1 Department of Physiology, University of Bern, CH-3012 Bern, Switzerland; 2 Departments of Physiology and of Molecular Biology and Biophysics and University of Maryland Biotechnology Institute, and 3 Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland 21201
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The mechanism of Ni2+ block of the Na+/Ca2+ exchanger was examined in Sf 9 cells expressing the human heart Na+/Ca2+ exchanger (NCX1-NACA1). As predicted from the reported actions of Ni2+, its application reduced extracellular Na+-dependent changes in intracellular Ca2+ concentration (measured by fluo 3 fluorescence changes). However, contrary to expectation, the reduced fluorescence was accompanied by measured 63Ni2+ entry. The 63Ni2+ entry was observed in Sf 9 cells expressing the Na+/Ca2+ exchanger but not in control cells. The established sequential transport mechanism of the Na+/Ca2+ exchanger could be compatible with these results if one of the two ion translocation steps is blocked by Ni2+ and the other permits Ni2+ translocation. We conclude that, because Ni2+ entry was inhibited by extracellular Ca2+ and enhanced by extracellular Na+, the Ca2+ translocation step moved Ni2+, whereas the Na+ translocation step was inhibited by Ni2+. A model is presented to discuss these findings.
heart; nickel ion quench; fluo 3
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
THE SODIUM/CALCIUM EXCHANGER is an important mechanism for removing Ca2+ from diverse cells. In heart, it extrudes Ca2+ that has entered through Ca2+ channels to initiate contraction (3, 12). The Na+/Ca2+ exchanger transports three Na+ for every Ca2+ it moves in the opposite direction, and it is able to maintain the cytoplasmic Ca2+ concentration ([Ca2+]i) three to four orders of magnitude below the extracellular Ca2+ concentration ([Ca2+]o). Nevertheless, the direction of net Ca2+ transport depends on the electrochemical gradient of Na+ (19, 20, 25). Simultaneous and consecutive transport models have been suggested for Na+ and Ca2+ translocations (11, 22, 25, 26), and a bulk of evidence favors the latter. The individual reaction steps as well as the Na+/Ca2+ exchanger kinetics are largely unknown. One of the major problems for such studies is the absence of any specific pharmacological inhibitor of the Na+/Ca2+ exchanger (31, 32). In the absence of a specific inhibitor, blockade of the Na+/Ca2+ exchanger can be produced by heavy metal ions such as La3+, Mn2+, or Ni2+ (4). Extracellular Ni2+ is the divalent cation most widely used to block the current (INaCa) generated by the electrogenic Na+/Ca2+ exchanger in cardiac myocytes and in other cells (16, 23). To investigate how Ni2+ affects the Na+/Ca2+ exchanger function, an expression system was used. The expression system enabled us to separate the actions of Ni2+ on the Na+/Ca2+ exchanger from nonspecific effects (6). The cloned human cardiac Na+/Ca2+ exchanger (18) was expressed in an insect cell line derived from ovarian cells of Spodoptera frugiperda (Sf 9 cells) using a baculovirus expression vector. This expression system is particularly useful because high levels of expression are achieved (24). Preliminary results of this work have been previously presented (7).
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Expression of Human Na+/Ca2+ Exchanger in Sf 9 Cells
The cloned human Na+/Ca2+ exchanger (18) was subcloned into baculovirus for expression in an insect cell line from S. frugiperda, Sf 9 cells. The Sf 9 insect cell line was maintained to ~1-2 ×106 cells/ml in 100-ml spinner culture flasks at 28°C in Grace's insect medium (Life Science Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum and 50 mg/ml gentamicin (Sigma) with constant stirring at 80 rpm. For expression of the Na+/Ca2+ exchanger, 5 ml of the cell suspension were transferred into a 25-cm2 flask, and the cells were grown into a 70% confluent monolayer. The monolayer of cells was infected with recombinant baculovirus containing the human Na+/Ca2+ exchanger cDNA (18) in serum-free medium for 60 min at a multiplicity of infection of 5. The cultures were maintained for 1-3 days in complete medium after removal of the viral inoculum.Immunofluorescence for Detecting Na+/Ca2+ Exchanger Protein
The immunofluorescent labeling was carried out on uninfected Sf 9 cells and cells 3 days after infection with the recombinant baculovirus. The cells were freshly harvested, resuspended after centrifugation in PBS, and allowed to settle on glass coverslips. The cells were fixed and permeabilized with 95% methanol atConfocal [Ca2+]i Measurements
Membrane potential was controlled in the whole cell configuration of the patch-clamp technique (AxoPatch 200, Axon Instruments, Foster City, CA). For internal dialysis, the cells were patch clamped with pipettes made from filamented glass on a horizontal puller (DMZ Universal Puller, Zeitz Instrumente, Augsburg, Germany). When filled with pipette solution, the pipettes had resistances that ranged between 1.2 and 2.5 M
|
For
[Ca2+]i
measurements, the Sf 9 cells were allowed to settle onto a coated
(Celltak, Becton Dickinson, Heidelberg, Germany) glass surface of a
superfusion chamber mounted on an inverted microscope (Nikon Diaphot)
for 20-30 min. The microscope was equipped with a Zeiss Neofluar
lens (63× oil numerical aperture 1.25). The
Ca2+ indicator fluo 3 (0.1 mM;
Molecular Probes, Eugene, OR) was dialyzed into the cell via the patch
pipette for at least 2 min. During loading with the
Ca2+ indicator, the cells were
held at a membrane potential of 40 mV. Rapid (half time 300 ms)
changes of the extracellular solution [Table 1;
solution
2 with either 155 mM
Na+ or 155 mM
N-methyl-D-glucamine (NMDG) instead of
NaCl] were performed using a gravity-driven superfusion system.
The setup for fluorescence measurements was based on a confocal
laser-scanning microscope equipped with an argon ion laser (available
wavelengths 488 and 514 nm). For excitation of fluo 3, the 488-nm line
of the laser was used (~74 µW on preparation). The fluorescence was
measured at >515 nm. To yield the time course of
Ca2+ signals, images were
repeatedly acquired with a frequency of 1 Hz. The
[Ca2+]i
was computed using the self ratio method with published calibration parameters and assumption of a resting
[Ca2+]i
of 100 nM (5). Timing and data acquisition of scanning and the voltage clamp were controlled by a Apple PowerPC 8100/100 equipped
with a data acquisition board (software developed under LabView,
National Instruments) and synchronized by a custom-made electronic
device. The image analysis was performed with a customized version of
an image-processing software program (National Institutes of Health Image).
45Ca2+ Measurements
45Ca2+ influx. Three days after infection, Na+-dependent 45Ca2+ influx was measured at 32°C in Sf 9 cells. Immediately before the experiment, control and infected cells, grown as a monolayer, were resuspended (1 × 105 cells/ml) in the culture medium. Solution changes were done after spinning down the cells at 1500 rpm for 5 min in a Beckman TJ-6 centrifuge. The cells were preincubated with 140 mM Na+ solution (Table 1; solution 3) containing 100 µM ouabain for 30 min. Then the solution was changed to either Na+-free solution (140 LiCl replaced NaCl) or 140 mM Na+ solution, both containing 2 mCi/ml 45Ca2+ (total 30 µM Ca2+) and 100 µM ouabain (Amersham, Arlington Heights, IL). After 10 min, the cells were washed and 45Ca2+ was counted using a scintillation counter (LS-5000CE, Beckman, Palo Alto, CA). The total counts were normalized to the protein concentration measured with a standard protein assay (Bio-Rad protein assay, Bio-Rad Laboratories, Richmond, CA) at 595 nm using a spectrophotometer (DU 70, Beckman). To block the Na+/Ca2+ exchanger activity, 8 mM NiCl2 was added to 0 Na+ solution.
45Ca2+ efflux. Na+-dependent 45Ca2+ efflux was measured at 32°C in Sf 9 cells 3 days after infection. The cells, grown as a monolayer, were resuspended in 140 mM Na+ solution (solution 3). The cells were then incubated for 2.5 h at 32°C with 225 µM 45Ca2+ in solution 3 (140 LiCl replaced NaCl) to load the cells with isotopic Ca2+. The cells were washed, and the solution was changed to either 140 mM Na+ solution or Na+-free solution (140 LiCl replaced NaCl), both solutions containing nonradioactive Ca2+ at 225 µM. After 10 min, the remaining 45Ca2+ left in cells was determined by scintillation counting. The amount of 45Ca2+ efflux was calculated and normalized to the protein concentration. In some experiments, 8 mM NiCl2 was added to block the Na+/Ca2+ exchanger activity.
63Ni2+ influx measurement. A protocol similar to the 45Ca2+ influx experiments was used with the following differences. Both the 140 Na+ and Na+-free solutions contained 1 mM CaCl2 and, instead of 45Ca2+, 12 µCi/ml 63Ni2+ was used during 10 min of incubation at 32°C. No ouabain was included in these solutions, and 140 mM NMDG was used in the second Na+-free solution (NMDG instead of LiCl). To block the activity of the Na+/Ca2+ exchanger, 3',4'-dichlorobenzamil (DCB; 100 µM) was used. Isotopic 63Ni2+ was used to measure Ni2+ movement (total [Ni2+]o 3 mM). Because a trace amount of 63Ni2+ was supplied only on one side of the membrane, unidirectional flux was measured. When isotopic 63Ni2+ was placed on the side of high Ni2+ concentration and the other side of the membrane contained very little Ni2+, then the trace 63Ni2+ accurately measured net Ni2+ influx. Because there was no active transport of Ni2+, the back flux of Ni2+ was thus negligible. For statistical analysis, the results were calculated as means ± SE, and ANOVA was used to analyze the significance of differences between means.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Na+/Ca2+ Exchanger Expression in Sf 9 Cells
An Sf 9 expression system was used to study the Na+/Ca2+ exchanger transport properties because of the paucity of endogenous channels and transporters in these cells. Expression of the Na+/Ca2+ exchanger was localized in the plasma membrane of these cells, as evidenced by immunofluorescence labeling as shown in Fig. 1A. In addition to the location of the Na+/Ca2+ exchanger in the plasma membrane, some protein is found in intracellular compartments. This is evident in a profile of the fluorescence intensity distribution across an infected Sf 9 cell. Two maxima of fluorescence can be distinguished: 1) a small and narrow peak corresponding to fluorescence in the plasma membrane and 2) two large and broad peaks in the cytoplasmic region on both sides of the nucleus. As observed by others (21), a large amount of exchanger protein is found inside the cells. About 90% of the infected cells in a culture showed positive immunofluorescence labeling of the Na+/Ca2+ exchanger protein. A fraction of the expressed Na+/Ca2+ exchanger protein was also seen within intracellular compartments, and the uninfected cells showed no antibody staining (Fig. 1B).
|
45Ca2+ Uptake via Na+/Ca2+ Exchanger
To test the function of the Na+/Ca2+ exchanger expressed in Sf 9 cells, [Na+]o-dependent Ca2+ influx and Ca2+ efflux were measured. Figure 2A shows 45Ca2+ influx into Sf 9 cells 3 days after infection. Before measurement of Ca2+ influx, cells were placed in a solution containing 100 µM ouabain and normal [Na+]o (140 mM) for 30 min to increase [Na+]i. The cells were then placed in the test solution with 45Ca2+ for 10 min before the cells were washed and the 45Ca2+ activity was counted. When the test solution contained 140 mM Na+, the Ca2+ influx was substantially greater than in uninfected cells (15.1 ± 1.6 vs. 4.6 ± 1.9 nmol · mg protein
|
To study how extracellular Ni2+ affects Ca2+ extrusion by the Na+/Ca2+ exchanger, the cells were first loaded with 45Ca2+ by incubating the cells in 45Ca2+ for 2.5 h. Cells were then washed and placed in the test solution, and 45Ca2+ efflux was measured. Figure 2B shows that 45Ca2+ efflux was significantly greater in infected cells when the test solution contained 140 mM Na+ (162 ± 26 nmol, n = 4) compared with uninfected cells (8.3 ± 3.1 nmol, n = 4, P < 0.01) or compared with infected cells in Na+-free (140 mM Li+) test solution (10.5 ± 3.7 nmol, n = 4, vs. 3.1 ± 0.8 nmol, n = 4, P < 0.01). Importantly, Ni2+ (8 mM) blocked Ca2+ efflux in 140 mM external Na+ (12.7 ± 5.7 nmol, n = 4). The experiments shown in Fig. 2 demonstrate that Ni2+ blocks Na+-dependent Ca2+ influx and efflux.
Fluo 3 Measurements of Na+/Ca2+ Exchanger Activity in Sf 9 Cells
If extracellular Ni2+ blocks the Na+/Ca2+ exchanger in Sf 9 cells, thereby reducing net Ca2+ efflux in Na+-containing solutions, [Ca2+]i should increase when Ni2+ is applied (14). This was examined by measuring [Ca2+]i using the fluorescent Ca2+ indicator fluo 3. We used a confocal imaging method to measure [Ca2+]i, and the Sf 9 cells were patch clamped under whole cell configuration. The membrane potential was clamped at
|
Ni2+ Transport Hypothesis
To test the hypothesis that Ni2+ is transported by the Na+/Ca2+ exchanger as Ni2+ blocks the Na+/Ca2+ exchanger, we measured the 63Ni2+ influx in Sf 9 cells. Three days after infection, cells were quickly washed (2 min) in 140 mM Na+ before being exposed to a test solution containing 63Ni2+. The test solution contained 140 mM Na+, 150 Li+ (0 Na+), or 140 NMDG (0 Na+). When baculovirus-infected cells were incubated with 63Ni2+, in the presence of 140 mM external Na+, a significant amount of Ni2+ influx was measured (46.18 ± 8.6 nmol, n = 4) compared with uninfected cells (4.13 ± 1.1 nmol, n = 4, P < 0.01). Because this result supports the suggestion that Ni2+ is transported by the Na+/Ca2+ exchanger, it would be helpful to determine whether Ni2+ transport by the Na+/Ca2+ exchanger is affected by a different blocker. DCB is an organic blocker of the Na+/Ca2+ exchanger that appears to work by a mechanism completely independent from that of Ni2+ (25). When DCB (100 µM) was added to the test solution, ~35% of the Ni2+ influx was blocked in the 140 mM Na+ test solution, as shown in Fig. 4. However, DCB did not affect the Ni2+ influx in the test solutions containing no Na+ (140 mM Li+ or 140 mM NMDG). In all cases, Ni2+ influx was greater in Sf 9 cells expressing the Na+/Ca2+ exchanger than in the uninfected control cells. This result further supports the notion that Ni2+ influx is mediated by the Na+/Ca2+ exchanger.
|
Influence of External Ni2+ and Ca2+ on Ni2+ Uptake
Enhancement of Ni2+ influx by the presence of 140 mM Na+, as shown in Fig. 4, suggests that the Ni2+ transport into the cell is not competitive with extracellular Na+. This observation is consistent with the idea that Na+/Ca2+ exchanger-dependent Ni2+ influx may be facilitated by the Ni2+ binding to the Ca2+ translocation site on the Na+/Ca2+ exchanger rather than by binding to the Na+ translocation site (see also DISCUSSION). To test this hypothesis, Ni2+ influx was measured under a variety of conditions. First, if Ni2+ binds to the Ca2+ translocation site and is transported in place of Ca2+, it should be inhibited by Ca2+. This hypothesis was tested using 3-day-infected cells. After a brief rinse (2 min) in an extracellular solution containing 140 mM Na+, the cells were exposed to a test solution containing 63Ni2+, 140 mM Na+, and a variable amount of Ca2+ (between 1 µM and 10 mM) for 10 min. Figure 5A shows that external Ca2+ inhibits Ni2+ influx and reveals that 54% of Ni2+ influx is blocked by [Ca2+]o of 10 mM. Although it is clear from Fig. 5A that the Ni2+ influx decreases as [Ca2+]o increases, no clear mechanistic model accounting quantitatively for the inhibition is apparent. A single Michaelis-Menten process cannot explain the data in Fig. 5A. The data are, nevertheless, consistent with Ni2+ being transported via the Ca2+ translocation mechanism of the Na+/Ca2+ exchanger if a more complex inhibitory mechanism is assumed (see Fig. 5B). Future experiments may provide data to distinguish complex competition models composed of, for example, multiple independent Michaelis-Menten processes (see Fig. 5B). Figure 5C shows that increase in [Ni2+]o does enhance the 63Ni2+ influx. Three-day-infected Sf 9 cells were briefly washed (2 min) before being exposed to various Ni2+ concentrations containing 63Ni2+ in the presence of 140 Na+ and 1 mM Ca2+ for 10 min. Half-maximal Ni2+ influx required [Ni2+]o of ~1.2 mM, and the data were fitted with the dose-response equation 63Ni2+ = 40/1 + 10(0.66
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ni2+ has been widely used to block the INaCa in cardiac myocytes since Kimura et al. (16) initially showed that the INaCa was eliminated completely in the presence of 5 mM extracellular Ni2+. Subsequently, Ni2+ has been widely used by others to block the INaCa in many cell types (8, 10, 15, 29). The role of INaCa during the cardiac action potential has been investigated with Ni2+ as a tool to inhibit INaCa (1, 27). Despite its widespread use, the mechanism by which Ni2+ blocks the Na+/Ca2+ exchanger has remained elusive. Indeed, recent experiments by Iwamoto and Shigekawa (14) show that Ni2+ blocks the gene products of NCX1, NCX2, and NCX3, but with varying efficacy. It was assumed that Ni2+ blocked the Na+/Ca2+ exchanger by binding to one of the cation binding sites in the protein and by immobilizing the transport cycle. Detailed knowledge about the reaction steps affected by this inhibitor would provide information about the molecular operation of the Na+/Ca2+ exchanger itself. In this study, we have determined that, although the Na+/Ca2+ exchanger is blocked by Ni2+, the transporter is not fully immobilized. On the contrary, Ni2+ flux occurs across the cell membrane and is due to Ni2+ transport by the Na+/Ca2+ exchanger.
Ni2+ Transport by Na+/Ca2+ Exchanger
When assessing the activity of the Na+/Ca2+ exchange in rat neonatal cardiac myocytes by measuring changes in [Ca2+]i, we noticed that the fluorescence of fluo 3 was reduced below the resting [Ca2+]i during application of high concentrations of Ni2+ (6). It is known that some divalent cations (e.g., Mn2+) (9, 17, 34) can quench the fluorescence of fluo 3. Ni2+ transport in frog skeletal and ventricular muscle cells has been reported, and the Na+/Ca2+ exchange has been implicated in this observation (4, 13). Therefore, we suspected that Ni2+ might be transported into the cardiac cell via some transmembrane pathway, possibly by the Na+/Ca2+ exchange itself, and then quench fluo 3. Insect cells expressing the cloned Na+/Ca2+ exchanger are a suitable preparation to identify and characterize the Na+/Ca2+ exchange as a pathway for Ni2+ entry, because only a few Ca2+ signaling pathways are present in native cells. Infected Sf 9 cells showed abundant expression of the Na+/Ca2+ exchanger protein both in the plasma membrane and in intracellular compartments, as visualized with immunocytochemical techniques using a monoclonal antibody. More importantly, 45Ca2+ flux methods revealed substantial functional activity of the Na+/Ca2+ exchanger proteins in the plasmalemma of infected cells. Uninfected cells did not exhibit any significant 45Ca2+ influx, indicating that there was no detectable endogenous Na+/Ca2+ exchange activity in native Sf 9 cells. On the basis of these control experiments, we concluded that Sf 9 cells transiently expressing the human cardiac Na+/Ca2+ exchanger would be an ideal preparation to test the putative role of this transporter in Ni2+ uptake.We recorded the increase of [Ca2+]i in infected Sf 9 cells during removal of the external Na+, confirming the presence of the Na+/Ca2+ exchanger activity. Interestingly, when 10 mM Ni2+ was added to the superfusion solution, the fluorescence was reduced considerably below the initial intensity. There are three possible explanations: 1) Ni2+ enters the cells and quenches fluo 3 fluorescence, 2) Ni2+ entry powers the extrusion of Ca2+, and 3) both processes (explanations 1 and 2) occur simultaneously. We have shown that Ni2+ can quench fluo 3 fluorescence (6). How could Ni2+ entry power Ca2+ efflux? Let us assume that Ni2+ is transported via the Ca2+ translocation site (see Fig. 5A). If this is true, then a step increase in Ni2+ provides high [Ni2+]o with no [Ni2+]i. Ni2+/Ca2+ exchange will favor Ni2+ entry but not Ni2+ extrusion. Hence as Ni2+ enters, Ca2+ is extruded. Clearly such a mechanism can only work when [Ni2+]i is low. In the steady state, the entry and exit rates for Ni2+ must be in balance. Thus we argue that explanation 3 is correct. However, because of the rapidity of the reduction of fluorescence and its maintained low level, we think explanation 1 is the dominant process. Moreover, when Ni2+ is removed from the extracellular solution, Ni2+ is able to exit the cell by means of Ni2+/Ca2+ exchange and thus the fluorescence increases.
Ni2+ Paradox: Transport and Block
Although Ni2+ transport via the Ca2+ translocation site seems reasonable given the evidence, the question of how it blocks the INaCa is not answered. Fig. 6 shows a model of Na+/Ca2+ exchanger transport. Figure 6A shows how the Na+/Ca2+ exchanger normally transports ions. In contrast, Ca2+/Ca2+ exchange, a partial reaction of the Na+/Ca2+ exchange (Fig. 6B), occurs in the background during normal exchanger activity but is only seen as a measurable process when Na+ transport is blocked (e.g., by Na+ removal or possibly by DCB). Similarly, Ni2+/Ca2+ exchange should be simplest to understand when Na+ transport is blocked (see Fig. 6C). Figure 5 provides evidence that Ni2+/Ca2+ exchange is due to Ni2+ transport on the Na+/Ca2+ exchanger via the Ca2+ translocation site.
|
How can this understanding help us explain why Ni2+ transport is greater when [Na+]o is elevated as in Fig. 4? Figure 4 represents a surprising result that would be hard to interpret without the aid of the experiments shown in Fig. 5. If Ni2+ transport is due to its movement on the Ca2+ binding site on the Na+/Ca2+ exchanger as argued by the data shown in Fig. 5, then the results in Fig. 4 can be explained. The same amount of Ni2+ influx in Na+-free solution with and without DCB suggests that DCB does not interfere with the Ca2+ translocation step. This finding is in agreement with Niggli and Lederer (25) and with the idea that DCB interferes with Na+ binding or Na+ translocation. In voltage clamp experiments also, Ni2+ has been shown to block the INaCa (6, 16, 25). Additionally, the larger Ni2+ influx in Na+-containing solutions suggests that extracellular Na+ has a modulatory (not transport) function, since it is hard to devise a transport scheme binding Na+ influx with Ni2+ influx (on the Ca2+ transport site). Extracellular Na+ modulation of the Na+/Ca2+ exchanger has been reported by many investigators (2, 30). The significant inhibition by DCB of the action of the extracellular Na+ on Ni2+ influx suggests only that the modulation of the Na+/Ca2+ exchanger by extracellular Na+ is altered by DCB and does not provide further insight into the mechanism of action of DCB.
The remaining question is, How does Ni2+ block net transport on the Na+/Ca2+ exchanger? Or, How does Ni2+ block INaCa? Or, How does it block Ca2+ transport? The simplest explanation is that Ni2+ binds to the Ca2+ binding site and thus interferes with the "normal" transport of Na+ and Ca2+. The Na+/Ca2+ exchanger could be blocked if Ni2+ interactions become the rate-limiting translocation step and if this step became much slower than the normal rate-limiting transport step. However, a residual INaCa should be measurable but is not visible (6).
An alternative explanation is that Ni2+ has two actions: 1) it is transported via Ca2+/Ca2+ exchange, and 2) it has an additional action to block Na+ translocation. Reduced INaCa and Na+/Ca2+ exchanger activity and Ca2+ efflux would all be due to a significantly reduced turnover rate of the exchanger. This alternative possibility could be distinguished from the first suggestion by measuring Na+/Na+ exchange during Ni2+ block. The first hypothesis predicts that Ni2+ will not affect Na+/Na+ exchange. If it were inhibited by Ni2+, then the second explanation would be supported. The complete block of INaCa that we have observed favors the second hypothesis.
In conclusion, we have presented experimental evidence that the human cardiac Na+/Ca2+ exchanger can transport Ni2+ as Ni2+ is blocking the Na+/Ca2+ exchanger. Therefore, Ni2+ does not seem to simply lock or immobilize the exchanger in a tightly Ni2+-bound state.
![]() |
ACKNOWLEDGEMENTS |
---|
M. Egger and A. Ruknudin contributed equally to this work.
![]() |
FOOTNOTES |
---|
We are grateful to Dr. H. Porzig for the antibody and to Dr. B. Schwaller for the immunofluorescence staining.
This work was supported by a grant from the Swiss National Science Foundation to E. Niggli (31.50564.97), American Heart Association grants to A. Ruknudin, and National Institutes of Health grants to D. H. Schulze and W. J. Lederer.
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.
Address for reprint requests and other correspondence: D. H. Schulze, Dept. of Microbiology and Immunology, University of Maryland, 655 W. Baltimore St., Baltimore, MD 21201 (E-mail: dschulze{at}umaryland.edu).
Received 26 August 1998; accepted in final form 20 January 1999.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Beuckelmann, D. J.,
and
W. G. Wier.
Sodium-calcium exchange in guinea-pig cardiac cells: exchange current and changes in intracellular Ca2+.
J. Physiol. (Lond.)
414:
499-520,
1989[Abstract].
2.
Blaustein, M. P.,
and
J. M. Russell.
Sodium-calcium exchange and calcium-calcium exchange in internally dialyzed squid giant axons.
J. Membr. Biol.
22:
285-312,
1975[Medline].
3.
Bridge, J. H.,
J. R. Smolly,
and
K. W. Spitzer.
The relationship between charge movements associated with Ica and INa-Ca in cardiac myocytes.
Science
248:
376-378,
1990[Medline].
4.
Brommundt, G.,
and
F. Kavaler.
La3+, Mn2+, and Ni2+ effects on Ca2+ pump and on Na+-Ca2+ exchange in bullfrog ventricle.
Am. J. Physiol.
253 (Cell Physiol. 22):
C45-C51,
1987
5.
Cheng, H.,
W. J. Lederer,
and
M. B. Cannell.
Calcium sparks: elementary events underlying excitation-contraction coupling in heart muscle.
Science
262:
740-744,
1993[Medline].
6.
Egger, M.,
A. Ruknudin,
P. Lipp,
P. Kofuji,
W. J. Lederer,
D. H. Schulze,
and
E. Niggli.
Functional expression of the human cardiac Na+/Ca2+ exchanger in SF9 cells: rapid and specific Ni2+ transport.
Cell Calcium
25:
9-17,
1999[Medline].
7.
Egger, M.,
A. Ruknudin,
E. Niggli,
D. H. Schulze,
and
W. J. Lederer.
Ni2+ uptake mediated by the human Na/Ca exchanger (Abstract).
Biophys. J.
72:
A164,
1997.
8.
Eisenrauch, A.,
M. Juhaszova,
G. C. Ellis Davies,
J. H. Kaplan,
E. Bamberg,
and
M. P. Blaustein.
Electrical currents generated by a partially purified Na/Ca exchanger from lobster muscle reconstituted into liposomes and adsorbed on black lipid membranes: activation by photolysis of Ca2+.
J. Membr. Biol.
145:
151-164,
1995[Medline].
9.
Foder, B.,
O. Scharff,
and
O. Thastrup.
Ca2+ transients and Mn2+ entry in human neutrophiles induced by thapsigargin.
Cell Calcium
10:
477-490,
1989[Medline].
10.
Goldhaber, J. I.
Free radicals enhance Na+/Ca2+ exchange in ventricular myocytes.
Am. J. Physiol.
271 (Heart Circ. Physiol. 40):
H823-H833,
1996
11.
Hilgemann, D. W.,
D. A. Nicoll,
and
K. D. Philipson.
Charge movement during Na+ translocation by native and cloned cardiac Na+/Ca2+ exchanger.
Nature
352:
715-718,
1991[Medline].
12.
Hilgemann, D. W., K. D. Philipson, and G. Vassort (Editors). Sodium-calcium exchange. Ann.
NY Acad. Sci. 779, 1996.
13.
Hoya, A.,
and
R. A. Venosa.
Characteristics of Na+-Ca2+ exchange in frog skeletal muscle.
J. Physiol. (Lond.)
486:
615-627,
1995[Abstract].
14.
Iwamoto, T.,
and
M. Shigekawa.
Differential inhibition of Na/Ca exchanger isoforms by divalent cations and isothiourea derivative.
Am. J. Physiol.
275 (Cell Physiol. 44):
C423-C430,
1998
15.
Kappl, M.,
and
K. Hartung.
Rapid charge translocation by the cardiac Na+-Ca2+ exchanger after a Ca2+ concentration jump.
Biophys. J.
71:
2473-2485,
1996[Abstract].
16.
Kimura, J.,
S. Miyamae,
and
A. Noma.
Identification of sodium-calcium exchange current in single ventricular cells of guinea-pig.
J. Physiol. (Lond.)
384:
199-222,
1987[Abstract].
17.
Kleyman, T. R.,
and
E. J. Cragoe, Jr.
Amiloride and its analogs as tools in the study of ion transport.
J. Membr. Biol.
105:
1-21,
1988[Medline].
18.
Kofuji, P.,
R. W. Hadley,
R. S. Kieval,
W. J. Lederer,
and
D. H. Schulze.
Expression of the Na-Ca exchanger in diverse tissues: a study using the cloned human cardiac Na-Ca exchanger.
Am. J. Physiol.
263 (Cell Physiol. 32):
C1241-C1249,
1992
19.
Leblanc, N.,
and
J. R. Hume.
Sodium current-induced release of calcium from cardiac sarcoplasmic reticulum.
Science
248:
372-376,
1990[Medline].
20.
Lederer, W. J.,
J. R. Berlin,
N. M. Cohen,
R. W. Hadley,
D. M. Bers,
and
M. B. Cannell.
Excitation-contraction coupling in heart cells: roles of the sodium-calcium exchange, the calcium current and the sarcoplasmic reticulum.
Ann. NY Acad. Sci.
588:
190-206,
1990[Abstract].
21.
Li, Z.,
C. D. Smith,
J. R. Smolley,
J. H. B. Bridge,
J. S. Frank,
and
K. D. Philipson.
Expression of the cardiac Na+-Ca2+ exchanger in insect cells using a baculovirus vector.
J. Biol. Chem.
267:
7828-7833,
1992
22.
Luger, P.
Voltage dependence of sodium-calcium exchange: predictions from kinetic models.
J. Membr. Biol.
99:
1-11,
1987[Medline].
23.
Matsuoka, S.,
and
D. W. Hilgemann.
Inactivation of outward Na+-Ca2+ exchange current in guinea-pig ventricular myocytes.
J. Physiol. (Lond.)
476:
443-458,
1994[Abstract].
24.
Miller, L. K.
Insect baculoviruses: powerful gene expression vectors.
Bioessays
11:
91-95,
1989[Medline].
25.
Niggli, E.,
and
W. J. Lederer.
Molecular operations of the sodium-calcium exchanger revealed by conformation currents.
Nature
349:
621-624,
1991[Medline].
26.
Niggli, E.,
and
P. Lipp.
Voltage dependence of Na-Ca exchanger conformational currents.
Biophys. J.
67:
1516-1524,
1994[Abstract].
27.
Noble, D.,
S. J. Noble,
G. C. L. Bett,
Y. E. Earm,
W. K. Ho,
and
I. K. So.
The role of sodium-calcium exchange during the cardiac action potential.
Ann. NY Acad. Sci.
639:
334-353,
1991[Medline].
28.
Porzig, H.,
Z. Li,
D. A. Nicoll,
and
K. D. Philipson.
Mapping of the cardiac sodium-calcium exchanger with monoclonal antibodies.
Am. J. Physiol.
265 (Cell Physiol. 34):
C748-C756,
1993
29.
Rasgado-Flores, H.,
and
M. D. Blaustein.
Na/Ca exchange in barnacle muscle has a stoichiometry of 3Na+/1Ca2+.
Am. J. Physiol.
252 (Cell Physiol. 21):
C499-C504,
1987
30.
Reeves, J. P.,
and
J. L. Sutko.
Competitive interactions of sodium and calcium with the sodium-calcium exchange system of cardiac sarcolemmal vesicles.
J. Biol. Chem.
258:
3178-3182,
1983
31.
Siegl, P. K.,
E. J. Cragoe, Jr.,
M. J. Trumble,
and
G. J. Kaczorowski.
Inhibition of Na+/Ca2+ exchange in membrane vesicle and papillary muscle preparations from guinea pig heart by analogs of amiloride.
Proc. Natl. Acad. Sci. USA
81:
3238-3242,
1984[Abstract].
32.
Slaughter, R. S.,
M. L. Garcia,
E. J. Cragoe, Jr.,
J. P. Reeves,
and
G. J. Kaczorowski.
Inhibition of sodium-calcium exchange in cardiac sarcolemmal membrane vesicles. 1. Mechanism of inhibition by amiloride analogues.
Biochemistry
27:
2403-2409,
1988[Medline].
33.
Stauderman, K. A.,
and
R. M. Pruss.
Dissociation of Ca2+ entry and Ca2+ mobilization responses to angiotensin II in bovine adrenal chromaffin cells.
J. Biol. Chem.
264:
18349-18355,
1989
34.
Trosper, T. L.,
and
K. D. Philipson.
Effects of divalent cations on Na+-Ca2+ exchange in cardiac sarcolemmal vesicles.
Biochim. Biophys. Acta
731:
63-68,
1983[Medline].
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Visit Other APS Journals Online |