Department of Pharmacology and Physiology, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07103
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ABSTRACT |
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Transfected Chinese hamster ovary cells stably expressing the bovine cardiac Na+/Ca2+ exchanger (CK1.4 cells) were used to determine the range of cytosolic Ca2+ concentrations ([Ca2+]i) that activate Na+/Ca2+ exchange activity. Ba2+ influx was measured in fura 2-loaded, ionomycin-treated cells under conditions in which the intracellular Na+ concentration was clamped with gramicidin at ~20 mM. [Ca2+]i was varied by preincubating ionomycin-treated cells with either the acetoxymethyl ester of EGTA or medium containing 0-1 mM added CaCl2. The rate of Ba2+ influx increased in a saturable manner with [Ca2+]i, with the half-maximal activation value of 44 nM and a Hill coefficient of 1.6. When identical experiments were carried out with cells expressing a Ca2+-insensitive mutant of the exchanger, Ba2+ influx did not vary with [Ca2+]i. The concentration for activation of exchange activity was similar to that reported for whole cardiac myocytes but approximately an order of magnitude lower than that reported for excised, giant patches. The reason for the difference in Ca2+ regulation between whole cells and membrane patches is unknown.
giant patch; barium uptake; fura 2; ethylene
glycol-bis(-aminoethyl
ether)-N, N, N', N'-tetraacetic
acid-acetoxymethyl ester
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INTRODUCTION |
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SODIUM/CALCIUM EXCHANGE is a carrier-mediated transport
process that moves three Na+
across the membrane in exchange for a single
Ca2+ in the other direction. It is
found in the plasma membrane of many, but not all, cells and is
particularly abundant in cardiac myocytes, where it serves as the
principal Ca2+ efflux mechanism
(1, 22). It has a relatively low affinity for cytosolic
Ca2+ [Michaelis-Menten
constant (Km) = 4 µM] (21, 28) but has a high turnover number (~5,000
s1) (3, 17). The
exchanger was cloned initially by Philipson and his colleagues (30). It
is a protein of 938 amino acids, with an amino-terminal signal
sequence, 11 putative transmembrane segments, and a large (520 residue)
hydrophilic domain between the fifth and sixth transmembrane segments.
The hydrophilic domain is essential for normal regulatory behavior, but
it can be deleted by mutagenesis or by chymotrypsin treatment, without
altering the basic kinetic behavior of the exchanger (28).
Exchange activity is regulated by ATP-dependent and Ca2+-dependent processes. The former involves, at least in part, the synthesis of phosphatidylinositol 4,5-bisphosphate (18), which reduces or eliminates an inactivation process dependent on the presence of cytosolic Na+ (5, 20). Roles for ATP-dependent regulation through phosphorylation (10, 11, 23) and by cytoskeletal actin (7) have also been suggested. The Ca2+-dependent regulatory process involves the activation of transport activity through the binding of cytosolic Ca2+ to regulatory sites in the central hydrophilic domain (8, 16, 19). Philipson and his colleagues (25, 27) have recently identified the Ca2+-binding regions within the hydrophilic domain that are responsible for this behavior. Under normal physiological conditions, regulatory activation by cytosolic Ca2+ is required for all three manifestations of exchange activity: forward and reverse Na+/Ca2+ exchange (8, 16, 24, 27), Ca2+/Ca2+ exchange (24), and Na+/Na+ exchange (9, 14).
Reports differ as to the affinity of the exchanger for Ca2+ at its regulatory sites. Measurements of outward exchange currents in giant membrane patches (15) from cardiac myocytes, or from Xenopus oocytes expressing the cardiac exchanger, indicated that half-maximal activation (K1/2) of exchange activity typically occurs within a range of 300-600 nM for the cytosolic Ca2+ concentration ([Ca2+]i), with little or no cooperativity (Hill coefficients of 1-1.5) (19, 27, 33). Occasionally, much higher values for K1/2 by Ca2+ have been observed (5). On the other hand, measurements of outward exchange currents in whole cell studies with guinea pig myocytes suggest that Ca2+ activation occurs at much lower concentrations. Miura and Kimura (29) reported a K1/2 of 22 nM [Ca2+]i and Noda et al. (31) found K1/2 to be 47 nM. The reasons for the difference between the whole cell and giant patch studies are presently unknown.
Our laboratory has focused on the properties of the bovine cardiac
Na+/Ca2+
exchanger when expressed in transfected Chinese hamster ovary (CHO)
cells. We have recently described the use of fura 2-based measurements
of cytosolic Na+-dependent
Ba2+ influx to assay
Na+/Ca2+
exchange activity in these cells (4, 6). The use of
Ba2+ offers several experimental
advantages over Ca2+:
Ba2+ is not sequestered by
intracellular organelles, it is not transported by ionomycin, and, at
concentrations of 1 mM, Ba2+ does
not enter these cells through store-dependent
Ca2+ channels. Furthermore, the
exchanger's affinity for Ba2+ at
its regulatory Ca2+ binding sites
is extremely low [dissociation constant
(Kd) 10 µM] (33), so that the influence of
[Ca2+]i
on Ba2+ influx can be assessed
without complications due to autoactivation of exchange activity by
cytosolic Ba2+. The use of
Ba2+ therefore permits one to
measure exchange activity under conditions in which corresponding
Ca2+ fluxes would be
uninterpretable due to the multiple pathways for
Ca2+ influx and internal
sequestration.
Previous studies have shown that release of Ca2+ from intracellular stores stimulated exchange-mediated Ba2+ influx, presumably by elevating [Ca2+]i. The results suggested that increases in [Ca2+]i through a range of 20-40 nM were associated with increases in Na+/Ca2+ exchange activity. It was not possible to estimate the K1/2 for activation of exchange activity by Ca2+ in these studies. In this report, we examined the acceleration of exchange activity over a broader range of [Ca2+]i using EGTA- and Ca2+-loading procedures in cells permeabilized with the Ca2+ ionophore ionomycin. The K1/2 for Ca2+ activation was determined to be 44 nM. Thus our results are in agreement with measurements of inward exchange currents in cardiac myocytes and suggest that activation of exchange activity by Ca2+ in intact cells occurs at much lower concentrations than in excised membrane patches.
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METHODS |
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Materials and solutions.
Fura 2-AM and EGTA-AM were purchased from Molecular Probes (Eugene, OR)
and stored at 20°C as frozen solutions (3 and 50 mM,
respectively) in DMSO. Gramicidin D (Sigma, St. Louis, MO) was stored
at
20°C as a 1 mM stock solution in DMSO. All other biochemicals and ionophores were purchased from Sigma. The compositions of the physiological salt solutions (PSS) used in these studies were as
follows: Na-PSS contained (in mM) 140 NaCl, 5 KCl, 1 MgCl2, 10 glucose, and 20 MOPS,
adjusted to pH 7.4 (37°C) with Tris. K-PSS had the same composition
as Na-PSS except that NaCl was omitted and the total concentration of
KCl was 140 mM. For 20/120 Na-K-PSS, Na-PSS and K-PSS were mixed in a
1:6 ratio to yield final Na+ and
K+ concentrations of 20 and ~120
mM, respectively.
Cells.
The CHO cells stably expressing the
Na+/Ca2+
exchanger (CK1.4 cells) have been fully described in several previous
publications (4, 6, 7, 32). The cells were prepared by transfecting the
parental CHO cells (CCL-61; American Type Culture Collection) with the
expression vector pcDNA I/Neo (Invitrogen, Carlsbad, CA) containing a
cDNA insert coding for the bovine cardiac
Na+/Ca2+
exchanger (32). CK138 cells were prepared from the same parental cells
by transfection with a deletion mutant of the exchanger, (241-680), missing 440 of the 520 amino acids of the
exchanger's central hydrophilic domain. The behavior of this
mutant in excised patches (28) and in the transfected CHO cells (7) has
been described previously. The cells were grown in Iscove's modified Dulbecco's medium containing 10% FCS and antibiotics as described (32).
Fura 2-based assay of Ba2+ influx. (6) Cells were grown to confluence in 75-cm2 culture flasks, washed three times with Na-PSS, and incubated for 1 min at 37°C with Na-PSS containing 5 mM EDTA to detach cells from the flask. The suspended cells were centrifuged at 700 g for 1 min, resuspended in Na-PSS + 1 mM CaCl2, centrifuged again, and resuspended in 4-5 ml Na-PSS + 1 mM CaCl2 containing 1% BSA. The cells were divided into 300-µl aliquots and incubated for 30 min at 37°C to recover from the isolation procedure. Individual aliquots of the cells were then incubated with 3 µM fura 2-AM and 0.25 mM sulfinpyrazone (to retard transport of fura 2 out of the cells) for an additional 30 min. The fura 2 and sulfinpyrazone were added as 1,000-fold concentrated stock solutions in DMSO.
After the fura 2 loading period, the cells were centrifuged for several seconds in an Eppendorf mini centrifuge, washed twice, and preincubated for 1 or 5 min in 100 µl of Na-PSS + 10 µM ionomycin, with other additions as indicated for the individual experiments. In some experiments, 50 µM EGTA-AM was included in the preincubation medium to load the cells internally with EGTA. The cells were then added directly to fluorescence cuvettes containing 3 ml of 20/120 Na-K-PSS + 0.3 mM EGTA. After fura 2 fluorescence was monitored for 30 s, BaCl2 (30 µl of 0.1 M solution) was added to the cuvette and fura 2 fluorescence was monitored for an additional 220 s. Fura 2 fluorescence was measured at 510 nm with alternate excitation at 350 and 390 nm using a Photon Technology International RF-M 2001 fluorometer (South Brunswick, NJ); data points were obtained at 1.8-s intervals. All fluorescence values were corrected for autofluorescence using cells that had not been loaded with fura 2. Data are presented as the ratio of fluorescence (R) for excitation at 350 and 390 nm (350/390 ratio) and represent the mean values (±SE; error bars shown in Figs. 1-5) for the number of experiments (n) indicated. Student's one-tailed t-test was used for significance testing; results were considered significant when P was <0.05. The fura 2 signal was calibrated for Ca2+ in digitonin-permeabilized cells according to the procedure of Grynkiewicz et al. (13) and yielded values for Rmax = 10.2, Rmin = 1.51, and fluorescence ratio at 390 nm excitation for free fura 2 over Ba2+-saturated fura 2 = 4.77. On the basis of an average of reported Kd values for the Ba2+-fura 2 complex of 1.51 µM (6), 350/390 ratios of 2, 2.5, 3, 4, and 5 correspond to intracellular Ba2+ concentration ([Ba2+]i) values of 0.4, 0.9, 1.5, 2.9, and 4.9 µM, respectively. With a Kd of 224 nM for the Ca2+-fura 2 complex (13), the corresponding values for [Ca2+]i are 63, 136, 219, 426, and 712 nM, respectively. ![]() |
RESULTS |
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EGTA loading and Na+/Ca2+ exchange. Suspensions of fura 2-loaded CK1.4 cells were incubated for either 1 or 5 min in Ca2+-free Na-PSS with ionomycin, a Ca2+ ionophore, to release Ca2+ from intracellular stores. The cells were then diluted 30-fold into a fluorometer cuvette containing Ca2+-free PSS with a Na+ concentration of 20 mM (20/120 Na-K-PSS; see METHODS). (The concentration of ionomycin following dilution of the cells, 0.33 µM, is sufficient to prevent internal stores from reaccumulating Ca2+; this is shown by the rapid Ca2+ release that occurs when this concentration of ionomycin is added directly to Ca2+-loaded cells.) Gramicidin (1 µM), a pore-forming ionophore for monovalent cations, was added to the cuvette to "clamp" the intracellular Na+ concentration at a value approximately equal to the extracellular concentration.
As shown in Fig. 1, trace a, the addition of 1 mM BaCl2 to CK1.4 cells that had been preincubated with ionomycin for 1 min produced a small initial jump in the fura 2 ratio, followed by a time-dependent increase in the ratio due to Ba2+ influx. The initial rapid jump in the fura 2 signal was due to extracellular fura 2 that had been released into the medium from the cells. Ba2+ influx under these conditions was due to Na+/Ca2+ exchange, since the removal of Na+ from the preincubation and cuvette media nearly abolished the rise in the fura 2 ratio (6). Moreover, when vector-transfected control cells, which do not exhibit Na+/Ca2+ exchange, were used in similar experiments, Ba2+ influx was practically undetectable (data not shown). Additional details on the use of Ba2+ to measure Na+/Ca2+ exchange activity in this kind of assay can be found in Condrescu et al. (6).
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Ca2+ loading and Na+/Ca2+ exchange. To examine the effects of a broad range of [Ca2+]i on Na+/Ca2+ exchange activity, CK1.4 cells were treated with ionomycin and preincubated for 1 min in Na-PSS with 0, 0.1, 0.3, or 1 mM added CaCl2. Figure 3A displays Ba2+ influx for each of the treatment protocols as well as the data for cells preloaded with EGTA-AM (Fig. 1). Each treatment produced a different level of [Ca2+]i in the cells before the addition of Ba2+, and the corresponding rates of Ba2+ influx correlated positively with the initial value of [Ca2+]i. The initial rates of Ba2+ influx, obtained from the slopes of the progress curves 10-30 s following the addition of Ba2+, are plotted in Fig. 3B against the values of [Ca2+]i computed from the average 350/390 ratios during the 10 s immediately before the addition of Ba2+. The data were fit to the Hill equation, yielding a Hill coefficient of 1.6 with a Km = 44 nM (continuous curve in Fig. 3B).
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Ca2+
dependence of exchange activity at high intracellular
Na+
concentration.
Matsuoka and Hilgemann (26) reported that outward exchange currents in
guinea pig cardiac myocytes were practically independent of cytosolic
Ca2+ when intracellular
Na+ was high (100 mM) and
extracellular Na+ was absent. To
examine this issue, fura 2-loaded CK1.4 cells were loaded with a high
concentration of internal Na+ by
preincubating them for 5 min in Na-PSS containing 2 µg/ml gramicidin
and 10 µM ionomycin under the conditions noted below. The cells were
then diluted 30-fold into fluorescence cuvettes containing K-PSS to
assay Ba2+ uptake. The data in
Fig. 5 compare the results for cells when [Ca2+]i
was varied by including either 0.1 mM
CaCl2 (condition
a) or 0.3 mM EGTA + 50 µM EGTA-AM
(condition b) in the preincubation medium. Ba2+ influx under these
conditions was considerably faster than in the previous experiments
because of the high intracellular
Na+ concentration
([Na+]i)
and the absence of extracellular
Na+. As shown in the
inset to Fig. 5, the initial rate of
Ba2+ influx under
condition b
([Ca2+]i = 8 nM) was 46% of that under condition
a
([Ca2+]i = 60 nM). For comparable
[Ca2+]i
values when extracellular Na+
concentration
([Na+]o) = 20 mM [Na+]i
(Fig. 4), Ba2+ influx at the low
[Ca2+]i
was <25% of that at the higher
[Ca2+]i.
The results suggest that exchange activity exhibits a reduced sensitivity to cytosolic Ca2+ when
[Na+]i
is high and
[Na+]o
is low, in accordance with the observations of Matsuoka and Hilgemann
(26).
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DISCUSSION |
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Previous experiments in transfected CHO cells expressing the cardiac exchanger indicated that intracellular Ca2+ stores played a major role in controlling the level of exchange activity (4, 34). Thus release of Ca2+ from the stores increased [Ca2+]i and activated exchange activity, whereas Ca2+ sequestration when the stores were partially depleted lowered [Ca2+]i to a level that blocked activation of exchange activity. The results indicated that quite low [Ca2+]i levels (20-40 nM) were capable of activating exchange activity. However, it was not possible to estimate the K1/2 for Ca2+ in these studies. Kinetic modeling of Ca2+ efflux data in these cells provided independent, albeit indirect, support for regulatory activation at low Ca2+ concentrations (12). The model parameters that best fit the efflux data implied that the exchanger had a high turnover number and low affinity for Ca2+ (Km = 6 µM), with a K1/2 of 94 nM and a Hill coefficient of 4.2 for regulatory Ca2+ activation.
In this report, we directly measured the
Ca2+ sensitivity of exchange
activity over a broader range of
[Ca2+]i
values than examined previously. Exchange activity was half-activated at
[Ca2+]i = 44 nM, and the activation appeared to be slightly cooperative (Hill
coefficient of 1.6). Autoactivation of exchange activity by
Ba2+ entering the cells did not
appear to influence the results. The Kd for
Ba2+ activation of the exchanger
in giant membrane patches was reported to be ~10 µM (33), and
internal Ba2+ concentrations did
not exceed 1-2 µM during the time period over which the rate of
Ba2+ influx was measured
(10-30 s following Ba2+
addition). Autostimulation of exchange activity by
Ba2+ might be a factor in
trace c in Fig. 1 and
trace f in Fig. 3, in which rates of
Ba2+ influx showed a tendency
toward progressive acceleration (slight upward curvature) during the
later portions of the time course (>150 s). These deviations from
linearity were small and were only seen at 350/390 ratios above 2.5 ([Ba2+]i 1 µM). Given these considerations, only the highest measured rates of Ba2+ influx would be
influenced by autostimulation of exchange activity, and any effect
would be very modest in comparison to the
Ca2+-stimulated rate.
Autostimulation could be more important for the high rates of
Ba2+ influx under the high
[Na+]i,
low
[Na+]o
conditions of Fig. 5; even under these conditions, however, there is no
evidence for an accelerating rate of
Ba2+ influx (upward curvature)
during the initial period following Ba2+ addition (cf. Fig. 5,
inset). We conclude that
autostimulation of exchange activity by
Ba2+ has little or no effect on
the rates of Ba2+ influx measured
here.
The K1/2 for Ca2+ activation of exchange activity agreed reasonably well with that measured in guinea pig cardiac myocytes by Miura and Kimura (22 nM; Ref. 29) and by Noda et al. (47 nM; Ref. 31) but was an order of magnitude less than that found in excised membrane patches (300-600 nM) (19, 27, 33). Miura and Kimura (29) observed strong cooperativity for Ca2+ activation (Hill coefficient of 3.7), but this was not confirmed by Noda et al. (31), who reported a Hill coefficient of 1.0. For giant membrane patches in the absence of ATP, Hill coefficients for Ca2+ activation between 1 and 1.5 are generally reported (19, 27, 33). In the presence of ATP, variable results have been obtained and Hill coefficients as high as 3.0 have been observed (5). In general, the results point to an increased affinity of the exchanger for regulatory Ca2+ in whole cells compared with excised membrane patches. There is little agreement, however, as to the degree of cooperativity for Ca2+ activation.
The reasons for the differences in Ca2+ activation of exchange activity in whole cells vs. excised patches are not known. Two obvious considerations involve possible effects of ATP, which is absent in most giant patch studies, and cytoskeletal interactions. Collins et al. (5) examined the effects of ATP on Ca2+ activation in experiments with giant patches from guinea pig myocytes. Although ATP shifted the Ca2+ activation curve somewhat to the left, the magnitude of the shift varied considerably and activating Ca2+ concentrations remained far above the range observed in whole cells. In preliminary experiments in which CK1.4 cells were depleted of ATP using metabolic inhibitors, the rate of exchange-mediated Ba2+ influx was not markedly affected (unpublished data). Thus ATP-dependent effects do not appear to be a plausible explanation for the differences between the patch and whole cell data. Cytoskeletal interactions also seem to be poor determinants of the exchanger's Ca2+ sensitivity, at least in CK1.4 cells. Thus activation of Ba2+ influx by cytosolic Ca2+ in these cells was not greatly altered by treatments that disrupt microfilaments (cytochalasin D or latrunculin B) and/or microtubules (nocodazole) (unpublished observations).
Another possibility is that differences in transmembrane ion gradients between typical whole cell and excised patch experiments may affect the exchanger's sensitivity to Ca2+. In most giant patch studies, the extracellular membrane surface is continuously exposed to millimolar concentrations of Ca2+, conditions that would shift the exchanger's transport sites to the cytosolic surface (the E1 configuration in Hilgemann's terminology; Refs. 19, 20). In many whole cell studies, on the other hand, the extracellular medium is Na+ and Ca2+ free until transport is initiated, and these conditions would shift the transport sites to the extracellular membrane surface (E2 configuration). Matsuoka and Hilgemann (26) have suggested that the E2 configuration might facilitate recovery from the inactivated state produced by the absence of Ca2+, thereby shifting the Ca2+ dependence for activation to lower concentrations. The data in Fig. 5 provide support for this interpretation, since Ba2+ influx was not strongly inhibited by cytosolic EGTA when [Na+]i was high and [Na+]o was low, conditions that would favor the E2 configuration. However, this cannot be the entire explanation for the disparity in Ca2+ activation between the patch and whole cell studies. Thus exchange-mediated Ca2+ efflux in CK1.4 cells is activated at low [Ca2+]i (see the introduction) (12) despite conditions that would favor the E1 configuration (high [Na+]o, low [Na+]i). Moreover, Noda et al. (31) observed a K1/2 for Ca2+ activation of 47 nM in guinea pig myocytes, although 140 mM Na+ was present in the extracellular medium in their experiments ([Na+]i = 20 mM).
A final possibility is that Ca2+ leakage from intracellular stores close to the plasma membrane elevates the local [Ca2+]i above bulk cytosolic levels measured with fura 2. Although the use of ionomycin to deplete intracellular Ca2+ stores would seem to preclude this possibility in our experiments, there may be intracellular compartments that are relatively resistant to ionomycin (e.g., because of internal acidification). Recent results from our laboratory (unpublished observations) suggest that such stores exist in CHO cells. Their possible involvement in modulating Ca2+-dependent activation of exchange activity is currently being investigated.
An explanation for the differences between the Ca2+ activation properties of the exchanger in patches and intact cells therefore remains elusive. Perhaps additional regulatory factors are present in the cells that have been lost when preparing excised patches. The issue is of considerable importance, since identifying the factors that modulate the "set point" for Na+/Ca2+ exchange could prove crucial to understanding the physiological regulation of this important transporter.
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ACKNOWLEDGEMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-49932.
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FOOTNOTES |
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Address for reprint requests: J. P. Reeves, Dept. of Pharmacology and Physiology, New Jersey Medical School, Univ. of Medicine and Dentistry, 185 S. Orange Ave., Newark, NJ 07103
Received 29 December 1997; accepted in final form 30 March 1998.
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