1 Department of Molecular Physiology, National Cardiovascular Center Research Institute, Suita, Osaka 565-8565; and 2 Department of Physiology, School of Medicine, Fukuoka University, Fukuoka 814-0180, Japan
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Physiological
functions of the intracellular regulatory domains of the
Na+/Ca2+ exchanger NCX1 were studied by
examining Ca2+ handling in CCL39 cells expressing a
low-affinity Ca2+ regulatory site mutant (D447V/D498I), an
exchanger inhibitory peptide (XIP) region mutant displaying no
Na+ inactivation (XIP-4YW), or a mutant lacking most of the
central cytoplasmic loop (246-672). We found that D447V/D498I
was unable to efficiently extrude Ca2+ from the cytoplasm,
particularly during a small rise in intracellular Ca2+
concentration induced by the physiological agonist
-thrombin or
thapsigargin. The same mutant took up Ca2+ much less
efficiently than the wild-type NCX1 in Na+-free medium when
transfectants were not loaded with Na+, although it
appeared to take up Ca2+ normally in transfectants
preloaded with Na+. XIP-4YW and, to a lesser extent,
246-672, but not NCX1 and D447V/D498I, markedly accelerated the
loss of viability of Na+-loaded transfectants. Furthermore,
XIP-4YW was not activated by phorbol ester, whereas XIP-4YW and
D447V/D498I were resistant to inhibition by ATP depletion. The results
suggest that these regulatory domains play important roles in the
physiological and pathological Ca2+ handling by NCX1, as
well as in the regulation of NCX1 by protein kinase C or ATP depletion.
calcium flux; sodium loading; cell viability; cell death; protein kinase C
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
THE SODIUM/CALCIUM EXCHANGER catalyzes electrogenic exchange of three Na+ for one Ca2+ across the plasma membrane (4). It forms a multigene family of highly homologous proteins comprising three isoforms, NCX1, NCX2, and NCX3 (16, 22, 24). These isoforms presumably have similar molecular topologies consisting of multiple membrane-spanning segments and a large central cytoplasmic loop. NCX1 is highly expressed in cardiac muscle, brain, and kidney and at much lower levels in many other tissues, whereas the expression of NCX2 and NCX3 is limited mainly to the brain.
The physiological and pathological roles of the Na+/Ca2+ exchanger have been studied most extensively in cardiac muscle. During each excitation-contraction cycle, the exchanger extrudes Ca2+ from the cytoplasm (the forward-exchange mode) in an amount equivalent to that entering cardiomyocytes via the L-type Ca2+ channels to balance the intracellular Ca2+ (Cai2+) content (1, 32). The exchanger also operates in the reverse mode, contributing to influx of Ca2+ into cardiomyocytes during cardiac depolarization. However, it is unclear whether such Ca2+ influx via the exchanger is physiologically important in triggering the release of Ca2+ from the sarcoplasmic reticulum (26, 27). When the intracellular Na+ (Nai+) concentration ([Na+]i) is abnormally elevated under pathological conditions such as ischemia-reperfusion, the exchanger catalyzes a large influx of Ca2+, leading to Ca2+ overloading of cardiomyocytes (29).
The cardiac isoform of the Na+/Ca2+ exchanger NCX1 has been shown to be secondarily modulated by the transport substrates Cai2+ and Nai+ (6, 7, 18). Submicromolar Cai2+ enhances the exchange current by promoting recovery of the exchanger from the "I2 inactivation state," whereas high Nai+ enhances the entry of the exchanger into the "I1 inactivation state." Recent mutational analyses of NCX1 function have revealed that a high-affinity Ca2+ binding site comprising two clusters of acidic amino acids in the central cytoplasmic loop is required for Cai2+-dependent activation (15, 20), whereas the exchanger inhibitory peptide (XIP) region in the NH2 terminus of the same cytoplasmic loop is involved in the Nai+-dependent inactivation (19). The latter inactivation is antagonized by Cai2+ and MgATP (6) and probably also by phosphatidylinositol 4,5-bisphosphate (5). The physiological significance of the regulation of Na+/Ca2+ exchange by Cai2+ or Nai+, however, remains poorly understood, although these regulations were demonstrated to occur in intact cardiomyocytes (13, 18). In addition, it is not known how these regulations are related to other types of modulation of Na+/Ca2+ exchange, namely, activation by protein kinase C (PKC)-dependent phosphorylation and inhibition by cell ATP depletion (2, 3, 8, 9, 11, 17, 28).
In this study we examined handling of Cai2+ by
deregulated mutants of NCX1, i.e., D447V/D498I, XIP-4YW, and
246-672, expressed in CCL39 fibroblasts treated with
-thrombin and other agents or loaded with excess Na+. We
suggest that the Cai2+ regulatory site is important for
the efficient extrusion of Cai2+ by NCX1 during a
relatively small rise in intracellular Ca2+ concentration
[Ca2+]i induced by physiological agonists,
whereas the Nai+-dependent inactivation is important
for protecting cells from Ca2+ overloading by NCX1 during
the pathological Nai+ accumulation. We also showed that
the regulation by PKC-dependent phosphorylation or ATP depletion was
not observed in some of these deregulated mutants of NCX1.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials.
Chinese hamster lung fibroblasts (CCL39) were obtained from American
Type Culture Collection. 45CaCl2,
22NaCl, and [-32P]dATP were purchased from
Amersham. Ouabain, monensin, phorbol 12-myristate 13-acetate (PMA),
oligomycin, and BSA were obtained from Sigma Chemical. Fura 2-AM was
acquired from Dojindo Laboratories (Kumamoto, Japan). All other
chemicals were of the highest grade available.
Cell culture. CCL39 cells and their NCX1 mutant transfectants were maintained in DMEM supplemented with 7.5% heat-inactivated FCS, 50 U/ml penicillin, and 50 µg/ml streptomycin.
Construction and stable expression of NCX1 mutants.
Sac II-Hind III sites were used to subclone dog
heart NCX1.1 cDNA into pCRII (designated pCRII-NCX1), as described
previously (9). Mutations for Y224W/Y226W/Y228W/Y231W
(designated XIP-4YW) and D447V/D498I (amino acid numbers based on Ref.
23) were made by site-directed mutagenesis by the "fusion PCR"
method (8). In this procedure, two DNA fragments were
produced by PCR with pCRII-NCX1 as a template by use of Pfu
polymerase and two pairs of outer and inner primers, with the latter
inner primers containing an overlapping sequence with the same
mutations. The final PCR product was generated with these DNA fragments
as templates with use of the sense and antisense outer primers
5'-GGAGACCTAGGTCCCAGCACC-3' (the endogenous Avr
II restriction site is underlined) and
5'-ATTTCCTCGAGCTCCAGATGT-3' (the endogenous Xho
I restriction site is underlined), respectively. The final PCR
products were cut with Avr II and Xho I and then exchanged with the corresponding regions in pCRII-NCX1. Additionally, the mutant cDNA with amino acids 244-671 deleted (designated
244-671) was constructed as described previously
(8). Successful construction was verified by sequencing
(ABI PRISM, Perkin-Elmer). The full-length cDNAs containing NCX
constructs were inserted between Sac II and Hind
III sites of the mammalian expression vector pKCRH (9). To
stably express exchangers, Lipofectin (GIBCO BRL) was used to transfect
pKCRH plasmids into CCL39 cells. Cell clones were isolated from the
colonies grown in DMEM containing 500 µg/ml G-418 for 10 days and
tested for Na+/Ca2+ exchange activity and
exchanger protein expression. Single-cell clones expressing the highest
levels of exchangers were chosen and used for experiments.
Assay of Nai+-dependent 45Ca2+ uptake. For cell Na+ loading, confluent cells in 24-well dishes were incubated at 37°C for 20 min in 0.5 ml of modified balanced salt solution [BSS: 10 mM HEPES-Tris (pH 7.4), 146 mM NaCl, 4 mM KCl, 2 mM MgCl2, 0.1 mM CaCl2, 10 mM glucose, and 0.1% BSA] containing 1 mM ouabain and 10 µM monensin. 45Ca2+ uptake was then initiated by switching the medium to Na+-free BSS (NaCl replaced with equimolar choline chloride) or to normal BSS, both of which contained 45CaCl2 (1.5 µCi/ml) but not monensin. After a 30-s incubation, 45Ca2+ uptake was stopped by washing cells four times with an ice-cold solution containing 10 mM HEPES-Tris (pH 7.4), 120 mM choline chloride, and 10 mM LaCl3. Cells were solubilized with 0.1 N NaOH, and aliquots were taken for determination of radioactivity and protein. Nai+-dependent 45Ca2+ uptake was calculated by subtracting 45Ca2+ uptake in normal BSS from that in Na+-free BSS.
Measurements of [Ca2+]i and [Na+]i. [Ca2+]i was monitored using fura 2 as a fluorescent Ca2+ indicator. Cells cultured on glass coverslips were loaded with 4 µM fura 2-AM for 20 min at 37°C in BSS containing 1 mM CaCl2 and then washed twice with the same medium. In some experiments (see Fig. 6A), Na+ loading medium was used for the fura 2 loading. Glass coverslips were fixed to a mount that was diagonally inserted into a cuvette filled with 2.2 ml of an appropriate medium. The fluorescence signal was monitored, and [Ca2+]i was calculated as described previously (25).
For estimation of [Na+]i, cells were equilibrated with BSS containing 1 mM CaCl2 and 146 mM 22NaCl (10 µCi/ml) for 5 h and then incubated for 20 min in the above-described Na+ loading medium, which contained 146 mM 22NaCl. 22Na+ uptake was measured using the same procedure used for 45Ca2+ uptake. [Na+]i was calculated from values of 22Na+ uptake with 5 µl/mg protein used as the total intracellular water space (14). [Na+]i for the Na+-loaded CCL39 cells was estimated to be 86 ± 8.9 mM (n = 3). Essentially the same value was obtained for Na+-loaded cells expressing different NCX constructs.Measurement of whole cell outward current.
Outward exchange currents from NCX1 mutant transfectants were measured
using the whole cell patch-clamp technique, as previously described
(31). The extracellular solution contained 150 mM LiCl
(replacing NaCl), 1 mM MgCl2, 0 or 1 mM CaCl2,
20 µM ouabain, 2 µM nicardipine, 5 µM ryanodine, and 5 mM HEPES
(pH 7.2), whereas the pipette solution contained 20 or 100 mM NaOH, 20 mM CsOH, 1.1 mM MgCl2, 20 mM tetraethylammonium chloride, 2 mM MgATP, 2 mM creatine phosphate, 19.8 mM CaCl2, 50 mM
EGTA, and 50 mM HEPES (pH 7.2). The ionized Ca2+
concentration in the pipette solution was calculated to be 0.16 µM.
The outward current was activated by switching the external solution
from one without CaCl2 to one with CaCl2. All
experiments were performed at ~35°C, and the holding and test
potentials were 40 mV. All data were acquired and analyzed with
pCLAMP (Axon Instrument) software.
Assay of cell viability.
Cell viability was assessed by staining cells with 0.025% trypan blue
for 5 min. Cells with stained cytoplasm were counted at room
temperature on an inverted microscope (model IMT-2, Olympus) with a
×20 objective lens. Three or four microscope fields containing 100
cells each were examined.
Northern blot.
Total RNAs were extracted from cells transfected with NCX constructs
with TRIzol reagent (GIBCO BRL). RNAs (10 µg/lane) were separated by
electrophoresis on 1% agarose gels containing 1× MOPS buffer [10 mM
sodium acetate, 1 mM EDTA, and 40 mM MOPS (pH 7.2)] and 0.66 M
formaldehyde. After the gels were soaked in diethyl pyrocarbonate-treated H2O (5 min, 3 times), RNAs were
transferred to nylon membranes by capillary diffusion in 10×
saline-sodium citrate and fixed by ultraviolet cross-linking.
Hybridization and washing were performed according to the protocols for
Gene Screen (DuPont New England Nuclear). Nucleotides 1-878 of the coding region of NCX1 cDNA served as a template for the RNA probe synthesis with use of the RadPrime DNA labeling system (GIBCO BRL) and
[-32P]dATP. The signal from the
32P-labeled probe was visualized and quantified by a
Bioimage analyzer (BAS2000, Fuji Film).
Other procedures. Preparation of a rabbit polyclonal antibody against a glutathione S-transferase fusion protein containing amino acids 240-737 of NCX1 was described previously (8). Preparation of a chicken polyclonal antibody that recognizes the extracellular loops of the NCX1 protein has also been described (9). Cell membrane preparation, SDS-PAGE, and immunoblotting were performed according to the previously described methods (30). The immunoblots were visualized using the enhanced chemiluminescence detection system (Amersham). The immunocytochemistry was performed using the above-mentioned chicken polyclonal antibody, as described previously (10). Protein was measured with bicinchoninic acid protein assay reagent (Pierce) with BSA as a standard.
Statistical analysis. Values are means ± SE of 3 or 4 independent determinations. Differences for multiple comparisons were analyzed by unpaired t-test or one-way ANOVA followed by Dunnett's test. P < 0.05 was considered statistically significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Expression of wild-type or mutated NCX1 in CCL39 fibroblasts.
Recent mutational analysis has revealed that a high-affinity
Ca2+ binding site in the central cytoplasmic loop of NCX1
is required for the activation of Na+/Ca2+
exchange by Cai2+ (15, 20),
whereas the XIP region in the same loop is involved in the
Nai+-dependent inactivation of
Na+/Ca2+ exchange (19) (see the
introduction). We constructed deregulated mutants of NCX1, D447V/D498I,
XIP-4YW, and 246-672, which represent a mutant of the
Ca2+ regulatory site exhibiting a low Ca2+
affinity [Ca2+ concentration at half-maximal activation
(Kh)
1 µM] (20), a
mutant of the XIP region displaying minimum
Nai+-dependent inactivation (see Fig. 3), and a mutant
from which a large fraction of the cytoplasmic loop including the
Ca2+ binding site was deleted but which retains the XIP
region, respectively. We expressed these NCX1 mutants in CCL39 cells to
obtain information about the physiological roles of these regulatory
segments in Na+/Ca2+ exchange.
|
Nai+-dependent
45Ca2+ uptake and outward
current in cells expressing wild-type and mutated NCX1s.
We followed time courses of Nai+-dependent
45Ca2+ uptake into CCL39 cells stably
expressing different NCX constructs (Fig.
2). These cells, which had been loaded
with Na+ by treatment with ouabain and monensin in 146 mM
extracellular Na+ (Nao+), were estimated to
contain 86 mM Nai+ (see METHODS). In cells
expressing NCX1 or D447V/D498I, the uptake became markedly slow after
~30 s, whereas it continuously increased at least up to 6 min in
cells expressing XIP-4YW or 246-672. In contrast,
Nai+-dependent 45Ca2+ uptake
was not detected in control CCL39 cells, as we reported previously
(8, 10). We measured the initial rates of
Ca2+ uptake as a function of extracellular Ca2+
concentration ([Ca2+]o). All the double
reciprocal plots of uptake rates vs. [Ca2+]o
were linear for the wild-type and mutated exchangers, and from these
plots the Michaelis-Menten constant for Ca2+
(KCa) and the maximum velocity
(Vmax) were calculated (Table 1). KCa values for
D447V/D498I and
246-672 were similar to
KCa for the wild-type NCX1 (0.23 mM), whereas
KCa for XIP-4YW was significantly larger (0.53 mM). The order of Vmax was ranked as follows:
NCX1 = D447V/D498I > XIP-4YW
246-672.
|
|
|
[Ca2+]i transients in
CCL39 cells expressing wild-type and mutated NCX1s.
We compared [Ca2+]i transients elicited by
-thrombin (2 U/ml) or ionomycin (10 µM) in cells transfected with
various NCX constructs (Fig. 4). In the
presence of 146 mM Nao+,
[Ca2+]i transients in these cells were
suppressed to variable degrees compared with that in control CCL39
cells, although such suppression was not observed in the absence of
Nao+ (Fig. 4, A-D, and data not shown).
In control CCL39 cells, [Ca2+]i transients
were not different in the presence or absence of Nao+.
The summary data for the
-thrombin experiments are shown in Fig.
4D, in which average values for the peak and the resting [Ca2+]i measured in the presence and absence
of 146 mM Nao+ are presented for each NCX construct. In
146 mM Nao+, the wild-type NCX1 was able to reduce the
peak [Ca2+]i to a level close to the resting
[Ca2+]i (87-113 nM). In contrast,
D447V/D498I reduced the peak [Ca2+]i much
less efficiently to ~400 nM under the conditions used. The order of
the effectiveness in reducing peak [Ca2+]i
was ranked as follows: NCX1 > XIP-4YW >
246-672 > D447V/D498I. The same order of effectiveness
was observed for these NCX1 mutants when 1 µM thapsigargin or 10 µM
ionomycin was used to elicit [Ca2+]i
transients (Fig. 4, A-C, and data not shown). In the
absence of Nao+, the peak
[Ca2+]i transients elicited by thapsigargin
were similar to those elicited by
-thrombin, but those elicited by
ionomycin were much higher (
1 µM).
|
|
[Ca2+]i rise and cell
damage in Na+-loaded cells expressing
wild-type and mutated NCX1s.
CCL39 cells expressing different NCX constructs were loaded with
Na+ as in Fig. 2, and a [Ca2+]i
rise was elicited by changing medium containing 146 mM Na+
and 0.1 mM Ca2+ to one containing no Na+ and 1 mM Ca2+ (Fig. 6A).
Removal of Nao+ produced a marked increase in
[Ca2+]i in these cells, although the same
procedure did not influence [Ca2+]i in
control CCL39 cells (Fig. 6A). In the wild-type NCX1 or D447V/D498I transfectants, [Ca2+]i quickly
reached a peak and then declined rapidly to a steady-state level. On
the other hand, the declining phase of the
[Ca2+]i change was not observed or diminished
in XIP-4YW or 246-672 transfectants.
|
PKC- or ATP depletion-dependent regulation of
Na+/Ca2+
exchange in cells expressing wild-type and mutated NCX1s.
Phorbol ester and growth factors stimulate the rate of
Nai+-dependent 45Ca2+ uptake
into cardiomyocytes, smooth muscle cells, and NCX1-transfected cells,
whereas cell ATP depletion inhibits it (2, 3,
8, 9, 11, 17,
28). We compared the effects of PMA (0.01-0.3 µM)
and ATP depletion on the rate of Nai+-dependent
45Ca2+ uptake into cells expressing different
NCX constructs. PMA significantly accelerated the uptake into cells
expressing NCX1 or D447V/D498I (Fig.
7A). Intriguingly, such an
effect of PMA was not observed in cells expressing XIP-4YW or
246-672 (Fig. 7A). On the other hand, treatment with
2.5 µg/ml oligomycin and 10 mM 2-deoxy-D-glucose for
5-20 min resulted in inhibition of the uptake into cells
expressing the wild-type NCX1 (Fig. 7B). However, the effect
of metabolic inhibitors on the uptake was not observed in cells
expressing XIP-4YW, D447V/D498I, or
246-672 (Fig.
7B).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Previous electrophysiological studies (6, 7, 18-21) have provided clear evidence that activity of the Na+/Ca2+ exchanger is allosterically regulated by Cai2+ and Nai+. Cai2+ activates the exchanger by binding to a Ca2+ regulatory site in the large central cytoplasmic loop with high affinity (Kh ~ 0.3 µM) (15, 20), whereas relatively high Nai+ causes the inactivation of the exchanger in which the XIP region seems to play an essential role (19). To clarify the physiological significance of these regulatory systems, we compared the properties of the wild-type and deregulated mutants of NCX1 expressed in CCL39 fibroblasts that are virtually devoid of endogenous exchange activity (8, 10).
We studied kinetic properties of mutant exchangers by measuring the
time-dependent changes of the whole cell outward exchange current (Fig.
3), Nai+-dependent 45Ca2+
uptake (Fig. 2), and [Ca2+]i in transfectants
(Figs. 4-6). The quantitative comparison of the results obtained,
however, was difficult for several reasons. 1) It was
difficult for us to correctly estimate the expression level of
functional exchangers in the plasma membrane. 2) The regulatory site mutants had additional complications. For example, the
Ca2+ site mutant (D447V/D498I), which was demonstrated to
have a low affinity for regulatory Ca2+
(Kh1 µM) (20), also exhibited
enhanced Na i+-dependent inactivation (Fig.
3C). 3) The time resolution of the methods used
for these measurements is different. For example, a fast decline in the
exchange activity seen in Fig. 3C could be directly
monitored by the electrophysiological technique but not by other manual
procedures. Furthermore, 45Ca2+ uptake
measurement gave a time-integrated value for the exchange activity.
This method is not sensitive, and thus a certain amount of time is
needed to detect a change in the rate of exchange activity. 4) A change in [Ca2+]i is
influenced by activities not only of the
Na+/Ca2+ exchanger, but also of other
Ca2+ transporters in the endoplasmic reticulum, plasma
membrane, and mitochondria. Therefore, we obtain qualitative
conclusions from this comparative study.
We found that the transport activity of D447V/D498I as estimated from
the Vmax for the initial rate of
Nai+-dependent 45Ca2+ uptake in
cells preloaded with high Nai+ was similar to that of
the wild-type NCX1, whereas those of XIP-4YW and 246-672
measured under equivalent conditions were ~80 and ~35% of the
wild-type control, respectively (Table 1). The expression level of
D447V/D498I in cells as detected by Northern blot or immunoblot
analysis was slightly higher than that of the wild-type NCX1 (Fig. 1),
which might explain the apparent absence of the effect of the enhanced
Nai+-dependent inactivation on the overall amount of
Ca2+ taken up by this mutant during the exchange reaction
(Fig. 2). The low transport activity of
246-672, on the other
hand, is likely to arise from the low expression of this mutant in the plasma membrane possibly due to its decreased delivery to the membrane,
as suggested by the reduced immunofluorescence staining in the plasma
membrane and the normal mRNA level (Fig. 1; see RESULTS).
In contrast, XIP-4YW had a relatively high Vmax,
despite its low level of expression suggested by Northern blot and
immunoblot analyses and immunofluorescence staining (Fig. 1, Table 1;
see RESULTS). It seems likely that this mutant exchanger
has an increased turnover rate. The relatively slow
45Ca2+ uptake by this mutant measured at 0.1 mM
Cao2+ (Fig. 2) may be explained by the present finding
that this mutant exhibited a 2.5-fold lower apparent affinity for
Cao2+ than the wild-type NCX1 (Table 1).
We evaluated the abilities of these mutants to extrude
Ca i2+ from cells not loaded with
Nai+ in response to stimulation with the
physiological Ca2+-mobilizing agonist
-thrombin (Fig. 4, A-D). In medium containing 146 mM
Na+, the wild-type NCX1 was able to reduce the peak
[Ca2+]i to a level close to resting
[Ca2+]i, whereas D447V/D498I under the same
conditions reduced it only slightly (Fig. 4D). XIP-4YW and
246-672 exerted intermediate effects. The qualitatively similar
results were obtained when 1 µM thapsigargin or 10 µM ionomycin was
used to elicit [Ca2+]i transients in place of
-thrombin (Fig. 4, A-C; see RESULTS). Thus D447V/D498I was unable to rapidly extrude Cai2+
during a relatively small rise in [Ca2+]i
induced by the physiological agonist
-thrombin, as well as during a
similar or much larger [Ca2+]i rise induced
by thapsigargin or ionomycin. On the other hand, when cells maintained
in BSS containing 1 mM Ca2+ were transferred to
Na+-free medium, the wild-type NCX1 induced a slow
[Ca2+]i transient with a peak value of ~500
nM, whereas D447V/D498I caused only a minimal increase in
[Ca2+]i (Fig. 5). XIP-4YW and
246-672
exerted intermediate effects. Thus D447V/D498I functions much less
efficiently than the wild-type NCX1 also in the reverse-exchange mode
under conditions where [Ca2+]i and
[Na+]i are maintained at normal low levels.
The observed inefficiency of D447V/D498I in Ca2+ extrusion
(Fig. 4) or Ca2+ uptake (Fig. 5) is likely due to its low
affinity for regulatory Ca2+ (20), because
enhanced Nai+-dependent inactivation in this mutant
(Fig. 3C) would not influence Na+/Ca2+ exchange significantly in cells with
normal low Nai+. Interestingly, 246-672 was
more efficient in these Ca2+ transports than D447V/D498I,
although it is expressed at a lower level (Figs. 4 and 5, Table 1; see
RESULTS). It might be possible that
246-672, from
which a large fraction of the cytoplasmic domain, including the
Ca2+ regulatory site, is deleted, has a higher affinity for
the transport substrate Cai2+ or Nai+
than D447V/D498I, because it was shown previously that the apparent affinity of D447V/D498I for the transport substrate
Cai2+ increased ninefold when it was digested with
chymotrypsin from the cytoplasmic side (20). On the other
hand, the less efficient Ca2+ extrusion (Fig. 4) or
Ca2+ uptake (Fig. 5) by XIP-4YW than by the wild-type NCX1
might be explained partly by the decreased affinity of this mutant for the transported ions, as evidenced by its lower apparent affinity for
Cao2+ (Table 1). The decreased affinity of the
Ca2+ regulatory site might also contribute to lower
activity of this mutant, because Matsuoka et al. (19)
reported that some XIP mutants exhibit significantly reduced apparent
affinity for regulatory Ca2+.
We compared Nao+ removal-induced changes in
[Ca2+]i in various NCX transfectants loaded
with high Nai+ (Fig. 6A). In the wild-type
NCX1 and D447V/D498I transfectants, [Ca2+]i
increased rapidly to similar high peak values and then rapidly declined
to steady-state levels. In XIP-4YW and 246-672 transfectants, in contrast, [Ca2+]i reached lower peak
values and the declining phase of [Ca2+]i was
absent or diminished (Fig. 6A). At the peak
[Ca2+]i, Nai+-dependent
Ca2+ influx via the Na+/Ca2+
exchanger appears to balance against Ca2+ removal from the
cytoplasm via Ca2+ removal systems such as the plasma
membrane and the endoplasmic reticulum Ca2+ pumps. The
subsequent reduction in the rate of Nai+-dependent
Ca2+ influx would therefore result in the reduction of
[Ca2+]i. The fast decline of
[Ca2+]i in the wild-type NCX1 and D447V/D498I
transfectants (Fig. 6A) is consistent with the rapid
slowdown of Nai+-dependent
45Ca2+ uptake observed in these transfectants
(Fig. 2). The absence or decrease of the declining phase of
[Ca2+]i in XIP-4YW or
246-672
transfectants in Fig. 6A is also consistent with the near
absence or reduction of the slowdown of Nai+-dependent
45Ca2+ uptake observed in the corresponding
mutant transfectants (Fig. 2). It appears that the reduction of
[Ca2+]i after the peak and the slowdown of
Nai+-dependent 45Ca2+ uptake in
the NCX transfectants reflect the extent of expression of
Nai+-dependent inactivation of
Na+/Ca2+ exchange in these transfectants,
because decay of the outward exchange current in respective NCX
transfectants showed correspondingly similar differences under
comparable experimental conditions (Fig. 3, A-D),
although direct comparison of these data is difficult, as noted above.
Interestingly, D447V/D498I and the wild-type NCX1 transfectants loaded with high Nai+ exhibited similar high Ca2+ uptake activities (Figs. 2 and 6A, Table 1), whereas D447V/D498I took up Ca2+ much less efficiently than did the wild-type NCX1 in transfectants not loaded with Nai+ (Fig. 5). We have no clear-cut explanation for this difference. One possible explanation could be that [Ca2+]i immediately below the plasma membrane remained elevated to a level sufficient to fully activate D447V/D498I in cells loaded with high Nai+ in the presence of 0.1 mM Cao2+ (see METHODS), although the average [Ca2+]i before the Nao+ removal was not elevated, as monitored by fura 2 fluorescence (Fig. 6A).
We followed a change in viability of Nai+-loaded NCX
transfectants placed in Nao+-free medium containing 1 mM CaCl2. The viability evaluated on the basis of the
exclusion of trypan blue decreased time dependently in cells expressing
XIP-4YW or 246-672, although cells expressing the wild-type
NCX1 or D447V/D498I remained viable under the same conditions (Fig.
6B). The observed morphological changes (Fig. 6C)
suggest that cell death was due to prolonged Ca2+
overloading. We obtained similar results when cells were treated with 1 mM ouabain for 3 h in the physiological medium containing high
Nao+ (see RESULTS). These results suggest
that the Nai+-dependent inactivation is able to
function as a protective mechanism by which cells protect themselves
from damage caused by Ca2+ overloading via the reverse mode
of Na+/Ca2+ exchange during high cell
Na+ loading. This function of the
Nai+-dependent inactivation would be particularly
important in the heart, in which pathological Na+
accumulation in cardiomyocytes activates the reverse mode of Na+/Ca2+ exchange during ischemia-reperfusion
(29).
We previously provided evidence that vasoactive agonists such as
platelet-derived growth factor-BB and endothelin-1 stimulate the rate
of Nai+-dependent 45Ca2+ uptake
into cardiomyocytes, smooth muscle cells, and NCX1- or NCX3-transfected
fibroblasts via a mechanism involving protein phosphorylation by PKC
(8, 9, 11). Such PKC-dependent regulation does not require the direct phosphorylation of the exchanger
itself but apparently requires the large central intracellular loop of
the exchanger, because 246-672 was not activated by phorbol ester and other agonists (8). We confirmed the absence of
the effect of PMA on Nai+-dependent
45Ca2+ uptake by
246-672 (Fig.
7A). Intriguingly, PMA also failed to enhance the uptake by
XIP-4YW, whereas it activated the uptake by D447V/D498I, as it did for
the wild-type NCX1 (Fig. 7A). These results suggest that the
PKC-dependent regulation of the exchanger requires the intact XIP
region, but not the intact Ca2+ regulatory site. Our
present hypothesis is that, on stimulation of cells with PKC
activators, PKC or its downstream protein kinase phosphorylates
unidentified regulatory factor(s), which then enhances Na+/Ca2+ exchange through its interaction with
the XIP region of the exchanger. How the XIP-4YW region is involved in
the PKC-dependent regulation is unknown.
Cell ATP depletion, on the other hand, has been shown to inhibit
Na+/Ca2+ exchange in many cell types
(2, 8, 9, 28). We
found that Nai+-dependent
45Ca2+ uptake by XIP-4YW, D447V/D498I, or
246-672 was minimally affected by ATP depletion under
conditions in which the uptake by the wild-type NCX1 was inhibited
significantly (by ~30% after ATP depletion for 20 min; Fig.
7B). The result was somewhat surprising, because the XIP
mutant and the Ca2+ regulatory site mutant were resistant
to the inhibition by ATP depletion. ATP depletion has been reported to
cause inhibition of many other ion transporters (5).
However, the underlying mechanisms for such inhibitions are poorly
understood, although ATP depletion is known to affect many aspects of
cell metabolism, including the phosphorylation of proteins and active
metabolites such as phosphatidylinositol 4,5-bisphosphate, the
cytoskeleton structure, and the induction of various stress responses
(5).
In summary, this study suggests that the high-affinity Ca2+ regulatory site and the XIP region displaying the Nai+-dependent inactivation play essential roles in the physiological and pathological handling of [Ca2+]i by the Na+/Ca2+ exchanger NCX1. It further suggests that these same domains are also important in the regulation of NCX1 by PKC or ATP depletion. Further studies are required to clarify the molecular mechanisms by which these domains modulate exchange activity in response to PKC activation or ATP depletion.
![]() |
ACKNOWLEDGEMENTS |
---|
This work was supported by Ministry of Education, Science and Culture of Japan Grants-in-Aid 09273101 and 10770048, a grant for Research on Health Sciences Focusing on Drug Innovation from the Japan Health Science Research Foundation, and a grant from the Uehara Foundation.
![]() |
FOOTNOTES |
---|
Address for reprint requests and other correspondence: M. Shigekawa, Dept. of Molecular Physiology, National Cardiovascular Center Research Institute, Fujishiro-dai 5, Suita, Osaka 565-8565, Japan (E-mail: shigekaw{at}ri.ncvc.go.jp).
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. §1734 solely to indicate this fact.
Received 5 July 1999; accepted in final form 4 February 2000.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Bridge, JH,
Smolley JR,
and
Spitzer KW.
The relationship between charge movements associated with ICa and INa-Ca in cardiac myocytes.
Science
248:
376-378,
1990[ISI][Medline].
2.
Condrescu, M,
Gardner JP,
Chernaya G,
Aceto JF,
Kroupis C,
and
Reeves JP.
ATP-dependent regulation of sodium-calcium exchange in Chinese hamster ovary cells transfected with the bovine cardiac sodium-calcium exchanger.
J Biol Chem
270:
9137-9146,
1995
3.
Haworth, RA,
Goknur AB,
Hunter DR,
Hegge JO,
and
Berkoff HA.
Inhibition of calcium influx in isolated adult rat heart cells by ATP depletion.
Circ Res
60:
586-594,
1987[Abstract].
4.
Hilgemann, DW.
The cardiac Na-Ca exchanger in giant membrane patches.
Ann NY Acad Sci
779:
136-158,
1996[ISI][Medline].
5.
Hilgemann, DW.
Cytoplasmic ATP-dependent regulation of ion transporters and channels: mechanisms and messengers.
Annu Rev Physiol
59:
193-220,
1997[ISI][Medline].
6.
Hilgemann, DW,
Collins A,
and
Matsuoka S.
Steady-state and dynamic properties of cardiac sodium-calcium exchange. Secondary modulation by cytoplasmic calcium and ATP.
J Gen Physiol
100:
905-932,
1992[Abstract].
7.
Hilgemann, DW,
Matsuoka S,
Nagel GA,
and
Collins A.
Steady-state and dynamic properties of cardiac sodium-calcium exchange. Sodium-dependent inactivation.
J Gen Physiol
100:
905-932,
1992[Abstract].
8.
Iwamoto, T,
Pan Y,
Nakamura TY,
Wakabayashi S,
and
Shigekawa M.
Protein kinase C-dependent regulation of Na+/Ca2+ exchanger isoforms NCX1 and NCX3 does not require their direct phosphorylation.
Biochemistry
37:
17230-17238,
1998[ISI][Medline].
9.
Iwamoto, T,
Pan Y,
Wakabayashi S,
Imagawa T,
Yamanaka HI,
and
Shigekawa M.
Phosphorylation-dependent regulation of cardiac Na+/Ca2+ exchanger via protein kinase C.
J Biol Chem
271:
13609-13615,
1996
10.
Iwamoto, T,
Wakabayashi S,
Imagawa T,
and
Shigekawa M.
Na+/Ca2+ exchanger overexpression impairs calcium signaling in fibroblasts: inhibition of the [Ca2+] increase at the cell periphery and retardation of cell adhesion.
Eur J Cell Biol
76:
228-236,
1998[ISI][Medline].
11.
Iwamoto, T,
Wakabayashi S,
and
Shigekawa M.
Growth factor-induced phosphorylation and activation of aortic smooth muscle Na+/Ca2+ exchanger.
J Biol Chem
270:
8996-9001,
1995
12.
Iwamoto, T,
Watano T,
and
Shigekawa M.
A novel isothiourea derivative selectively inhibits the reverse mode of Na+/Ca2+ exchange in cells expressing NCX1.
J Biol Chem
271:
22391-22397,
1996
13.
Kimura, J,
Noma A,
and
Irisawa H.
Na-Ca exchange current in mammalian heart cells.
Nature
319:
596-597,
1986[ISI][Medline].
14.
L'Allemain, G,
Paris S,
and
Pouysségur J.
Growth factor action and intracellular pH regulation in fibroblasts. Evidence for a major role of the Na+/H+ antiporter.
J Biol Chem
259:
5809-5815,
1984
15.
Levitsky, DO,
Nicoll DA,
and
Philipson KD.
Identification of the high-affinity Ca2+-binding domain of the cardiac Na+-Ca2+ exchanger.
J Biol Chem
269:
22847-22852,
1994
16.
Li, Z,
Matsuoka S,
Hryshko LV,
Nicoll DA,
Bersohn MM,
Burke EP,
Lifton RP,
and
Philipson KD.
Cloning of the NCX2 isoform of the plasma membrane Na+-Ca2+ exchanger.
J Biol Chem
269:
17434-17439,
1994
17.
Linck, B,
Qui Z,
He Z,
Tong Q,
Hilgemann DW,
and
Philipson KD.
Functional comparison of three different isoforms of the Na+/Ca2+ exchanger (NCX1, NCX2, NCX3).
Am J Physiol Cell Physiol
274:
C415-C423,
1998
18.
Matsuoka, S,
and
Hilgemann DW.
Inactivation of outward Na+-Ca2+ exchange current in guinea-pig ventricular myocytes.
J Physiol (Lond)
476:
443-458,
1994[Abstract].
19.
Matsuoka, S,
Nicoll DA,
He Z,
and
Philipson KD.
Regulation of the cardiac Na+-Ca2+ exchanger by the endogenous XIP region.
J Gen Physiol
109:
273-286,
1997
20.
Matsuoka, S,
Nicoll DA,
Hryshko LV,
Levitsky DO,
Weiss JN,
and
Philipson KD.
Regulation of the cardiac Na+-Ca2+ exchanger by Ca2+. Mutational analysis of the Ca2+-binding domain.
J Gen Physiol
105:
403-420,
1995[Abstract].
21.
Matsuoka, S,
Nicoll DA,
Reilly RF,
Hilgemann DW,
and
Philipson KD.
Initial localization of regulatory regions of the cardiac sarcolemmal Na+-Ca2+ exchanger.
Proc Natl Acad Sci USA
90:
3870-3874,
1993[Abstract].
22.
Nicoll, DA,
Longoni S,
and
Philipson KD.
Molecular cloning and functional expression of the cardiac sarcolemmal Na+-Ca2+ exchanger.
Science
250:
562-565,
1990[ISI][Medline].
23.
Nicoll, DA,
and
Philipson KD.
Molecular studies of the cardiac sarcolemmal sodium-calcium exchanger.
Ann NY Acad Sci
639:
181-188,
1991[Abstract].
24.
Nicoll, DA,
Quednau BD,
Qui Z,
Xia Y-R,
Lusis AJ,
and
Philipson KD.
Cloning of a third mammalian Na+-Ca2+ exchanger, NCX3.
J Biol Chem
271:
24914-24921,
1996
25.
Ohshima, N,
Iwamoto T,
and
Shigekawa M.
Regulation of Ca2+ entry in rat aortic smooth muscle cells in primary culture.
J Biochem (Tokyo)
116:
274-281,
1994[Abstract].
26.
Sham, JSK,
Cleeman L,
and
Morad M.
Functional coupling of Ca2+ channels and ryanodine receptors in cardiac myocytes.
Proc Natl Acad Sci USA
92:
121-125,
1995[Abstract].
27.
Sipido, KR,
Maes M,
and
de Werf FV.
Low efficiency of Ca2+ entry through the Na+-Ca2+ exchanger as trigger for Ca2+ release from the sarcoplasmic reticulum. A comparison between L-type Ca2+ current and reverse-mode Na+-Ca2+ exchange.
Circ Res
81:
1034-1044,
1997
28.
Smith, JB,
and
Smith L.
Energy dependence of sodium-calcium exchange in vascular smooth muscle cells.
Am J Physiol Cell Physiol
259:
C302-C309,
1990
29.
Tani, M.
Mechanisms of Ca2+ overload in reperfused ischemic myocardium.
Annu Rev Physiol
52:
543-559,
1990[ISI][Medline].
30.
Tawada-Iwata, Y,
Imagawa T,
Yoshida A,
Takahashi M,
Nakamura H,
and
Shigekawa M.
Increased mechanical extraction of T-tubule/junctional SR from cardiomyopathic hamster heart.
Am J Physiol Heart Circ Physiol
264:
H1447-H1453,
1993
31.
Uehara, A,
Iwamoto T,
Shigekawa M,
and
Imanaga I.
Whole-cell currents from the cloned canine cardiac Na+/Ca2+ exchanger NCX1 overexpressed in a fibroblast cell CCL39.
Pflügers Arch
434:
335-338,
1997[ISI][Medline].
32.
Wier, WG.
Cytoplasmic [Ca2+] in mammalian ventricle: dynamic control by cellular processes.
Annu Rev Physiol
52:
467-485,
1990[ISI][Medline].