (49, 50). The elimination of SOCC inhibition by Zn2+ and Cd2+, under similar reducing conditions, suggests that both metal ions interact with cysteine residues on the SOCC. On the other hand, the striking difference in the affinity of Cd2+ or Zn2+ to the SOCC may indicate that their binding sites do not completely overlap. Resolving this issue will involve the scanning of cysteine mutagenesis along the SOCC followed by affinity analysis of Cd2+ and Zn2+ inhibition.

It has been argued that chelation of heavy metals by DTT may play a role in elimination of Zn2+ inhibition by DTT of NMDA channels, rather than a reducing effect on the cysteine residues (51). In contrast, other studies on the purinergic receptors (44) and on NMDA receptors (17) have suggested that redox potential regulates the interaction of Zn2+ with cysteines in Zn2+ inhibition of SOCC. Several findings of the present study would appear to lend strong support for a mechanism involving redox regulation of the Zn2+ binding site on SOCC by DTT. First, although DTT may also chelate La3+ and Gd3+, it does not have any effect on the inhibition of SOCC by these metals (Fig. 5a). Second, {beta}-mercaptoethanol, a potentially weaker chelator of Zn2+, eliminated zinc inhibition of SOCC as effectively as DTT (Fig. 4a). Third, application of zinc at widely varying concentrations (i.e. 10–1000 µM), which would have dramatically changed its free concentration, did not attenuate the capacity of DTT to eliminate Zn2+ inhibition (Figs. 4, 5). Also, a range of La3+ concentrations (2–20 µM) in the presence of DTT did not change its effectiveness viz. SOCC inhibition (Fig. 5). Thus, we suggest that the effect of the reducing agents on zinc inhibition is related to a change in redox and is not mediated by zinc chelation.

Both characterization of Zn2+ binding sites and insertion of an artificial Zn2+ site into the sequence of membrane proteins are powerful approaches available today to those studying their structure-function aspects (31, 52). This may be particularly useful for SOCC since very little is known about domains or residues playing a functional role in ion permeation through this pathway (53). To this end, our results can provide a working model, illustrated in Fig. 7, on the nature of the Zn2+ inhibitory site of SOCC. According to our results, Zn2+ acts as a competitive inhibitor of Ca2+ permeation, indicating that the Zn2+ inhibitory site is at, or near, the permeation pathway and is not an allosteric site. In contrast to the LTCC, studies by this (Fig. 3b) and other laboratories (1) indicate that SOCC is impermeable to zinc. Thus, we propose that Zn2+ interacts with an extracellular site, perhaps at a vestibule preceding the cation selectivity domain of SOCC. Zinc is often coordinated in its binding site by cysteines, histidines, or both. Our data, supporting a role for cysteines in the Zn2+ inhibition site (Figs. 4, 5), do not exclude the participation of other residues such as histidines. The properties of the zinc inhibitory site on the SOCC and the CIC chloride channel are similar. On the latter, zinc ions interact with 3 cysteines on the extracellular site of the ion permeation pathway (35). It will be interesting to determine whether there are common structural motives on CIC channel and Trps, related to the zinc inhibitory site. Future studies, combining chemical modification and site-directed mutagenesis of susceptible cysteines, will be required for the identification of the residue coordinating Zn2+ to its inhibitory site in the various Trp proteins. Such analysis may be helpful not only in identifying the Zn2+ binding site but also in providing a clue to the specific role played by various Trps in SOCC activity, a question that is still hotly debated (54).



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  		FIG. 7.
Schematic model for the zinc inhibitory site on SOCC. Zinc interacts with cysteines and may also coordinate with other residues at an extracellular domain along the SOCC permeation pathway, thereby inhibiting Ca2+ permeation into the cells.

 
The redox-dependent regulation of the SOCC by zinc is a novel functional aspect of this channel. Such regulation may be of particular physiological interest in salivary glands because of the dramatic effects that changes in redox potential trigger there, e.g. after exposure to secretagogues such as isoproterenol (55, 56). This, together with the pivotal role played by sustained calcium rise in regulating ionic content and pH of salivary secretions (57, 58), points toward the potential importance of this novel mode of regulation. Interestingly, although duct and HSG cells express similar Trp proteins, they exhibit altered sensitivity to zinc; the mechanism for this striking difference is not understood (26, 59). A mechanism involving redox potential may provide a plausible explanation.


   FOOTNOTES
 
* This work was supported by German-Israeli Foundation for Scientific Research and Development Grant 10588099.01/98, Israeli Science Foundation (ISF) Grant 456/02.1 (to I. S. and A. M.), and ISF equipment Grant 456/02.2 (to I. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ To whom corresponding may be addressed. Tel.: 972-8-6477331; Fax: 972-8-6477628; E-mail: arie{at}bgumail.bgu.ac.il. || To whom corresponding may be addressed. Tel.: 972-8-6477328; Fax: 972-8-6477628; E-mail: sekler{at}bgumail.bgu.ac.il.

1 The abbreviations used are: SOCC, store-operated calcium channel(s); LTCC, L-type calcium channel(s); DTT, dithiothreitol; GABA, {gamma}-aminobutyric acid, Type A; NMDA, N-methyl-D-aspartate; TG, thapsigargin. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Ze'ev Silverman for critical reading of the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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