In squid nerves intracellular Mg2+
promotes deactivation of the ATP-upregulated
Na+/Ca2+ exchanger
Reinaldo
DiPolo1,2,
Graciela
Berberián3, and
Luis
Beaugé2,3
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 |
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 |
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 |
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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Fig. 1.
MgATP interactions in the upregulation of the
Na+/Ca2+ exchanger in dialyzed squid axons.
, 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 , -methylene-ATP (AMP-PCP) is
used as an Mg2+ chelating agent. Notice that the exchanger
can be maintained in this activated state for >2 h. D:
deactivation by Mgi2+ of the ATP-stimulated
Na+/Ca2+ exchange. Nos. refer to free
Mg2+ in mM. See RESULTS for more details.
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Fig. 2.
Reversibility of Mg2+ deactivation of the
ATP-dependent upregulation of Na+/Ca2+
exchanger in dialyzed squid axons.
Nao+-dependent Ca2+ efflux
in the presence of 1 mM intracellular Mg2+
concentration ([Mg2+]i) was stimulated by 4 mM ATP. Notice the complete inhibition of the ATP-upregulated exchange
fluxes by 10 mM Mgi2+ at constant ATP
concentration ([ATP]). Return to the initial 1 mM
[Mg2+]i produced a reactivation of the
ATP-dependent fluxes to their original levels. Temperature: 17.5°C.
For more details see RESULTS.
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Fig. 3.
Dose-response relationship between the ATP-stimulated
Nao+-dependent Ca2+ efflux and
[Mg2+]i at constant [ATP].
Nao+-dependent Ca2+ efflux in the presence
of 4 mM ATP was measured in 5 different dialyzed squid axons in which
Mg2+ concentration ([Mg2+]) in the dialysis
was varied between 0.2 and 10 mM. Points correspond to the mean ± SE normalized to the Ca2+ efflux values obtained with 1 mM
[Mg2+]. Nos. in parentheses refer to different axons.
Notice the belt shape of the ATP activation curve. Temperature:
17.5°C.
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Fig. 4.
ATP activation curve of Na+
gradient-dependent Ca2+ uptake in squid optic nerve plasma
membrane vesicles in the presence ( ) and absence
( ) of soluble cytosolic regulatory protein (SCRP).
Broken line shows the ATP activation curve for
Nao+-dependent Ca2+ efflux in dialyzed
squid axons. The Michaelis constant value for ATP in vesicles with SCRP
(0.17 mM) is very close to that determined in dialyzed axons [0.2 mM
(15)]. Notice that in the absence of SCRP no ATP
activation of Na+/Ca2+ exchange is observed.
Each point is the mean ± SE of triplicate determinations.
Experiments were carried out at 20°C. See METHODS for
details.
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Fig. 5.
Intracellular 32P labeling from
[ -32P]ATP of phosphoinositides in membrane
vesicles from squid optic nerve and bovine heart. A:
phosphoimage of 1-dimension TLC plates. Lanes 1-3 are
from nerve membrane vesicles. Lanes 5 and 6 are
from bovine heart membrane vesicles. Lane 1, control;
lane 2, vesicles with addition of SCRP; lane 3,
nerve vesicles treated with phospholipase C (PLC)-specific
phosphatidylinositol (PtdIns; 200 U/ml); lane 4, SCRP
without nerve membrane vesicles; lanes 5 and 6,
control or PtdIns-PLC-treated (20 U/ml) bovine heart membrane vesicles;
lane 7, negative control without membranes. Positions of
phosphatidylinositol 4-phosphate [PtdIns(4)P; PIP] and
phosphatidylinositol 4,5-bisphosphate
[PtdIns(4,5)P2; PIP2] are
indicated. B: absolute values of 32P
incorporation in PtdIns(4,5)P2 spots. Data are
means ± SE of triplicate determinations expressed as pmol/mg
protein present in the initial solution extracted with
chloroform/methanol. Note 1) the effective reduction in
PtdIns(4)P and PtdIns(4,5)P2 levels
in PtdIns-PLC-treated nerve and heart vesicles, 2) the lack
of effect of the SCRP on PtdIns(4)P and
PtdIns(4,5)P2 synthesis, and 3) the
absence of phosphoinositide production in the presence SCRP only.
[PIP2], PIP2 concentration.
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Fig. 6.
PtdIns(4)P (A) and
PtdIns(4,5)P2 (B) synthesis in
membrane vesicles from squid optic nerve with (filled bars) and without
(open bars) Ca2+ at two [ATP]. Membrane vesicles in the
presence of SCRP were incubated at room temperature in a solution
containing (presence of Na+ gradient; in mM): 200 N-methyl-D-glucamine (NMG)-MOPS (pH 7.3), 85 NMG-Cl, 30 NaCl, 0.15 EGTA, 0.2 vanadate, 0.25 or 0.5 [ -32P]ATP, and 1 Mg2+ in the presence or
absence of 1 µM Ca2+. After 1 min, the reaction was
stopped with chloroform-methanol-HCl, and the lipids of all assays were
extracted in the organic phase. The TLC plates were developed in a
mobile phase of chloroform-methanol-H2O-concentrated
NH4OH (40:48:10:5 vol/vol/vol/vol) using 20 × 10 cm
HP-TLC (Merck). The positions of the PtdIns(4)P and
PtdIns(4,5)P2 were detected using commercial
standards by submitting the plates to an atmosphere of saturated iodine
vapor. The 32P-labeled polyphosphoinositides were
visualized and quantified by the storm system. Values are means ± SE. Notice that, as is the case in bovine cardiac sarcolemmal vesicles
(5), in squid nerve membranes production of
PtdIns(4)P is Ca2+ independent, whereas that of
PtdIns(4,5)P2 is strongly dependent on
Ca2+.
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Fig. 7.
Effects of low and high [Mg2+] on
PtdIns(4,5)P2 synthesis (A) and
ATP-stimulated Na+ gradient-dependent 45Ca
uptake (B) in squid optic nerve membrane vesicles. Control,
net synthesis of PtdIns(4,5)P2 after 1 min
incubation; chase, levels of PtdIns(4,5)P2
measured 20 s after [32P]ATP was removed.
45Ca uptake was determined during the 20-s chase period.
Notice 1) high free [Mg2+] (10 mM) does not
influence either the net synthesis (control) nor induces a breakdown
(chase) of PtdIns(4,5)P2 and 2) high
free Mg2+ completely blocks the ATP-stimulated
Na+ gradient-dependent Ca2+ uptake. All values
are means ± SE of triplicate determinations. See
METHODS and RESULTS for details.
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Fig. 8.
Effect of
PtdIns(4,5)P2 incorporation in membrane vesicles
from squid nerve (A) and bovine heart (B) on the
Na+ gradient-dependent 45Ca uptake. Vesicles
were preincubated for 5 min at 0°C and for an additional minute at
20°C with PtdIns(4,5)P2 liposome (0.25 mg/mg
vesicles) protein vesicle. 45Ca uptake was measured for
15 s at 20°C. Notice 1) the usual MgATP stimulation
observed in both preparations and 2) the complete lack of
effect of PtdIns(4,5)P2 in the squid
Na+/Ca2+ exchange fluxes in contrast to the
marked stimulation in the bovine heart. All values are means ± SE
of quadruplicate determinations. See METHODS and
RESULTS for details.
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Fig. 9.
Lack of effect of PtdIns(4,5)P2
antibody [Ab-PtdIns(4,5)P2] and PtdIns-PLC on
MgATP-activated Na+/Ca2+ exchange in dialyzed
squid axons. , Ca2+ efflux in the presence
of external Na+; , Ca2+ efflux
in the absence of external Na+. A: intracellular
injection of Ab-PtdIns(4,5)P2 during internal
dialysis after addition of ATP; B: intracellular injection
of Ab-PtdIns(4,5)P2 during internal dialysis
before addition of ATP; C: intracellular injection of
PtdIns-PLC (final concentration in the axon of ~200 units) before ATP
addition. Notice the lack of effect of these compounds on the ATP
stimulation of the Nao+-dependent Ca2+
efflux.
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Fig. 10.
Effect of alkaline phosphatase on ATP stimulation of
Na+/Ca2+ exchange in bovine heart sarcolemmal
and squid optic nerve vesicles. A: squid nerve membranes.
Uptake of 45Ca was estimated without or with 50 and 200 U/ml of alkaline phosphatase. B: bovine heart sarcolemmal
membranes. Uptake of 45Ca was estimated without or with 200 U/ml of alkaline phosphatase. Before starting 45Ca uptake,
concentrated membrane vesicles were incubated for 2 min at 20°C in
the absence of Na+ gradient with no Ca2+ but
with Mg2+ and ATP, with and without alkaline phosphatase.
In the squid vesicles, all experiments were done in the presence of
SCRP. Values are means ± SE of triplicate determinations. See
METHODS for details.
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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 |
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 · s
1), 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).
From the reported time constant for MgATP washout in dialyzed squid
axons (14), fast and complete removal of intracellular Mgi2+ can only be achieved by the use of
Mg2+-chelating agents. We have taken advantage of the
Mg2+-binding capacity of AMP-PCP, a nonhydrolyzable ATP
analog that does not interact with the Na+/Ca2+
exchanger (12), to rapidly and entirely deplete
Mgi2+. Figure 1C shows that the
simultaneous withdrawal of Mgi2+ and ATP
together with the addition of 5 mM AMP-PCP keeps the ATP-stimulated
exchanger fully active. That this is indeed the forward mode of
Na+/Ca2+ exchange is demonstrated by its total
sensitivity to external Na+. An important result shown in
Fig. 1D is that the exchangers that remained upregulated
after the removal of Mg2+ can be fully deactivated by the
readdition of Mgi2+. This deactivation is not a
result of competition between Mgi2+ and
intracellular Ca2+ at intracellular sites since removal of
Mgi2+, as shown at the end of the experiment,
produces only a small increment in Ca2+ efflux. However,
that increment amounts to double the ATP-independent Ca2+
efflux, which is to be expected from the release of the reported noncompetitive partial inhibition of the exchanger (11).
The complete reversibility of the Mgi2+
deactivation of the ATP upregulation of the exchanger is shown in Fig.
2 where, after the
Mgi2+-induced deactivation at constant [ATP],
the upregulated fluxes return to the original levels when the initial
low [Mg2+]i is restored.
To evaluate the Mgi2+ dependence of the ATP
stimulation of Na+/Ca2+ exchange, we performed
a series of experiments in which the magnitude of the ATP stimulation
was followed at constant [ATP] (4 mM), whereas the concentration of
Mgi2+ was varied between 0.5 and 10 mM. As shown
in Fig. 3, there is a biphasic response:
stimulation up to ~1 mM Mgi2+ followed by a
progressive decline which at 10 mM Mgi2+ reaches
the values of the unstimulated Nao+-dependent
Ca2+ efflux.
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.
 |
DISCUSSION |
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.
 |
ACKNOWLEDGEMENTS |
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).
 |
FOOTNOTES |
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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