Physiological functions of the regulatory domains of the cardiac Na+/Ca2+ exchanger NCX1

Yan Pan1, Takahiro Iwamoto1, Akira Uehara2, Tomoe Y. Nakamura1, Issei Imanaga2, and Munekazu Shigekawa1

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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 (Delta 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 alpha -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, Delta 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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 Delta 246-672, expressed in CCL39 fibroblasts treated with alpha -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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Chinese hamster lung fibroblasts (CCL39) were obtained from American Type Culture Collection. 45CaCl2, 22NaCl, and [alpha -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 Delta 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.

In the current records shown in Fig. 3, we observed that the outward current remained for ~2 s after removal of Cai2+, whereas it lasted somewhat longer in some experiments. It is difficult to completely rule out the possibility that this residual current arises from Ca2+-activated current(s) other than Na+/Ca2+ exchange. However, this is likely not a possibility for the following reasons: 1) In these experiments we used a very high concentration of intracellular EGTA (50 mM) to minimize a possible change in [Ca2+]i (18). 2) The outward current due to activation of Cl- or K+ currents could not occur, because the reversal potential for the Cl- current (-24 mV) calculated from intracellular and extracellular Cl- concentrations was more positive than the test potential (-40 mV) and because the pipette solution contained Cs+ and tetraethylammonium but not K+. 3) The outward current was not evoked when ionomycin (0.3-3 µM) was added extracellularly to raise [Ca2+]i up to 0.5 µM in cells placed under the same intracellular and extracellular ionic conditions as those shown above, except extracellular CaCl2 and intracellular CaCl2/EGTA were replaced by nominally free Ca2+ (data not shown). We considered that the residual current observed after the solution change was probably due to the Na+/Ca2+ exchange and speculate that complete Ca2+ removal from the surface of cultured CCL39 cells might take place rather slowly after the solution change, although the reason for the slow removal is not clear.

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 [alpha -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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 Delta 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.

We examined expression levels of the wild-type and mutated NCX1s in CCL39 cells by Northern blot (Fig. 1A) or immunoblot analysis (Fig. 1B). In cells transfected with NCX1, XIP-4YW, or D447V/D498I, we detected a strongly hybridizing mRNA band of ~3.1 kb (Fig. 1A). In contrast, a shorter band of 2.0 kb was detected in cells transfected with Delta 246-672, whereas there was no detectable signal in cells transfected with vector alone. By densitometry, we found that the transcripts of D447V/D498I and Delta 246-672 were somewhat more abundant but that of XIP-4YW was 50% less abundant than the wild-type NCX1. On the other hand, immunoblot analysis with a rabbit anti-NCX1 antibody revealed that a 130- to 150-kDa protein was expressed in cells transfected with the wild-type NCX1, XIP-4YW, or D447V/D498I, whereas no protein bands were detected in cells transfected with vector or Delta 246-672. The latter finding can be explained by the facts that CCL39 cells express an extremely low level of endogenous NCX1 (9, 10) and that Delta 246-672 lacks the epitope region for the antibody used (see METHODS). The relative amounts of these proteins, as estimated by densitometry, were comparable to the amounts of the transcripts (Fig. 1).


View larger version (51K):
[in this window]
[in a new window]
 
Fig. 1.   Expression of the wild-type and mutated NCX1s in CCL39 cells. A: Northern blot. Total RNA (10 µg) isolated from control CCL39 cells or transfectants with each NCX construct was loaded onto individual lanes. The blot was hybridized with a probe for NCX1 (top) and then with a probe for beta -actin (middle). The position of the rRNA band is indicated. The expression level of each NCX construct estimated by densitometry was normalized to that of beta -actin and is shown as a percentage of the level for the wild-type NCX1 (bottom). B: Western blot. Microsomes (20 µg/lane) prepared from transfected and nontransfected cells were subjected to immunoblot analysis with anti-NCX1 polyclonal antibody (top). The position of the molecular mass marker is indicated. Densitometric estimates of bands for NCX mutants are presented as a percentage of the level for wild-type NCX1 (bottom).

We further evaluated the expression of the exchangers by immunofluorescence staining with a chicken antibody that recognizes the extracellular loops of the NCX1 protein (10). We found that plasma membrane staining was similar in cells expressing the wild-type NCX1 and D447V/D498I but was significantly less (<= 50%) in cells expressing XIP-4YW or Delta 246-672 (data not shown).

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 Delta 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 Delta 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 Delta 246-672.


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 2.   Time courses of intracellular Na+ (Nai+)-dependent 45Ca2+ uptake into control CCL39 cells and CCL39 cells expressing wild-type and mutated NCX1s. 45Ca2+ uptake into Na+-loaded cells was measured for indicated periods of time. Na+-loaded cells were prepared as described in METHODS. Values are means ± SE (n = 3).


                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Kinetic parameters determined from [Ca2+]o dependences of 45Ca2+ uptake by wild-type and mutant NCX1s

We used the whole cell patch-clamp technique (see METHODS) to measure the outward exchange currents from CCL39 cells expressing various NCX constructs. In our previous study (31) we showed that the whole cell outward current evoked by external application of 2 or 5 mM Ca2+ to NCX1-expressing cells preloaded with 100 mM Nai+ exhibited properties very similar to those of the whole cell current and the giant excised-patch current from the native exchanger of the cardiac myocytes (7, 18). We also showed that this outward current was completely suppressed in the presence of the Na+/Ca2+ exchanger blocker 5 mM Ni2+ or 3 µM KB-R7943 (12) and was not evoked in nontransfected cells (31).

In Fig. 3, the outward current was evoked for 15 s by external application of 1 mM Ca2+ to cells preloaded with 100 mM Nai+. In cells expressing the wild-type NCX1, the outward current increased rapidly and then decayed even in the presence of external Ca2+ (Cao2+) (Fig. 3A). The current decay could not be fitted by a single exponential but fitted well with two exponentials, as in the case of cardiac myocytes (18). In cells expressing D447V/D498I, the generated outward current decayed at a much faster rate (Fig. 3C). In contrast, the decline of the outward current was reduced in cells expressing XIP-4YW or Delta 246-672 (Fig. 3, B and D). We repeated these measurements and found that the percent decline of the outward current over 15 s was 59 ± 4%, 9 ± 3% (P < 0.05 vs. NCX1), 81 ± 4% (P < 0.05 vs. NCX1), and 36 ± 3% (n = 4, P < 0.05 vs. NCX1) for NCX1, XIP-4YW, D447V/D498I, and Delta 246-672, respectively. We similarly measured the outward exchange current in the wild-type NCX1 transfectants that had been loaded with 20 mM Na+ (Fig. 3E). The current decline after the initial peak was 25 ± 4% (n = 4, P < 0.05 vs. NCX1 with 100 mM Nai+), which is much smaller than that in Fig. 3A. It is thus likely that the observed current decline is due to the expression of Nai+-dependent inactivation of the exchanger (7).


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 3.   Typical records of outward exchange currents from CCL39 cells expressing wild-type and mutated NCX1s. Outward exchange currents were evoked by external application of 1 mM CaCl2 for 15 s. A-D: 100 mM Nai+; E: 20 mM Nai+. Cao2+, external Ca2+.

[Ca2+]i transients in CCL39 cells expressing wild-type and mutated NCX1s. We compared [Ca2+]i transients elicited by alpha -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 alpha -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 > Delta 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 alpha -thrombin, but those elicited by ionomycin were much higher (>= 1 µM).


View larger version (28K):
[in this window]
[in a new window]
 
Fig. 4.   Ca2+ transients induced by alpha -thrombin or ionomycin in control CCL39 cells and CCL39 cells expressing wild-type and mutated NCX1s. A-C: fura 2-loaded cells were treated with 2 U/ml of alpha -thrombin (T) or 10 µM ionomycin (IM) in balanced salt solution containing 1 mM Ca2+ in the presence or absence of 146 mM extracellular Na+ (Nao+). A: vector alone; B: wild-type NCX1; C: D447V/D498I. D: summary data for peak values of alpha -thrombin-induced intracellular Ca2+ concentration ([Ca2+]i) transients (solid and hatched bars) and the resting [Ca2+]i (open and stippled bars). Open and solid bars, 146 mM Nao+; stippled and hatched bars, without Nao+.

In Fig. 5, cells expressing NCX constructs were equilibrated in physiological medium containing 146 mM Na+ and 1 mM CaCl2, and a change in [Ca2+]i was induced by exposing cells to Nao+-free medium containing 1 mM CaCl2. In these cells we observed slow transient increases in [Ca2+]i that occur presumably via the reverse mode of Na+/Ca2+ exchange, because an increase in [Ca2+]i was not induced in control CCL39 cells (Fig. 5). The peak values of the [Ca2+]i rise in cells expressing NCX constructs were ordered as follows: wild-type NCX1 > XIP-4YW >=  Delta 246-672 > D447V/D498I.


View larger version (27K):
[in this window]
[in a new window]
 
Fig. 5.   Typical traces of [Ca2+]i transients induced by Nao+ removal in control CCL39 cells and CCL39 cells expressing wild-type and mutated NCX1s. Fura 2-loaded cells were equilibrated in normal balanced salt solution containing 1 mM Ca2+ and then transferred to Na+-free balanced salt solution containing 1 mM Ca2+.

[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 Delta 246-672 transfectants.


View larger version (37K):
[in this window]
[in a new window]
 
Fig. 6.   Nao+ removal-induced changes in [Ca2+]i, viability, and morphology of Na+-loaded cells expressing wild-type and mutated NCX1s. A: cells were loaded with Na+ and fura 2 for 20 min. Changes in [Ca2+]i were induced by switching Na+ loading medium to Na+-free balanced salt solution containing 1 mM Ca2+ and 1 mM ouabain. B: Na+-loaded cells were transferred to Na+-free balanced salt solution containing 1 mM Ca2+ and 1 mM ouabain as in A, and time-dependent changes in cell viability were assayed using trypan blue staining. Viable cells are shown as a percentage of the total cell population (means ± SE, n = 3). * Significantly different from each control. C: morphology of cells expressing wild-type NCX1 (left) or XIP-4YW (right) observed under phase-contrast optics at 0 and 80 min after Nao+ removal. Scale bar, 20 µm.

We examined the viability of cells expressing different NCX constructs that were maintained under conditions identical to those used in Fig. 6A except for fura 2 loading. In the population of cells expressing XIP-4YW or Delta 246-672, the number of cells stained with trypan blue increased in a time-dependent manner, reaching 95 and 60%, respectively, 80 min after removal of Nao+ (Fig. 6B). During this time, cells exhibited morphological changes, such as bleb formation, which were followed by breakdown of the plasma membrane (Fig. 6C and data not shown). Such morphological changes resembled those observed in control CCL39 cells after treatment with ionomycin for a similar amount of time (10), suggesting that cell damage may be caused by prolonged Ca2+ overloading. Consistent with such an interpretation, XIP-4YW or Delta 246-672 transfectants did not exhibit reduced viability when placed in Ca2+-free medium (data not shown). On the other hand, the wild-type NCX1 or D447V/D498I transfectants or control CCL39 cells mostly remained viable at least for 80 min under the conditions used (Fig. 6B), although cells became slightly round (Fig. 6C and data not shown).

We further examined the viability of cells under more physiological salt conditions. Cells expressing different NCX constructs were treated with 1 mM ouabain for 3 h in normal DMEM, and their viability was accessed by trypan blue staining. We found that 96 and 58% of the cell populations expressing XIP-4YW and Delta 246-672, respectively, became trypan blue positive, although 92 and 84% of cells expressing the wild-type NCX1 and D447V/D498I, respectively, remained trypan blue negative. The observed morphological changes were similar to those in Fig. 6C (data not shown).

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 Delta 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 Delta 246-672 (Fig. 7B).


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 7.   Effects of phorbol 12-myristate 13-acetate and ATP depletion on Nai+-dependent 45Ca2+ uptake into cells expressing wild-type and mutated NCX1s. 45Ca2+ uptake into Na+-loaded cells was measured as described in METHODS. A: cells were pretreated with 0 (control) or 0.3 µM phorbol 12-myristate 13-acetate for 20 min. B: cells were pretreated with 10 mM D-glucose (control) or 10 mM 2-deoxy-D-glucose and 2.5 mg/ml oligomycin for 0-20 min. In cells expressing wild-type and mutated NCX1s, uptake activity of control cells was taken as 100%. Values are means ± SE of 3 independent experiments. * Significantly different from each control.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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+ (Kh>= 1 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 Delta 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 Delta 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 alpha -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 Delta 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 alpha -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 alpha -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 Delta 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, Delta 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 Delta 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 Delta 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 Delta 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 Delta 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 Delta 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 Delta 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 Delta 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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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].


Am J Physiol Cell Physiol 279(2):C393-C402
0363-6143/00 $5.00 Copyright © 2000 the American Physiological Society