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1 Laboratorio de Permeabilidad Iónica, Centro de Biofísica y Bioquímica, Instituto Venezolano de Investigaciones Cientificas, Caracas 1020-A, Venezuela; 3 Laboratorio de Biofísica, Instituto de Investigación Médica M. y M. Ferreyra, Consejo Nacional de Investigaciones Científicas y Técnicas, 5000 Córdoba, Argentina; and 2 Marine Biological Laboratory, Woods Hole, Massachusetts 02543
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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We investigated the role of intracellular Mg2+ (Mgi2+) on the ATP regulation of Na+/Ca2+ exchanger in squid axons and bovine heart. In squid axons and nerve vesicles, the ATP-upregulated exchanger remains activated after removal of cytoplasmic Mg2+, even in the absence of ATP. Rapid and complete deactivation of the ATP-stimulated exchange occurs upon readmission of Mgi2+. At constant ATP concentration, the effect of intracellular Mg2+ concentration ([Mg2+]i) on the ATP regulation of exchanger is biphasic: activation at low [Mg2+]i, followed by deactivation as [Mg2+]i is increased. No correlation was found between the above results and the levels of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] measured in nerve membrane vesicles. Incorporation of PtdIns(4,5)P2 into membrane vesicles activates Na+/Ca2+ exchange in mammalian heart but not in squid nerve. Moreover, an exogenous phosphatase prevents MgATP activation in squid nerves but not in mammalian heart. It is concluded that 1) Mgi2+ is an essential cofactor for the deactivation part of ATP regulation of the exchanger and 2) the metabolic pathway of ATP upregulation of the Na+/Ca2+ exchanger is different in mammalian heart and squid nerves.
sodium ion/calcium ion exchange; adenosine 5'-triphosphate; intracellular magnesium ion; phosphorylation-dephosphorylation; phosphoinositides
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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THE ELECTROGENIC Na+/Ca2+ exchange mediates the coupled movement of Na+ and Ca2+ across the plasma membrane of most animal cells, thus playing an important role in Ca2+ clearance in excitable tissues (23). One of the main features of this transporter is its modulation by different intra- and extracellular physiological ligands, including Ca2+, H+, Na+, K+, ATP, and phosphagens (7, 9, 17, 23). In squid axons, as well as in other preparations, including cardiac and smooth muscle cells, the activity of this transporter is upregulated by MgATP (7, 15, 21). Although there is general consensus that a phosphorylation-dephosphorylation process controls the state of activation of the exchanger, the mechanism(s) of this upregulation seem(s) to vary not only among different species but also within tissues of the same species (7, 15, 19, 21, 25). In squid nerves, ATP modulation of the Na+/Ca2+ exchanger ("on effect") not only requires intracellular Mg2+ (Mgi2+) but also a soluble cytosolic regulatory protein (SCRP) of low molecular weight (4, 16). Conversely, in the mammalian cardiac Na+/Ca2+ exchanger, ATP stimulation needs Mgi2+, but, at least in sarcolemmal vesicles (5, 22), it takes place without a soluble regulatory protein (however, see Ref. 24). In the heart exchanger, MgATP stimulation is linked to the phosphatidylinositide metabolism (5, 22) and, when expressed in fibroblasts, to protein kinase C activity (25).
Most studies on ATP stimulation of Na+/Ca2+ exchange have focused on the "on-activating" effect (2, 6, 9, 18), whereas little is know about the process responsible for the "off-deactivation" part. Reversal phosphorylation is widely accepted as a key mechanism for regulation of several intracellular events that occur in eukariotes in which the dephosphorylation reactions are carried out mostly by phosphatases that require Mg2+ (26, 29). In this study, we have used in vivo and in vitro squid nerve preparations to explore whether Mgi2+ influences the rate at which the ATP-stimulated fraction of the Na+/Ca2+ exchanges deactivates ("off effect"). We found that, after activation by MgATP, rapid removal of Mgi2+ locked the exchanger in a highly activated state both in the absence and presence of ATP. Readmission of Mgi2+ in the absence of the nucleotide rapidly and fully deactivates the stimulated exchanger. The effect of intracellular Mg2+ concentration ([Mg2+]i) on the ATP upregulated Na+/Ca2+ exchanger may have important implications under physiopathological conditions such as ischemia and reoxigenation in which ATP concentration ([ATP]) and [Mg2+]i suffer drastic changes. Part of this work has been previously reported in abstract form (3, 13).
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Intracellular dialysis of squid giant axons. As described previously (12), squid axons from the Marine Biological Laboratory (Woods Hole, MA; Loligo pealei) and the Instituto Venezolano de Investigaciones Cientificas (L. plei) were dialyzed with highly permeable capillaries of regenerated cellulose fibers (210-µm OD; 200-µm ID; molecular weight cutoff of 18 kDa; Spectra/Por 132226; Spectrum, Houston, TX). The standard dialysis medium had the following composition (in mM): 385 Tris-MOPS, 40 NaCl, 5 MgCl2, 285 glycine, and 1 Tris-EGTA, pH 7.3 and temperature between 17 and 18°C. The standard external solution had the following composition (in mM): 440 NaCl, 0.3 CaCl2, 60 MgCl2, and 10 Tris · Cl, pH 7.6. The osmolarity of all solutions was adjusted to 940 mosmol/l. Removal of external sodium was compensated with lithium. To stop any endogenous production of ATP and to effectively control the intracellular ATP concentration, 1 mM NaCN was always present in the external media. The Ca2+ pump component of Ca2+ efflux was eliminated by adding 0.1 mM vanadate to all dialysis solutions.
Preparation of squid nerve and bovine heart membrane vesicles. Membrane vesicles from squid optic nerve (Sepiotheutis sepioidea and L. pealei) and bovine heart were prepared by differential centrifugation as described elsewhere (5, 16) and loaded with 300 mM NaCl (nerve) or 160 mM NaCl (heart), 0.1 mM EDTA, and 30 mM MOPS-Tris (pH 7.3 at 20°C for nerve and pH 7.4 at 37°C for heart). Membrane vesicles are ~35% inside out in squid nerve (16) and 40% in mammalian heart (5). Because the ATP regulatory site of the exchanger is located intracellularly (7, 12), all MgATP-stimulated Na+ gradient-dependent Ca2+ uptake takes place in inside out vesicles only. This population of vesicles also has a powerful MgATP-dependent Ca2+ pump that was inhibited by adding vanadate to the incubation solutions.
Partial purification of the SCRP. Squid optic ganglia (brain) were homogenized (in a 1:1 vol/vol ratio) in 20 mM MOPS-Tris (pH 7.3 at 20°C), 0.1 mM EDTA, and an antiprotease cocktail (0.5 mM phenylmethylsulfonyl fluoride plus 10 µg/ml of aprotinin, leupeptin, and pepstatin A), followed by centrifugation at 12,000 g for 10 min. This supernatant was centrifuged further at 100,000 g for 30 min (postmicrosomal fraction). The 100,000-g supernatant fraction went through 100- and 30-kDa cut-off filters (Amicon Centricon). Aliquots of 200 µl (~1.2 mg of total protein) of the 30-kDa fraction suspended in 30 mM Tris · HCl (pH 7.3 at 20°C) were passed through an HPLC system using a Superdex-75 column (Pharmacia). The runs were performed with the same buffer at a flow rate of 0.4 ml/min, collecting fractions of 0.25 ml. Fraction 65, which corresponded to a molecular weight of ~13 kDa contained the SCRP (16).
Na+ gradient-dependent 45Ca uptake in
membrane vesicles.
45Ca uptake in squid membrane vesicles (16)
was measured at 20-22°C by incubating the vesicles (50-60
µg protein) for 10 or 20 s in media with high (300 mM) or low
(30 mM) Na+ (200 µl total volume). In addition, all
extravesicular solutions contained 0.2 mM vanadate and 20 mM MOPS-Tris
(pH 7.3 at 20°C) and the Mg2+ concentration
([Mg2+]) and [ATP] indicated in Figs. 1-10. In
low-Na+ medium, the osmolarity was compensated with
N-methyl-D-glucamine (NMG)-Cl. The reaction was
stopped with 0.8 ml of an ice-cold solution containing 20 mM MOPS-Tris,
300 mM KCl, and 1 mM EGTA and was filtered through Whatman GF/F glass
filters. The filters were washed with 5 ml of the same solution,
immersed in 5 ml of scintillation fluid, and counted in a liquid
scintillation counter. To obtain steady counts after addition of the
scintillator, the filters were left standing for 4 h and then were
counted. The effects of [Mg2+] on Ca2+
transport and phosphoinositide synthesis (see below) were studied in
chase experiments. 45Ca uptake in bovine heart membrane
vesicles was measured at 37° or at 20°C as indicated
(5). Aliquots of 2 µl (5.5 mg/ml) were diluted in 100 µl of solutions of the following composition: 160 or 10 mM NaCl, 20 mM MOPS-Tris (pH 7.4), 0.1 mM EGTA, 0.1 mM digitoxigenin, 0.2 mM
vanadate, 0.8 µM Ca2+, and the concentrations of ATP and
Mg2+ indicated in Figs. 1-10. Low-Na+
solutions contained osmotically equivalent amounts of NMG-Cl. Incorporation of phosphatidylinositol 4,5-bisphosphate
[PtdIns(4,5)P2] into vesicles was done by
incubating concentrated vesicles with PtdIns(4,5)P2 liposomes (0.250 mg/mg vesicle
protein) for 5 min at 0°C and then for 1 min at 20°C (1,
5). 45Ca uptake measurements were carried out at
20°C in both preparations. In the experiments with alkaline
phosphatase (type VII-NLA from bovine intestinal mucous) before
starting Ca2+ uptake, concentrated vesicles were
preincubated for 2 min at room temperature in the absence of
Na+ gradient with no Ca2+ but with ATP,
Mg2+, and 0, 50, or 200 U/ml of alkaline phosphatase.
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Extraction and TLC separation of phospholipids. Aliquots of 60 µg membrane vesicles were incubated for the indicated times, and solutions are specified in RESULTS. The reactions were stopped with ice-cold chloroform-methanol-HCl, and the lipids were extracted in the organic phase. The TLC plates were developed in a mobile phase of chloroform-methanol-water-concentrated NH4OH (40:48:10:5 vol/vol/vol/vol) using 20 × 10 cm HP-TLC (Merck). The positions of phosphatidylinositol 4-phosphate [PtdIns(4)P] and PtdIns(4,5)P2 were detected using commercial standards submitting the plates to saturated iodine vapor (5). The 32P-labeled phospholipids were visualized in an phosphor screen autoradiography of the plates and were analyzed quantitatively using Imagequant software of the Storm System (Storm-840).
Solutions.
All solutions were made with deionized ultrapure (18 M
) water,
(Milli-Q; Millipore). NaCl, KCl, MgCl2, and
CaCl2 were from Baker. Ouabain, digitoxigenin, Tris-OH,
NMG, MOPS, alkaline phosphatase (type VII-Nla from bovine intestinal
mucosa), phospholipase C (PLC)-specific phosphatidylinositol (PtdIns;
PLC-PtdIns from Bacillus cereus), PtdIns(4)P, and
PtdIns(4,5)P2 were obtained from Sigma. Tris-ATP
and 
,
-methylene-ATP (AMP-PCP) were from Boheringer Mannheim;
45Ca as chloride salt and [-32P]ATP were
purchased from New England Nuclear. Monoclonal
PtdIns(4,5)P2 antibody was obtained from
Perceptive Biosystems (Framingham, MA). The estimations of free
Ca2+ concentration ([Ca2+]) were carried out
with the Maxchelator program (Chris Patton Hopkins Marine Station). The
dissociation constants for the MgAMP-PCP complex were estimated with
Arsenazo III in the same solutions in which the experiments were
performed; the values obtained were 0.159 mM at low and 0.65 mM at high
ionic strength.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Reversibility and Mgi2+ dependence of ATP
stimulation of Na+/Ca2+ exchange in internally
dialyzed squid axons.
Figure 1A shows that, in the
presence of 1 mM Mgi2+, 3 mM ATP stimulates the
efflux of Ca2+ solely through the extracellular
Na+ (Nao+)-dependent (forward
Na+/Ca2+ exchange) component. At a constant
[Mg2+]i of 1 mM, this effect is reversible
upon removal of ATP. For a phosphorylation-dephosphorylation requiring
system, one would expect that removal of either ATP or Mg2+
would deactivate the exchanger. This is indeed the case in the experiment of Fig. 1A where removal of ATP in the presence
of Mg2+ completely deactivates the ATP-stimulated
exchanger. On the other hand, as shown in Fig. 1B, if the
removal of ATP takes place after Mgi2+ has been
taken away, no deactivation of the ATP-stimulated exchanger occurs.
Figure 1B also shows that removal of
Mgi2+ in the presence of ATP produces a small
but significant increase in Nao+-dependent
Ca2+ efflux. When compared with the ATP-independent
Nao+-dependent Ca2+ fluxes (~25
fmol · cm
2 · s1), that
increase is of the order expected for the noncompetitive partial
inhibition of Mgi2+ reported earlier in this
preparation (11). These results indicate that the
exchanger can be maintained in an upregulated state provided Mg2+ is taken away before the removal of ATP. In other
experiments (data not shown), as long as the
[Mg2+]i was virtually zero, the exchanger
could be maintained activated for long periods of time (up to 2 h).
Ca2+ exchange fluxes and biochemical studies in squid nerve and bovine heart plasma membrane vesicles: ATP, Mgi2+, and phosphoinositides. The use of plasma membrane vesicles from squid nerve and bovine heart allowed us to perform parallel Ca2+ transport and biochemical assays related to the role of Mg2+ on the ATP modulation on the Na+/Ca2+ exchanger. Figure 4 shows that, in squid nerve vesicles, MgATP in the presence of SCRP stimulates an Na+ gradient-dependent Ca2+ uptake in a hyperbolic fashion with a Michaelis constant of 0.17 mM. This value is very close to the 0.2 mM reported for dialyzed squid axons (15), indicating that ATP acts in intact nerves and membrane vesicles in a similar fashion. Also shown in Fig. 4 is the absolute requirement of the SCRP for the ATP stimulation of Na+ gradient-dependent Ca2+ uptake (see also Refs. 4 and 16).
In the next group of experiments, we followed the phosphoinositide synthesis in both squid nerve and bovine heart under different experimental conditions. Figure 5, A and B, shows that both preparations contain the metabolic machinery to synthesize PtdIns(4)P and PtdIns(4,5)P2 from PtdIns and MgATP. Moreover, pretreatment of the vesicles with PtdIns-PLC completely abolished PtdIns(4,5)P2 production in both preparations (Fig. 5A, lanes 3 and 6). Interestingly, as shown in Fig. 5A, lanes 1 and 2, in squid nerve vesicles, the SCRP does not modify the patterns of phosphatidylinositol synthesis and does not, by itself, lead to the production of either PtdIns(4)P or PtdIns(4,5)P2. The absolute values of 32P incorporation in PtdIns(4,5)P2 under different conditions are shown in Fig. 5B. The data indicate that the PtdIns(4,5)P2 production in both squid and bovine heart is quite comparable. We have previously reported that, in the bovine heart, PtdIns(4)P production is independent of Ca2+, whereas that of PtdIns(4,5)P2 is strongly dependent on this divalent cation (5). Figure 6, A and B, demonstrates that the Ca2+ dependence of PtdIns(4,5)P2 production in squid nerve is similar to that seen in bovine heart, thus suggesting that a comparable metabolic polyphosphoinositide machinery is present in both preparations. From the above results, we then pursued to look into the effects of Mg2+ on both the Na+/Ca2+ exchange fluxes and the PtdIns(4,5)P2 synthesis in squid nerve membranes. The membrane vesicles in the presence of SCRP were subjected to 1 min of preincubation in the absence of Na+ gradient (100 mM NaCl, 200 mM Na-MOPS, 0.15 mM EGTA, 0.2 mM vanadate, and enough CaCl2 to attain 0.8 µM Ca2+) with 0.25 mM nonradioactive or radioactive ([-32P]ATP) ATP,
10 µl of the SCRP, and either 0.5 or 10 mM Mg2+.
Afterward, one aliquot of vesicles incubated with
[32P]ATP was used to estimate the levels of net
PtdIns(4,5)P2 synthesis (Fig.
7A). The other aliquot was
used to determine the PtdIns(4,5)P2 levels after
20 s of incubation in solutions identical to those where
Na+/Ca2+ exchange was assayed. To that aim,
vesicles preincubated with radioactive
[PtdIns(4,5)P2 synthesis] and cold
(Ca2+ uptake) ATP were diluted 15 times in the presence of
Na+ gradient (Na+ substituted for NMG) in
solutions free of ATP with final concentrations of either 0.5 or 10 mM
Mg2+. There are two points to be stressed in these
experimental protocols. First, 0.5 mM Mg2+ instead of free
Mg2+ solutions were used since the vesicles become leaky in
the absence of this divalent cation. Nevertheless, as shown in Fig. 3,
0.5 mM Mg2+ has no inhibitory effect on the ATP-stimulated
Na+/Ca2+ exchanger. Second, the final [ATP]
attained during the chase period was only 16 µM, a concentration
unable to promote either PtdIns(4,5)P2 synthesis
(data not shown) or stimulation of the Na+/Ca2+
exchanger (see Fig. 4). The results in Fig. 7A demonstrate
that 10 mM [Mg2+] does not interfere with the synthesis
of PtdIns(4,5)P2. On the other hand (Fig.
7B), that concentration of Mg2+ completely
blocks the ATP-stimulated Na+ gradient-dependent
Ca2+ uptake. Therefore, the deactivation of the
ATP-stimulated Na+/Ca2+ exchanger by high
[Mg2+] in squid membrane vesicles is similar to that
reported here in dialyzed squid axons (see Fig. 3) and apparently is
unrelated to the levels of PtdIns(4,5)P2.
Figure 8 shows the differential effect of
PtdIns(4,5)P2 incorporation in membrane
vesicles (27) on the Na+ gradient-dependent
Ca2+ uptake in squid and heart under similar experimental
conditions. As shown, the typical activation by MgATP is observed in
both preparations (1, 5, 16). On the other hand, although
PtdIns(4,5)P2 causes a large stimulation of the
Na+/Ca2+ exchanger in the heart, it is
completely ineffective in activating the squid exchanger.
Effects of anti-PtdIns(4,5)P2 antibody and PtdIns-PLC in dialyzed squid axons. Experiments in cardiac excised patches demonstrated that the PtdIns(4,5)P2 antibody and treatment with PtdIns-PLC completely block ATP stimulation of the Na+/Ca2+ exchange current (22). Although the PtdIns-PLC effectively reduces the PtdIns(4,5)P2 synthesis both in bovine heart and squid nerve (Fig. 5, A and B), this treatment renders squid nerve membrane vesicles highly leaky; this makes it difficult to assess the effect of Na+/Ca2+ exchange on in vitro ATP stimulation. We have therefore performed experiments of this kind using dialyzed squid giant axons in which these compounds were intracellularly injected. As shown in Fig. 9, A and B, injection of the PtdIns(4,5)P2 antibody at a final concentration 200 times that required to inhibit the ATP-stimulated exchanger in cardiac giant patches (22) fails to block the ATP-stimulated Nao+-dependent Ca2+ efflux. No inhibition was observed whether the antibody was injected after (Fig. 9A) or before (Fig. 9B) the addition of ATP. The injected PtdIns-PLC was expected to reach a final concentration in the axon 10 times higher (200 units) than that required to completely inhibit the ATP stimulation of the exchanger in the heart (22). In examining the effect of PtdIns-PLC (see Fig. 9C), we found that, in the absence of ATP, this enzyme causes a small but significant increase in the unspecific Ca2+ leak; nevertheless, addition of ATP still causes stimulation of the Nao+-dependent Ca2+ efflux that is indistinguishable from that seen in control axons. Although in experiments of this kind there are no positive controls, the induction of an unspecific Ca2+ leak may be taken as evidence that the injected PLC has reached the membrane. Indirect positive controls are the experiments in which the injection of a large protein such as alkaline phosphatase completely blocks ATP and phosphoarginine regulation of the squid Na+/Ca2+ exchanger (14).
Effects of exogenous alkaline phosphatase on the ATP-stimulated Na+/Ca2+ exchanger in squid nerve and bovine heart vesicles. Previous efforts from our laboratories have shown that injection of alkaline phosphatase in dialyzed squid axons causes a complete deactivation of the ATP-stimulated Na+/Ca2+ exchange (14). In contrast, in bovine heart membrane vesicles, it had no effect (5). Figure 10A demonstrates that alkaline phosphatase, in a dose-dependent manner, also blocks the ATP stimulation of an Na+ gradient-dependent 45Ca uptake in squid nerve vesicles. Figure 10B shows that the same phosphatase has no effect on the MgATP stimulation of the bovine cardiac Na+/Ca2+ exchanger. As a control, the same experimental solutions containing ATP were incubated with 200 U/ml phosphatase. No significant hydrolysis of ATP was detected after 30 min.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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This work demonstrates the key role played by Mgi2+ in the deactivation of the ATP upregulated squid Na+/Ca2+ exchanger. The complete reversibility of these Mg2+ effects rules out any unspecific deleterious action of this divalent cation. If phosphorylation by ATP is responsible for activation of the exchanger, the most economical conclusion is that an Mgi2+-stimulated dephosphorylation accounts for its deactivation. The biphasic response of the Nao+-dependent Ca2+ efflux to Mgi2+ concentrations at constant [ATP] concur with the general notion that a phosphorylation-dephosphorylation process is involved in ATP modulation of Na+/Ca2+ exchange. The [Mg2+]i required to prevent ATP stimulation indicates that it acts with low apparent affinity (Fig. 3). Furthermore, this Mgi2+ deactivation cannot be accounted for by the reported noncompetitive partial inhibition already described in this preparation (11). This is evident in Fig. 1D where removal of Mgi2+ in the absence of ATP does not restore the levels of the ATP upregulated Na+/Ca2+ exchange fluxes but only causes the expected release of the inhibition observed in the absence of ATP (11).
Our experiments also show that the influence of [Mg2+]i on the ATP regulation of the Na+/Ca2+ exchanger is similar in dialyzed squid axons and in inside out membrane vesicles from squid nerves. This has allowed us to investigate possible relationships between Na+/Ca2+ exchange fluxes in the activated (low Mg2+) and deactivated (high Mg2+) states and the synthesis of polyphosphoinositides. The results suggest that phosphoinositides, key in the ATP regulation of the exchanger in mammalian heart, do not seem to be responsible for ATP regulation in the squid. The evidence, although indirect, is given by the lack of correlation between maneuvers leading to ATP activation and deactivation of Na+/Ca2+ exchange and the levels of membrane PtdIns(4,5)P2. This lack of correlation occurs despite the fact that bovine heart and squid nerve membranes behave very similarly with respect to their phosphoinositide metabolism. On the one hand, the [Ca2+] dependence of PtdIns(4)P and PtdIns(4,5)P2 production is the same. On the other hand, a specific phospholipase (PtdIns-PLC) blocks phosphoinositide synthesis in both species (see Fig. 5). Furthermore, the low-molecular-weight cytosolic regulatory protein required for the MgATP stimulation of the squid exchanger does not modify the phosphatidylinositide production in nerve vesicles. Interestingly, as is the case in dialyzed squid axons (14), we found that MgATP modulation of the Na+/Ca2+ exchanger in squid nerve vesicles is completely abolished by an alkaline phosphatase in contrast to the lack of effect in the mammalian heart. One possible explanation is that regulation of the exchanger by phosphorylation/dephosphorylation processes involves mainly proteins in the squid and lipids in the heart.
Recently, it has been shown that the squid Na+/Ca2+ exchange clone, when expressed in frog oocytes, manifests an MgATP stimulation that depends on PtdIns(4,5)P2 (20). If indeed PtdIns(4,5)P2 participates in the regulation of the exchanger in the squid, we would have expected, at least in part, modifications of the PtdIns(4,5)P2 levels in parallel with those of the ATP-modulated Na+/Ca2+ exchange fluxes, and this did not occur. One possibility is that the phosphoinositide pathway is amplified in the oocyte-expressed clone at 32°C (20). Another possibility is that the clone, inserted in an alien cell, behaves differently and becomes sensitive to other membrane environments. In favor of this is the lack of phosphoarginine regulation of the squid clone when expressed in frog oocytes (20). More compelling evidence for the existence of different pathways for MgATP stimulation of Na+/Ca2+ exchange in squid nerve and mammalian heart are the results described in Fig. 8. Under similar experimental conditions, incorporation of PtdIns(4,5)P2 in membrane vesicles does not modify the exchange fluxes in the squid while inducing the reported activation of the exchanger in the heart (1, 5, 27).
The intimate mechanism by which Mg2+ deactivate the ATP-stimulated Na+/Ca2+ exchanger in the squid remains unknown. However, two possibilities are attractive. 1) The first possibility is that the endogenous SCRP is, or behaves as, part of a two-component signal transduction system (30). Therefore, as in most response regulators, the regulatory protein, in addition to being phosphorylated, would have an Mg2+-dependent [acting with low affinity (28)] autophosphatase activity. 2) The second possibility is that an Mg2+-dependent (also with low affinity) phosphatase, perhaps of the protein phosphatase-2C type (8), mediates the dephosphorylation step. Unfortunately, inhibitors of these phosphatases are not known yet (29).
Finally, it must be pointed out that under physiological conditions one would not expect large fluctuations in the cytosolic concentrations of Mg2+ and/or ATP. Therefore, in a normal cell, this divalent cation, although essential as a cofactor for ATP regulation of the Na+/Ca2+ exchanger, is not likely to play a regulatory role on the exchanger. However, Mgi2+ could become crucial in certain pathological conditions leading to changes in its concentration. In fact, alterations in the function of the Na+/Ca2+ exchanger have been implicated in the cytosolic Ca2+ changes that occur during tissue hypoxia and reoxigenation (see Refs. 31 and 32 and references therein). Besides acidification, a main feature of prolonged ischemia is the dramatic drop in [ATP] (31, 32). Because in most tissues ATP is the major Mgi2+ buffer (10), an increase in free [Mg2+]i is expected under these circumstances. This will create the conditions for an Mgi2+ inhibition of the ATP-stimulated Na+/Ca2+ exchanger, thus contributing to cytosolic Ca2+ load.
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ACKNOWLEDGEMENTS |
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This work was supported by Grants from the National Science Foundation (IBN-9631107), The Consejo Nacional de Investigaciones Científicas y Tecnólogicas (CONICIT-Venezuela S1-99000946 and CONICET-Argentina 4904/97), Fundación Polar (Venezuela), Fundaciencias-IVIC, the CONICOR-Argentina 4501/97, and FONCYT-Argentina (PICT-97 05-00000-01092).
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FOOTNOTES |
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Address for reprint requests and other correspondence: R. DiPolo, Laboratorio de Permeabilidad Ionica, Centro de Biofísica y Bioquímica, IVIC, Apartado Postal 21827, Caracas 1020-A, Venezuela (E-mail: rdipolo{at}ivic.ve).
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.
Received 7 March 2000; accepted in final form 8 June 2000.
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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, Ca2+ efflux in the presence of external
Na+; 
, Ca2+ efflux in the
absence of external Na+. Vanadate (0.1 mM) was included in
all dialysis solutions to inhibit the Ca2+ pump. Notice
that all Ca2+ efflux is extracellular Na+
(Nao+) dependent forward
Na+/Ca2+ exchange. A: reversibility
of the MgATP stimulation of forward Na+/Ca2+
exchange upon removal of ATP in the presence of 2 mM intracellular
Mg2+ (Mgi2+). B: removal
of Mg2+ in the presence of ATP or removal of ATP in the
absence of Mg2+ keeps the exchanger in the upregulated
state. C: sustained activation of the
Na+/Ca2+ exchanger during simultaneous removal
of Mg2+ and ATP when 
) and absence
(
