From the
Chinese hamster ovary cells expressing the bovine cardiac Na/Ca
exchanger were treated with ouabain to increase
[Na
The Na/Ca exchanger is a major Ca
The
mechanism by which ATP regulates exchange activity is unknown. DiPolo
and Beaugé
(8, 12, 13) have suggested
that in squid giant axons, the exchanger is regulated by a
phosphorylation process, although the kinases/phosphatases involved
have not been identified. Studies with cardiac sarcolemmal patches are
not consistent with the phosphorylation hypothesis, however, and
suggest that exchange activity is indirectly regulated by
aminophospholipid translocase activity
(2) . This ATP-dependent
enzyme maintains a high density of phosphatidylserine at the cytosolic
surface of the bilayer and it is thought that the asymmetric
distribution of this lipid is essential for maximal exchange activity
(2) .
The cardiac Na/Ca exchanger has been cloned by
Philipson and his colleagues
(14) . The exchanger is a protein
of 938 amino acids with 11 putative transmembrane regions and a large
hydrophilic domain between the fifth and sixth transmembrane segments.
Deletion of 440 out of the 520 amino acids of the hydrophilic domain
does not alter the kinetics of the exchange process, but eliminates
secondary activation by cytosolic Ca
We also
examined the effects of ATP depletion on the Ca
In contrast to
the behavior of wild-type cells, 10 min of ATP depletion did not affect
the concentration profile for inhibition of
When
[Na
In a second series of experiments, we
alkalinized ATP-depleted cells with 20 m
M NH
We also examined the effects of
various agents which alter protein phosphorylation. No effects on
exchange activity were observed after 30 min exposure of CK1.4 cells to
a nonspecific protein kinase inhibitor (staurosporine, 1
µ
M), or to calphostin C (1 µ
M), a relatively
specific inhibitor of protein kinase C (data not shown). Moreover, the
protein phosphatase inhibitors okadaic acid (1 µ
M) and
calyculin (50 n
M) had no affect on the decline in exchange
activity, or the alterations in
Na
Additional experiments were carried out with cytochalasin D at a
Ca
Previous reports have established that Na/Ca exchange
activity is regulated by ATP in various cell types
(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13) .
The results presented here indicate that ATP depletion reduces the rate
of [Na
The rates of
We attempted to minimize the effects of organellar
Ca
A second factor that must be
considered is an indirect modulation of exchange activity through
effects of ATP depletion on pH
The results with cytochalasin
D suggest that the displacement of the Na
An alternate interpretation is that the
cytoskeleton maintains the wild-type exchanger in close proximity to a
Ca
Another effect of ATP depletion on exchange activity is apparent in
the fura-2 studies, in which ATP-depleted cells show only a slight
decline in [Ca
There are two general hypotheses as to the
mechanism by which ATP regulates exchange activity. Studies with
internally dialyzed squid axons suggest that exchange activity is
regulated by a phosphorylation mechanism
(8, 12, 13) . Our results with the CK1.4 cells
do not support the phosphorylation hypothesis. A phosphorylated form of
the exchanger was not detected by immunoprecipitation of the exchanger
from either transfected CHO cells or COS cells. Moreover, inhibitors of
protein kinases and phosphatases did not modify exchange activity or
alter the response of cells to ATP depletion. Nevertheless, we cannot
rule out this possibility because it is conceivable that a
phosphorylated form of the exchanger escaped detection by our
procedures and/or that the inhibitors used did not act against the
kinases/phosphatases involved. The other mechanism that has been
proposed for ATP-dependent regulation invokes an ATP-dependent
aminophospholipid translocase to maintain an asymmetric distribution of
negatively charged phospholipids between the cytoplasmic and
extracellular membrane surfaces
(2) . We did not test the
phospholipid translocase hypothesis in this study. However, we note
that the asymmetric distribution of acidic phospholipids has been
suggested to involve interactions with the cytoskeleton as well as the
aminophospholipid translocase (reviewed in Ref. 34).
In summary, our
results describe several new characteristics of ATP-dependent
regulation of Na/Ca exchange activity. First, in agreement with
previous reports
(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13) ,
ATP depletion inhibits both Ca
]
and stimulate
Ca
influx by Na/Ca exchange. Depletion of cellular
ATP inhibited
Ca uptake by 40% or more and reduced the
half-maximal Na
concentration for inhibition of
Ca uptake from 90 to 55 m
M. ATP depletion also
reduced the rate of rise in
[Ca
]
when
[Na
]
was reduced and
inhibited the decline in
[Ca
]
when high
[Na
]
was restored. The
effects of ATP depletion were either absent or reduced in cells
expressing a mutant exchanger missing most of the cytosolic hydrophilic
domain. We were unable to detect a phosphorylated form of the exchanger
in immunoprecipitates from
P-labeled cells. ATP depletion
caused a breakdown in the actin cytoskeleton of the cells. Treatment of
the cells with cytochalasin D mimicked the effects of ATP depletion on
the [Na
] inhibition profile for
Ca uptake. Thus, ATP depletion inhibits both the
Ca
influx and Ca
efflux modes of
Na/Ca exchange, and may alter the competitive interactions of
extracellular Na
and Ca
with the
transporter. The latter effect appears to be related to changes in the
actin cytoskeleton.
efflux
mechanism in cardiac myocytes and several other cell types. Studies
conducted with cardiac sarcolemmal membrane patches
(1, 2, 3, 4) and squid giant axons
(5) have defined two different regulatory processes for the
exchanger. These appear to involve two distinct inactive states of the
carrier, the first promoted by the presence of cytosolic Na
(Na
-dependent inactivation) and the second
promoted by the absence of cytosolic Ca
(secondary
Ca
activation). The presence of cytosolic ATP
attenuates Na
-dependent inactivation and increases the
affinity of the secondary activation site for cytosolic
Ca
. ATP also alters the kinetics of the exchange
process in a variety of cell types. In dialyzed squid giant axons, the
presence of ATP markedly reduces the K
for cytosolic Ca
and for extracellular
Na
(6, 7, 8) . In adult heart
myocytes
(9, 10) and cultured vascular smooth muscle
cells
(11) , ATP depletion using metabolic inhibitors reduces
Ca
entry via Na/Ca exchange by more than 80%.
(15) . In
this report, we use transfected Chinese hamster ovary cells permanently
expressing either the native bovine cardiac exchanger (CK1.4 cells;
Ref. 16) or the deletion mutant described above to examine the effects
of cellular ATP depletion on Na/Ca exchange activity. ATP depletion
strongly inhibits the Ca
efflux mode of Na/Ca
exchange and potentiates the inhibitory effect of extracellular
Na
on Ca
influx. These effects are
absent or reduced in cells expressing the deletion mutant. This
suggests that ATP-dependent regulation, like secondary activation by
cytosolic Ca
, is mediated by regions within the
hydrophilic domain of the exchanger. Portions of these results were
presented previously in abstract form
(17, 18) .
Cells
CK1.4 cells were prepared by transfection
of dhfrChinese hamster ovary cells with a
mammalian expression vector (pcDNA-NEO; Invitrogen) containing a cDNA
insert coding for the bovine cardiac Na/Ca exchanger
(16) .
CK138 cells were prepared similarly except that the nucleotides coding
for amino acids 241-681 were deleted from the insert by cleavage
with BclI at positions 1079 and 2399 and religation; the
BclI site at position 1079 was introduced in clone p17
(19) by site-directed mutagenesis using the method of Kunkel
(20) , provided commercially as the Bio-Rad Muta Gene 2 kit.
Expression of the expected mutation in CK138 cells was verified by RNA
extraction and reverse transcriptase-polymerase chain reaction using a
commercial kit (Invitrogen cDNA Cycle and TA Cloning kits). The
properties of the canine exchanger with an identical deletion were
previously described
(15) . The cells were grown in
Iscove's modified Dulbecco's medium containing 10% fetal
calf serum and G418
(16) . In some of the immunoprecipitation
experiments, COS cells transiently transfected with the cardiac Na/Ca
exchanger were used as described
(19) .
The cells were
grown to confluence in 24-well plastic dishes coated with fibronectin;
the plates were preincubated overnight with culture medium containing
17 µg/ml fibronectin prior to adding the cells. The fibronectin
coating was required to prevent ATP-depleted cells from detaching from
the plate during the transport assay. The medium was removed and the
cells were preincubated for 30 min at 37 °C in nominally
CaCa Uptake Assay
-free PSS
(
)
containing (in
m
M) 140 NaCl, 5 KCl, 1 MgCl
, and 20 m
M
Mops, buffered to pH 7.4 (37 °C) with Tris (Mops/Tris); 0.4
m
M ouabain was also added to inhibit the Na,K-ATPase and load
the cells internally with Na
( cf. Ref. 16)
and 10 m
M glucose was either included or omitted as specified
in individual experiments. To assay
Ca uptake, the
preincubation medium was removed by aspiration and replaced with 200
µl of assay medium (see below) containing 10 µCi/ml
CaCl
. After the desired interval,
Ca uptake was terminated by adding 1 ml of quenching
medium (room temperature) containing (in m
M) 100
MgCl
, 10 LaCl
, and 10 Mops/Tris, pH 7.4. The
quenching medium was immediately aspirated and the wells were washed 3
additional times by the repeated addition and aspiration of quenching
medium (1 ml). The quenching and washing procedure was completed within
7-8 s. The normal assay medium contained (in m
M) 5 KCl,
1 CaCl
, 20 Mops/Tris (pH 7.4) and the amounts of NaCl and
NMDG (adjusted to pH 7.4 with HCl) described in individual experiments;
10 m
M glucose was included where specified. For assays lasting
longer than 1 min, 0.4 m
M ouabain was also included in the
assay medium. Occasional variations in this protocol are described in
the text. Data are presented as the mean values (± S.E.) for the
number of individual experiments given in the figure legends.
ATP Depletion
CK1.4 cells were preincubated in
Ca-free PSS containing 0.4 m
M ouabain for 30
min in either the presence (control) or absence of 10 m
M
glucose. The medium was replaced with fresh medium containing
oligomycin (2.5 µg/ml) or oligomycin plus 2 µ
M
rotenone and the incubation continued for an additional 10-30
min. Cellular ATP levels were assayed by the firefly luciferase method
(Calbiochem kit).
Fura-2 Measurements
Cells were grown to confluence
on coverslips and then preincubated for 30 min in PSS containing 1
m
M CaCl, 5 µ
M fura-2-AM, 0.125
m
M sulfinpyrazone (to reduce transport of fura-2 out of the
cell; Ref. 16), with or without 0.4 m
M ouabain as indicated.
For ATP-depleted cells, glucose was omitted from the medium and 2.5
µg/ml oligomycin was included; for control cells, oligomycin was
omitted and 10 m
M glucose was included. The coverslips were
mounted in a quartz cuvette and superfused with PSS containing 1
m
M CaCl
with or without ouabain, glucose, and/or
oligomycin as indicated. To initiate Ca
influx by
Na/Ca exchange, the perfusion medium was changed to one with the same
composition except that [Na
] was reduced to
40 m
M (K substitution). Fluorescence was monitored at 505 nm
on a SPEX Fluorolog CM3 spectrofluorimeter at excitation wavelengths of
340 and 380 nm. All fluorescence values were corrected for
autofluorescence. Data are presented as the ratio of fluorescence at
340/380 nm excitation.
BCECF Measurements
Cells were grown to confluence
on coverslips and then preincubated for 30 min in PSS containing 1
m
M CaCland 5 µ
M BCECF-AM. Control
cells and ATP-depleted cells were treated as described under
``Fura-2 Measurements.'' Coverslips were washed three times
with PSS, mounted in the quartz cuvette, and superfused with PSS
containing 1 m
M CaCl
and glucose or oligomycin as
indicated. Changes in pH
were produced by
including 20 m
M NH
Cl in both the low
Na
-PSS and the 140 m
M Na
-PSS
added subsequently. Fluorescence was monitored at 530 nm with
excitation wavelengths of 440 and 503 nm. Cytosolic pH was calculated
from the 503/440 ratio and a calibration curve
(21) established
in separate coverslips subjected to 5 µg/ml nigericin in
potassium-substituted (145 m
M) PSS (pH
6.4-7.8).
Immunoprecipitation
CK1.4 cells or transfected COS
cells in a 60-mm plate were incubated 1 h (37 °C) in 1.5 ml of
phosphate-free Dulbecco's modified Eagle's medium; the
medium was replaced with 1.5 ml of fresh medium and
[P]orthophosphate (1 mCi/ml, 8-9
Ci/µmol) and incubated for 4 h at 37 °C. The cells were then
washed in cold buffer containing (in m
M) 150 NaCl, 20 sodium
phosphate (pH 7.0), 50 NaF, 10 sodium pyrophosphate, 5 EDTA, 5 EGTA, 1
each ortho- and meta-vanadate, 0.2
phenylmethylsulfonyl fluoride, 1 benzamidine, 0.1% bovine serum
albumin, and 5 µg/ml each aprotinin, leupeptin, and pepstatin. The
cells were lysed with 2% Triton X-100 and 0.5% sodium deoxycholate in
the above buffer and sonicated on ice (3
15 s). After
centrifugation for 15 min in an Eppendorf centrifuge, the supernatant
was precleared with 1 µl of preimmune serum + 50 µl of 50%
protein A-agarose previously equilibrated in lysis buffer. After 1 h at
4 °C, the mixture was centrifuged; 3 µl of immune serum was
added to the supernatant and the mixture was incubated 4 h or overnight
at 4 °C. After centrifugation, 50 µl of 50% protein A-agarose
was added to the supernatant and the mixture was incubated 1 h (4
°C) and centrifuged. The protein A-agarose was washed extensively
with ice-cold lysis buffer, resuspended in 30 µl of sample buffer,
and applied to one lane of a gel for SDS-polyacrylamide gel
electrophoresis. Transfection of COS cells and
S
labeling/immunoprecipitation were carried out as described previously
(19) . The antibody used for immunoprecipitation was prepared
against a fusion protein containing the NH
-terminal portion
of the exchanger
(19) .
Cytochalasin D Treatment
Cells were incubated in
Ca-free PSS plus 0.4 m
M ouabain with or
without 1 µ
M cytochalasin D and then assayed for
Ca uptake as described above. In some experiments, the
cells were preincubated for an additional 30 min with 1 µ
M
cytochalasin D added directly to the culture media prior to the 30 min
preincubation period; no difference was observed between the effects of
the different protocols.
Fluorescein Isothiocyanate-Phalloidin
Staining
Cells were grown to 20-40% confluence on Aclar
plastic coverslips (Proplastics) and were subjected to cytochalasin D
treatment or ATP depletion. The cells were then washed 3 times with
phosphate-buffered saline (PBS) followed by fixation in 4%
paraformaldehyde solution in PBS for 10 min. Coverslips were washed 3
more times with PBS and extracted with acetone at -20 °C for
4 min. Samples were air-dried and 1.4 units of fluorescein
isothiocyanate-labeled phalloidin (Molecular Probes) previously
dissolved in 180 µl of PBS were placed on each coverslip for 30 min
at room temperature. Cells were then washed 3 times with PBS and the
coverslips were mounted on slides using the SlowFade antifade reagent
in glycerol/PBS (Molecular Probes).
Materials
Rotenone and cytochalasin D were
obtained from Sigma. The oligomycin was a mixture of oligomycins A, B,
and C (65% A) and was obtained from Sigma (catalog No. O4876). Fura-2,
BCECF, and SlowFade were obtained from Molecular Probes.
ATP Depletion and
To determine the effects of ATP depletion on Na/Ca
exchange activity, CK1.4 cells were preincubated for 30 min in the
absence of glucose and then treated with a combination of mitochondrial
inhibitors: rotenone (2 µ
M) and oligomycin (2.5
µg/ml). This treatment reduced cellular ATP levels by 95% within 10
min of adding the inhibitors (data not shown). The inhibitors were much
less effective in the presence of 10 m
M glucose; under these
conditions, ATP levels declined by 30% within the first 2 min of
incubation and remained at this level for the duration of the
incubation period. The effects of these inhibitors (with and without
glucose present) on Ca Uptake in CK1.4
Cells
Ca uptake are shown in Fig. 1. In
these experiments, the cells were loaded internally with Na
by including 0.4 m
M ouabain in the preincubation and
assay media;
Ca uptake was assayed in either 150
m
M NMDG ( circles) or 150 m
M NaCl
( squares). Under these conditions,
Ca uptake
reflects the activity of the Na/Ca exchanger operating in the
``reverse,'' or Ca
influx, mode
(16) . As shown, 10 min of ATP depletion ( open symbols,
Fig. 1
) reduced the rate of
Ca uptake in the NMDG
medium by approximately 30% ( left panel); a more pronounced
inhibition was observed after 30 min of ATP depletion ( right
panel). Despite the reduced initial
Ca uptake, the
total
Ca accumulation during the later portions of the
time course was higher in ATP-depleted than in control cells. In the
presence of 150 m
M extracellular Na
, total
Ca accumulation was reduced compared to the NMDG medium
and ATP depletion reduced uptake still further. The inhibition of
Ca uptake by extracellular Na
is
characteristic of Na/Ca exchange activity and reflects the competition
between Na
and Ca
for transport
sites on the exchange carrier
(22, 23) .
Figure 1:
Effect of ATP depletion on
Ca uptake by CK1.4 cells. Left panel,
ouabain-treated CK1.4 cells were depleted of ATP for 10 min using
oligomycin and rotenone as described under ``Experimental
Procedures.'' For the control cells, 10 m
M glucose was
present during the exposure to oligomycin and rotenone.
Ca
uptake ( n = 3) was assayed in either 150 m
M
NMDG ( circles) or 150 m
M NaCl ( squares)
containing 1 m
M
CaCl
, 5 m
M
KCl, and 20 m
M Mops buffered to pH 7.4 (37 °C) with Tris.
The mitochondrial inhibitors (±10 m
M glucose) were
included in the assay media. Right panel, CK1.4 cells were
depleted of ATP for 30 min as described under ``Experimental
Procedures.''
Ca was assayed as described above
( n = 3).
To examine
the effects of extracellular Nain more detail, we
measured the rates of
Ca uptake (15 s) at various
Na
concentrations after 10 min of ATP depletion. The
results, shown in Fig. 2 A, indicate that Na
inhibited Ca
uptake more effectively in
ATP-depleted cells than in control cells. The data in panel A are plotted as the percentage of the rate seen in the absence of
Na
and represent the mean (± S.E.) of
10-11 independent experiments. The IC
for inhibition
by Na
was 90 m
M in
ATP-replete cells versus 57 m
M for ATP-depleted
cells; the Hill coefficients for the Na
concentration profiles were similar for the ATP-replete (2.4) and
ATP-depleted (2.0) cells. The shift in the
Na
inhibition curve was not
simply a consequence of the lower rate of
Ca uptake in the
ATP-depleted cells.
Ca uptake could be inhibited to a
similar extent by treating the cells with 1 m
M N-ethylmaleimide, or by including 1 m
M CaCl
in the preincubation media
(24) , without producing a
shift in the Na
inhibition curve
(data not shown). The effects of ATP depletion were fully reversed
within 10 min of adding 10 m
M glucose to the ATP-depleted
cells; cellular ATP levels were also restored under these conditions,
although only to 40% of initial values (data not shown).
concentration profile for
Ca uptake. As shown in
Fig. 2 B, control cells exhibited a typical Michaelis-Menten
type of behavior, with a V
of 7.6 nmol/mg
protein/15 s and an apparent K
of 0.19
m
M, as determined from a Lineweaver-Burk plot (not shown). For
the ATP-depleted cells, the rates of
Ca uptake appeared to
saturate quite sharply at concentrations above 0.4 m
M
Ca
with a half-maximal
[Ca
] of approximately 0.1 m
M. It
is very unlikely that these rates of
Ca uptake represent
true initial rates of Na/Ca exchange (see ``Discussion'') and
so it is difficult to interpret these data in strict kinetic terms. It
is important to note, however, that ATP depletion was not associated
with a shift in the Ca
concentration profile to
higher concentrations, and so the increased effectiveness of
Na
as an inhibitor of
Ca uptake does not
appear to be due to a reduced affinity of the exchanger for
Ca
.
ATP Depletion and
A large (520 amino acid) hydrophilic domain lies
between the fifth and sixth putative transmembrane regions of the Na/Ca
exchange protein and appears to reside on the cytoplasmic side of the
membrane
(14) . The results of Matsuoka et al. (15) indicated that 440 of the 520 amino acids in the
hydrophilic domain could be deleted without obvious changes in the
kinetic characteristics of exchange activity. The deletion altered the
regulatory behavior of the exchanger, however, since secondary
activation of NaCa Uptake in CK138
Cells
-dependent
Ca
influx by
[Ca
]
was not observed
(15) . To determine whether ATP-dependent regulation of exchange
activity was also altered in this mutant, we prepared CHO cells that
express the deleted form of the exchanger (see ``Experimental
Procedures''), which we designate CK138 cells.
Ca uptake by
Na
(Fig. 3 A). The
characteristics of the mutant cells were similar to those of
ATP-depleted wild-type cells (IC
= 60 m
M).
The Ca
concentration profile (Fig. 3 B)
for the CK138 cells was also similar in ATP-replete versus ATP-depleted CK138 cells; half-maximal Ca
uptake
occurred at
0.1 m
M Ca
under both
conditions, again similar to the Ca
concentration
dependence of ATP-depleted wild-type cells (compare
Fig. 2B). Note that the rates of
Ca uptake
by the CK138 cells were uniformly lower than for the CK1.4 cells and
tended to be less affected by ATP depletion (compare Fig. 2 B and Fig. 3 B; see legends for Figs. 2 and 3 for
additional data). [Ca]
Measurements-With
Ca uptake measurements, it is difficult to distinguish an
effect on the exchanger itself from secondary effects due to loss of
Ca
sequestration by intracellular organelles.
Therefore, we examined the effects of ATP-depletion on
exchanger-mediated changes of
[Ca
]
in fura-2-loaded
cells (Fig. 4); in these experiments, an increase in the ratio of
fluorescence with excitation at 340 versus 380 nm reflects an
increase in [Ca
]
. ATP
depletion was carried out using CK1.4 cells ( upper panel) or
CK138 cells ( lower panel) grown on glass coverslips; the cells
were preincubated in glucose-free PSS for 30 min and then loaded with
fura-2 for an additional 30 min in glucose-free PSS containing 2.5
µg/ml oligomycin with or without 0.4 m
M ouabain as
indicated. For the control cells, 10 m
M glucose was present
throughout and oligomycin was omitted. In the presence of glucose, a
reduction in extracellular [Na
] to 40
m
M (potassium substitution) produced a rise in in the 340/380
ratio which was faster and more extensive in ouabain-treated ( trace
3) than in untreated ( trace 4) cells. Restoration of 140
m
M Na
produced a rapid
decline in the 340/380 ratio in both batches of cells. The results with
the CK1.4 cells ( upper panel) are similar to those reported
previously
(16) ; the CK138 cells ( lower panel) behaved
nearly identically to the CK1.4 cells.
Figure 3:
Effect of
ATP depletion on Na-dependent inhibition ( A)
and [Ca
] dependence ( B) of
Ca uptake in CK138 cells. Ouabain-treated CK138 cells were
incubated with oligomycin and rotenone for 10 min, with ( closed
circles) or without ( open circles) 10 m
M
glucose, as described under ``Experimental Procedures.'' For
the data in panel A, the rate of
Ca uptake in the
NMDG media were 2.5 ± 0.2 and 2.1 ± 0.2 nmol/mg protein/s
with and without ATP, respectively. For panel A, n = 5, and for panel B, n =
3.
Figure 2:
[Na] and
[Ca
] dependence of
Ca uptake
in ATP-depleted versus control CK1.4 cells. Left
panel, inhibition by Na
of
Ca
uptake. Ouabain-treated CK1.4 cells were incubated with oligomycin and
rotenone for 10 min in the presence ( closed circles) or
absence ( open circles) of glucose as described under
``Experimental Procedures.''
Ca uptake (15 s)
was assayed as described under ``Experimental Procedures'' in
mixtures of 150 m
M NMDG and 150 m
M NaCl to generate
the Na
concentrations shown ( n =
10-11). The data are presented as the % of Ca
uptake observed in the Na
-free medium. The
portion of Ca
uptake occurring independently of
exchange activity under these conditions cannot be reliably determined
and so no correction for this has been applied. The rates of
Ca uptake in the NMDG media were 7.2 ± 0.5 and 4.2
± 0.4 nmol/mg protein/s with and without ATP, respectively.
Right panel, [Ca
] dependence of
Na/Ca exchange in ATP-depleted versus control cells.
Ouabain-treated CK1.4 cells were treated with oligomycin and rotenone
for 10 min, with ( closed circles) or without ( open
circles) 10 m
M glucose present as described under
``Experimental Procedures.'' The cells were assayed for
Ca uptake (15 s) in 150 m
M NMDG, 5 m
M
KCl, 20 m
M Mops/Tris (pH 7.4) at the indicated concentrations
of
CaCl
( n =
6).
For both the wild-type and
mutant cells, ATP depletion ( traces 1 and 2) slowed
the rise in the 340/380 ratio when
[Na]
was reduced to 40
m
M. However, the 340/380 ratio did not reach a plateau, as
observed in control cells, but continued to rise throughout the
duration of the period in low
[Na
]
. This probably
reflects the loss of Ca
pump activity of both
intracellular organelles and the plasma membrane under the ATP-depleted
conditions. Another difference between ATP-depleted and control cells
was that ouabain treatment had no effect on the response of the
ATP-depleted cells. Presumably, ATP depletion itself inhibits
Na,K-ATPase activity sufficiently that any additional effects of
ouabain are negligible.
]
was restored from
40 to 140 m
M in the ATP-depleted CK1.4 cells, only a slight
decline in the 340/380 ratio was evident. With ATP-depleted CK138
cells, a more pronounced reduction in the 340/380 ratio was observed,
but the decline was still considerably slower than observed in the
presence of glucose. A large part of this effect probably reflects the
loss of intracellular Ca
sequestration and/or plasma
membrane Ca-ATPase activity due to lack of ATP. However, since the
decline in [Ca
]
does
not appear to be as severely compromised in the CK138 cells, the
results suggest that the exchanger continues to catalyze Ca
efflux in the CK138 cells but is inhibited by ATP depletion in
the CK1.4 cells.
ATP Depletion and pH
Since
acidic conditions inhibit Na/Ca exchange
(25) , we examined the
effects of ATP depletion on pHusing BCECF
fluorescence as an intracellular pH indicator. After 30 min of ATP
depletion with 2.5 µg/ml oligomycin under glucose-free conditions,
pH
declined from 7.46 ± 0.05 to 6.85
± 0.1 (mean ± S.E.; n = 4). To determine
whether the effects of ATP depletion could be explained by the
inhibitory effects of cytosolic acidification, we assayed
Ca uptake at various
[Na
]
during cytosolic
acidification induced by the NH
Cl prepulse technique
(26) . Cells that were preloaded with 20 m
M
NH
Cl and assayed in NH
-free media exhibited a
reduction in pH
of 0.3 pH units; this treatment
reduced the rate of
Ca uptake by 20% but had no effect on
the [Na
]
inhibition
profile (data not shown).
Cl
during Na/Ca exchange; the results are shown in Fig. 5. These
experiments are similar to those depicted in Fig. 4, except that
all the traces are for ouabain-treated cells and for some of the traces
(depicted in bold), 20 m
M NH
Cl was
included in the 40 m
M Na
medium and in the
140 m
M Na
medium added subsequently. In
ATP-replete cells, the NH
Cl treatment increased
pH
to a peak value of 7.82 ± 0.02 ( n = 3), followed by a gradual decline in pH
by less than 0.1 unit over the subsequent 8 min (data not shown).
In ATP-depleted cells, the NH
Cl treatment raised
pH
to a stable value of 7.32 ± 0.09 ( n = 4), close to that observed in ATP-replete cells in the
absence of NH
Cl. The changes in
[Na
]
during these
experiments did not evoke any detectable changes in
pH
.
Figure 4:
Na/Ca
exchange activity and [Ca] in ATP-depleted
and control CK1.4 and CK138 cells. Upper panel, CK1.4 cells
were grown on coverslips, and loaded with fura-2 in the presence
( traces 1 and 2) or absence ( traces 3 and
4) of 2.5 µg/ml oligomycin, with ( traces 1 and
3) or without ( traces 2 and 4) 0.4
m
M ouabain, as described under ``Experimental
Procedures.'' The data depict the ratio of the fluorescence
obtained by excitation at 340 and 380 nm as described under
``Experimental Procedures.'' At the times indicated by the
lower bar, the perfusion medium was changed from 140 m
M Na
-PSS to PSS containing 40 m
M Na/100
m
M KCl and back to 140 m
M Na
-PSS.
Each trace is the mean of eight to nine individual experiments.
Lower panel, the procedure was identical to that described for
the upper panel except that CK138 cells were used. Traces are
the mean values for three to five
experiments.
As shown in Fig. 5( upper
panel), cytosolic alkalinization ( traces 1 and
4) accelerated the increase in the 340/380 ratio upon
[Na]
reduction in both
ATP-depleted and ATP-replete cells, but had little effect on the rate
of decline in [Ca
]
when [Na
]
was
restored to 140 m
M; after an initial small decline,
[Ca
]
remained elevated
in ATP-depleted cells after restoration of 140 m
M
Na
, whether or not
NH
Cl was present. A similar pattern of results was observed
in the case of CK138 cells expressing the deleted form of the exchanger
( lower panel, Fig. 5); again, a less pronounced effect
of ATP depletion on Na
-dependent
Ca
efflux was observed in the CK138 cells compared to
the CK1.4 cells. Thus, cytosolic alkalinization accelerates the
increase in [Ca
]
resulting from exchange-mediated Ca
entry in
ATP-depleted cells but does not overcome the inhibitory effects of ATP
depletion on Ca
efflux.
Figure 5:
Effect of cytosolic alkalinization on
Na/Ca exchange and [Ca] in ATP-depleted and
control CK1.4 cells ( upper panel) and CK138 cells ( lower
panel). The procedure was identical to that described in Fig. 4
except that all cells were ouabain-treated and that, for traces 1 and 4 (indicated in bold), 20 m
M
NH
Cl was included in the 40 m
M Na
perfusion medium and in the 140 m
M Na
medium added subsequently.
Although the results in
Fig. 5
seem to imply that the Cainflux mode of
the exchanger recovered fully from the effects of ATP depletion upon
cytosolic alkalinization, this is not necessarily the case. The rise in
[Ca
]
during
exchange-mediated Ca
entry is likely to be buffered
by the Ca
sequestering activities of intracellular
organelles under ATP-replete conditions, but not, or much less so, in
ATP-depleted cells. Thus, the loss of intracellular Ca
sequestration probably understates the actual inhibition of
exchange activity in the fura-2 experiments, and may exaggerate the
loss of exchange activity in the
Ca experiments. In this
regard, it should be noted that the presence of NH
Cl during
Ca uptake assays after 30 min of ATP depletion did not
restore the rate of
Ca influx to the levels seen with
ATP-replete cells (data not shown).
Phosphorylation of the Exchanger?
The hydrophilic
cytoplasmic domain of the exchanger contains several possible sites for
phosphorylation by different protein kinases. Indeed, a bacterially
expressed protein containing the entire hydrophilic domain fused to the
Escherichia coli maltose binding protein was a substrate for
in vitro phosphorylation by either protein kinase A or casein
kinase II; the maltose binding protein itself was not phosphorylated
under these conditions (data not shown). To determine whether the
exchanger expressed in CK1.4 cells is phosphorylated, we incubated the
cells with [P]phosphate to label the
intracellular nucleotide pool. As shown in Fig. 6, we were unable to
detect labeled exchanger upon immunoprecipitation of the exchanger from
P-labeled CK1.4 cells ( left panel) although
several
P-labeled contaminating proteins could be detected
and
S-labeled exchanger was easily observed ( right
panel). As described previously
(16) , the expressed
exchanger appeared as 2 bands at 150 and 120 kDa when
immunoprecipitated from CK1.4 cells. Additional experiments (data not
shown) were carried out with transfected COS cells, which express
higher levels of
S-labeled exchanger than the CHO cells;
nevertheless, we were still unable to detect
P labeling of
the exchanger in the transfected COS cells (data not shown). The
results suggest that the exchanger is not phosphorylated in either
CK1.4 cells or COS cells. However, we cannot completely rule out the
possibility that the exchanger is dephosphorylated during our sample
preparation procedures (despite the presence of fluoride,
pyrophosphate, and vanadate to inhibit phosphatases), and so this
conclusion must remain tentative.
-dependent inhibition, when
CK1.4 cells were depleted of ATP (data not shown). Finally, the
recovery of exchange activity when 10 m
M glucose was restored
to CK1.4 cells after 30 min of ATP-depletion was not affected by the
presence of staurosporine (1 µ
M) (data not shown).
Effects of ATP Depletion and Cytochalasin D on the Actin
Cytoskeleton
ATP depletion produces extensive alterations in the
cellular cytoskeleton in a number of cells
(27, 28, 29) . To assess the effects of ATP
depletion on the actin cytoskeleton in the CK1.4 cells, they were
stained with fluorescein isothiocyanate-labeled phalloidin, a
fluorescent agent that interacts with polymerized actin
(30) .
As shown in Fig. 7, panel A, the cells exhibited a network of
actin filaments extending throughout the cytoplasm; stress fibers and
local concentrations of fibrous actin in cortical regions of the cell
were clearly apparent. After 10 min of ATP depletion (see
``Experimental Procedures''), the filamentous network seemed
less sharply defined and punctate aggregates of actin appeared
throughout the cell (Fig. 7, panel B). After 30 min of
ATP depletion (Fig. 7, panel C), much of the actin had
aggregated to a diffuse region in the center of the cell. The effects
of 30 min of ATP depletion were fully reversible within 15 min of
replacing the medium with fresh PSS containing 10 m
M glucose
(data not shown). The results indicate that ATP depletion produced a
profound reorganization of actin microfilaments in these cells.
Figure 7:
Effect
of ATP depletion on actin cytoskeleton. CK1.4 cells were grown on
plastic coverslips and stained with fluorescein
isothiocyanate-palloidin as described under ``Experimental
Procedures.'' The treatments shown are: A, control cells
treated with oligomycin + rotenone in the presence of 10
m
M glucose; B, cells treated for 10 min with
oligomycin + rotenone in the absence of glucose (10 min ATP
depletion); C, cells after 30 min of ATP depletion; and
D, cells after 1 h exposure to 1 µ
M cytochalasin
D. Magnification is approximately 500. We thank Dr. John Connor, Roche
Institute of Molecular Biology, for use of the
microscope.
CK1.4 cells were treated for 1 h with 1 µ
M cytochalasin
D, an agent that binds to the barbed end of actin microfilaments and
alters their state of polymerization in intact cells
(30) . As
shown in Fig. 7, panel D, the microfilament network was
no longer visible in cytochalasin D-treated cells and most of the actin
appeared to reside in intensely stained aggregates that were dispersed
throughout the cell. Thus, cytochalasin D also promoted redistribution
of cellular actin, although the effects were not precisely identical to
those produced by ATP depletion.
Effect of Cytochalasin D Treatment on Na/Ca Exchange
Activity
The data in Fig. 8 A indicate that the effects
of ATP depletion on the Nainhibition profile could be mimicked by treating cells with 1
µ
M cytochalasin D. As shown, the cytochalasin D treatment
reduced the IC
for extracellular Na
from
85 m
M to approximately 50 m
M, similar to the effects
of ATP depletion in the CK1.4 cells (compare with
Fig. 2A). As shown in panel B of Fig. 8,
cytochalasin D treatment reduced the rate of
Ca uptake at
all Ca
concentrations tested. This experiment was
carried out with a different clone of transfected CHO cells
(C16-3 cells) instead of CK1.4 cells; comparable effects of
cytochalasin D were observed with the CK1.4 cells.
Figure 8:
Effect of cytochalasin D on Na/Ca exchange
in C16-3 cells. A, cells were preincubated with
( closed circles) or without ( open circles) 1
µ
M cytochalasin D in Ca-free PSS +
0.4 m
M ouabain as described under ``Experimental
Procedures.'' The cells were then assayed for the rate (15 s) of
Ca uptake at various Na
concentrations
( n = 5). B, cells were preincubated with 1
µ
M cytochalasin D (see ``Experimental
Procedures'') and assayed for the initial rate of
Ca
uptake at the indicated Ca
concentrations ( n = 5).
The effects of
cytochalasin D required a minimum of 30 min to develop, and were
manifest at concentrations as low as 50 n
M. Cytochalasin D did
not alter cellular ATP levels or pH(data not
shown). Moreover, agents that affect microtubule organization (10
µ
M nocodazole or colchicine) had no effect on Na/Ca
exchange activity in these cells although staining with
anti-
-tubulin antibodies revealed clear evidence of microtubular
disruption. The results suggest that the change in the concentration
profile for inhibition of Ca
uptake by
Na
is related to the breakdown of
the actin cytoskeleton. As in the case of ATP depletion, the effects of
cytochalasin D on the Na
inhibition profile were not observed in cells expressing the
deletion mutant of the Na/Ca exchanger (CK138 cells; data not shown).
concentration of 0.2 m
M instead of 1.0
m
M. As shown in Fig. 9, left panel, the
Na
-inhibition curve for CK1.4 cells was again
displaced to lower concentrations in cytochalasin D-treated cells. At
the lower Ca
concentration, half-maximal inhibition
occurred at approximately 40 m
M Na
in control
cells versus 27 m
M Na
in the
cytochalasin D-treated cells. These concentrations are about half of
those observed at 1 m
M Ca, reflecting the increased
effectiveness of Na
as a competitor of Ca
influx at the lower Ca
concentrations. As shown
in the right panel of Fig. 9, cytochalasin D had no
significant effect on the Na
-inhibition curve for the
CK138 cells, which exhibited half-maximal inhibition at approximately
28 m
M Na, i.e. the same as for the cytochalasin
D-treated CK1.4 cells. The results are consistent with those obtained
at higher Ca
concentrations (Fig. 8), and
illustrate the competitive nature of the inhibitory effect of
Na
on Ca
uptake.
Figure 9:
Effect of cytochalasin D on
Ca uptake in CK1.4 and CK138 cells at 0.2 m
M
Ca
. The experiment was conducted as described in the
legend to Fig. 8, except that that Ca
concentration
in the assay media was 0.2 m
M rather than 1.0 m
M.
Results are the means of five experiments for the CK1.4 cells and six
experiments for the CK138 cells. The rate of
Ca uptake (15
s) in the absence of Na
, with and without cytochalasin
D treatment, were 3.1 ± 0.1 and 4.2 ± 0.2 nmol/mg protein
for the CK1.4 cells, and 0.55 ± 0.04 and 0.75 ± 0.06
nmol/mg protein for the CK138 cells.
Surprisingly,
cytochalasin D treatment had no discernible effect on the changes in
[Ca]
induced by
Na
removal and restoration in
ouabain-treated CK1.4 cells (Fig. 10). Thus, despite the inhibition of
Ca uptake by cytochalasin D, the rate of rise in the
340/380 ratio upon [Na
]
reduction was essentially identical with and without cytochalasin
D. More importantly, disruption of the actin cytoskeleton did not
inhibit the decline in [Ca
]
upon restoration of Na
, as
was observed in ATP-depleted cells. We speculate that the
Ca uptake experiments are more sensitive to changes in
Ca
sequestration than the
[Ca
]
experiments, and
that changes in the actin cytoskeleton may alter the intracellular
distribution of Ca
sequestering organelles. This
might partially explain why exchange-mediated
Ca uptake is
inhibited by cytochalasin D but the increase in
[Ca
]
is not. The
decline in [Ca
]
when
high Na
is restored in the fura-2 experiments
(Fig. 10) reflects the combined effects of several different
processes, including decreased Ca
entry by Na/Ca
exchange, Ca
efflux by the plasma membrane Ca-ATPase
as well as the Na/Ca exchanger, and Ca
sequestration
by intracellular organelles. Thus, the absence of an effect does not
necessarily imply that
Na
-dependent Ca
efflux via the exchanger was unaffected by cytochalasin D, since
intracellular Ca
sequestration and Ca
efflux via the plasma membrane Ca
ATPase could
make any effects on Na
-dependent
Ca
efflux difficult to detect.
Figure 10:
Na/Ca exchange activity and
[Ca] in cytochalasin D-treated cells. CK1.4
cells were grown on coverslips and incubated for 30 min in PSS
containing 0.2 m
M Ca
, 0.4 m
M
ouabain, 5 µ
M fura-2-AM, and 0.25 m
M
sulfinpyrazone in the presence ( trace 2) or absence ( trace
1) of 1 µ
M cytochalasin D. Coverslips were placed in
the cuvette and perfused with 140 Na
-PSS + 0.2
m
M Ca
; at the times indicated by the lower
bar, the perfusion medium was changed to PSS containing 40 m
M
Na
+ 100 m
M KCl and then back to 140
m
M Na
-PSS. The traces shown are the average
of four individual traces. Exchange activity had increased markedly in
the CK1.4 cells used for these experiments in comparison to those used
in Fig. 4, probably due to the use of a different batch of fetal calf
serum in culturing the cells. In order to obtain results that were
comparable to those in Fig. 4, [Ca
] was
reduced from 1 m
M to 0.2 m
M for these
experiments.
]
-dependent
Ca
influx mediated by the exchanger and shifts the
concentration profile for inhibition of Ca
uptake to
lower Na
concentrations. The increased sensitivity of
the ``reverse mode'' of the exchanger to inhibition by
Na
was an unexpected finding
since studies with dialyzed squid axons have shown that the
concentration of Na
required to
activate Ca
efflux by Na/Ca exchange increases in the absence of ATP
(7, 8) . The increased
Na
inhibition, combined with the
reduced turnover of the exchanger, may be a protective mechanism to
prevent excessive Ca
entry via the exchanger during
periods of ATP depletion ( e.g. during cardiac ischemia). The
effectiveness of high [Na
]
in this regard can be inferred from the fura-2 results in Figs. 4
and 5. Despite the absence of cellular ATP and the loss of
Ca
efflux mechanisms,
[Ca
]
does not
substantially increase in ATP-depleted cells until
[Na
]
is reduced from
140 to 40 m
M.
Ca uptake in these
experiments reflect Na/Ca exchange activity but should not be taken as
true initial rate measurements. The accumulated Ca
represents a large increase in cellular Ca
content and the uptake process therefore involves intracellular
Ca
sequestration and other Ca
buffering processes as well as Na/Ca exchange. Thus, with an
intracellular water volume of
6 µl/mg protein
(16) ,
the amount of Ca
accumulated in 15 s by ATP-replete
cells (
7.5 nmol/mg protein; Fig. 2) corresponds to >1
mmol/liter cell water. The increase in
[Ca
]
under these
conditions is in the micromolar or submicromolar range
(16) ,
indicating that nearly all of the Ca
entering the
cell is bound by intracellular Ca
buffers or
sequestered by internal organelles. Therefore, it is important to
determine whether the effects of ATP depletion reflect changes in Na/Ca
exchange activity or changes in intracellular Ca
handling.
sequestration in the
Ca experiments
by blocking mitochondrial Ca
uptake with oligomycin
+ rotenone for both control (glucose present) and ATP-depleted
cells. In other experiments, which will be described in a separate
publication, we found that thapsigargin, an agent which inhibits
SERCA-type ATPases and blocks Ca
sequestration by the
endoplasmic reticulum, did not inhibit exchange-mediated
Ca uptake in the CK1.4 cells
(31) and did not
displace the Na
-inhibition curve.
Thus, the effects of ATP depletion on
Ca uptake cannot be
explained solely by the loss of Ca
sequestration by
mitochondria and/or the endoplasmic reticulum. Furthermore, cells
expressing the deletion mutant of the exchanger (CK138 cells) exhibited
an altered response to ATP depletion compared to the CK1.4 cells; this
indicates that some of the effects of ATP depletion on
Ca
uptake involve the exchanger itself, since effects on organellar
Ca
sequestration ought to be the same in both cell
types ( cf. below). Despite these considerations, we do not
claim that organellar Ca
sequestration has been
eliminated in our studies or that it plays no role in the observed
responses to ATP depletion. Indeed, the loss of intracellular
Ca
sequestration with ATP depletion probably
exaggerates the apparent decline in exchange activity in the
Ca experiments, while understating the actual loss of
exchange activity in the fura-2 experiments. Thus, after 30 min of ATP
depletion, the rate of
Ca uptake was profoundly inhibited
(Fig. 1 B), while the fura-2 results (Fig. 4)
suggested that the rise in
[Ca
]
due to exchange
activity was inhibited by only 50%.
. Thus,
pH
decreased by 0.6 units after 30 min of ATP
depletion and this could significantly inhibit Na/Ca exchange activity
(25) , alter intracellular Ca
buffering,
and/or inhibit organellar Ca
sequestration. The
experiments with NH
-induced acidification and
alkalinization (Fig. 5) support the idea that exchange activity
is inhibited by cellular acidification. However, the decline in
pH
seems less likely to have contributed to the
shift in the Na
inhibition curve
since no tendency in this direction was noticed in cells acidified by
the NH
-prepulse technique.
-inhibition
curve during ATP depletion is related to the breakdown of the actin
cytoskeleton. The precise mechanism(s) involved are uncertain. One
possibility is that the competition between external Na
and Ca
for transport sites on the exchanger is
modified by ATP depletion and/or cytoskeletal breakdown. This
modulation of transport kinetics could occur directly, through altered
contacts between the exchanger itself and elements of the cytoskeleton,
or indirectly through structural or ionic changes in the
exchanger's cytosolic environment. The former alternative is
compatible with a recent report that the exchanger binds to the
cytoskeletal protein ankyrin
(32) . The second alternative is
comprised of a large number of possibilities, including changes in the
local Na
or Ca
concentrations, and
alterations in secondary Ca
activation; these
changes, acting singly or in concert, could alter the kinetics of the
Na/Ca exchanger at the extracellular transport sites. Whatever the
precise mechanisms, the interactions which modify the
Na
inhibition profile are absent
in cells expressing the exchanger deletion mutant. This suggests that
these interactions are mediated by the exchanger's central
hydrophilic domain.
sequestering organelle, thereby promoting an
efficient coupling between Ca
influx and
Ca
sequestration. This spatial relationship would
presumably be lost during cytoskeletal breakdown, or in the exchanger
deletion mutant, thereby reducing
Ca influx and perhaps
altering the Na
inhibition
profile as well. For example, a reduced coupling between Ca
influx and local Ca
sequestration could lead to
a buildup of [
Ca] beneath the cytoplasmic
surface which would reduce net
Ca entry by promoting
Ca/
Ca exchange or
Na
-dependent
Ca
efflux. While we cannot eliminate this possibility, we believe that an
alteration in Na/Ca competition at the exchanger's external
surface provides a more straightforward explanation for the shift in
the Na
-inhibition profile.
]
when
140 m
M Na
is restored to the cells after a
period of [Na
]
reduction (Figs. 4 and 5). In part, this probably reflects the
loss of organellar Ca
sequestration and plasma
membrane Ca
pump activity due to the absence of ATP.
However, it seems likely that the rate of
Na
-dependent Ca
efflux is inhibited by ATP depletion as well. Previous studies
have shown that ATP depletion increases the K
for Ca
transport at the cytosolic surface and
decreases the affinity of the secondary Ca
activation
site for Ca
(1, 2, 3, 4, 5, 6, 7, 8) .
It has recently been shown, through the use of mutants defective in
secondary Ca
activation, that filling of the
activation site with Ca
regulates Ca
efflux as well as Ca
influx
(33) . Thus,
the apparent absence of
Na
-dependent Ca
efflux in ATP-depleted CK1.4 cells could reflect the reduced
affinity of the exchanger for Ca
ions at either the
transport sites, the secondary Ca
activation sites,
or both. The absence of secondary Ca
activation in
the exchanger deletion mutant
(15) may partly explain the
reduced sensitivity of Ca
efflux to ATP depletion in
the CK138 cells.
influx and
Ca
efflux mediated by the exchanger. Second, the
potency of extracellular Na
as an inhibitor of
exchange-mediated Ca
influx is increased in
ATP-depleted cells. Third, the effects of ATP on exchange activity
probably do not involve direct phosphorylation of the exchanger.
Fourth, exchange activity is modulated by changes in the actin
cytoskeleton. And finally, the effects of the actin cytoskeleton on the
Na
-inhibition profile require the
presence of the exchanger's central hydrophilic domain. While the
precise mechanism(s) of ATP-dependent regulation of exchange activity
remain to be determined, the actin cytoskeleton is likely to emerge as
a physiologically important regulator of this transport activity, as it
is for several other carrier-mediated transport processes
(35) .
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.