Functional comparison of the three isoforms of the Na+/Ca2+ exchanger (NCX1, NCX2, NCX3)

Bettina Linck1, Zhiyong Qiu1, Zhaoping He1, Qiusheng Tong2, Donald W. Hilgemann2, and Kenneth D. Philipson1

1 Departments of Physiology and Medicine and Cardiovascular Research Laboratories, University of California, Los Angeles, School of Medicine, Los Angeles, California 90095-1760; and 2 Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9040

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Three distinct mammalian Na+/Ca2+ exchangers have been cloned: NCX1, NCX2, and NCX3. We have undertaken a detailed functional comparison of these three exchangers. Each exchanger was stably expressed at high levels in the plasma membranes of BHK cells. Na+/Ca2+ exchange activity was assessed using three different complementary techniques: Na+ gradient-dependent 45Ca2+ uptake into intact cells, Na+ gradient-dependent 45Ca2+ uptake into membrane vesicles isolated from the transfected cells, and exchange currents measured using giant patches of excised cell membrane. Apparent affinities for the transported ions Na+ and Ca2+ were markedly similar for the three exchangers at both membrane surfaces. Likewise, generally similar responses to changes in pH, chymotrypsin treatment, and application of various inhibitors were obtained. Depletion of cellular ATP inhibited NCX1 and NCX2 but did not affect the activity of NCX3. Exchange activities of NCX1 and NCX3 were modestly increased by agents that activate protein kinases A and C. All exchangers were regulated by intracellular Ca2+. NCX1-induced exchange currents were especially large in excised patches and, like the native myocardial exchanger, were stimulated by ATP. Results may be influenced by our choice of expression system and specific splice variants, but, overall, the three exchangers appear to have very similar properties.

antiporters; membrane proteins; sodium; calcium

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

THE COUNTERTRANSPORT of three Na+ for one Ca2+ across the plasma membrane of a wide variety of cells is catalyzed by the Na+/Ca2+ exchanger (NCX). The primary role of the exchanger is to extrude Ca2+ from cells, although in some cases the exchanger may also mediate Ca2+ influx. The role of the exchanger is best understood in cardiac muscle, where exchange is the dominant Ca2+ efflux mechanism (32). The Na+/Ca2+ exchanger is also clearly important in Ca2+ homeostasis in tissues such as brain, kidney, and smooth muscle.

Three mammalian isoforms of the Na+/Ca2+ exchanger have been cloned. These isoforms, NCX1 (30), NCX2 (22), and NCX3 (31), are products of distinct genes (31). NCX1 is expressed at high levels in the heart but is also present in most other tissues in varying amounts (17, 19, 35). Several splice variants of NCX1 and NCX3 have also been described (18, 20, 28, 35). Recently, we proposed a nomenclature for the exchanger gene products and associated splice variants (35). The splice variants are expressed in a tissue-specific manner, although the functional consequences of alternative splicing have not been explored. NCX2 and NCX3 are expressed primarily in brain and skeletal muscle (22, 31). At the amino acid level the three exchangers are ~70% identical to one another (31). A distantly related exchanger, a Na+/Ca2+/K+ exchanger has been cloned from the outer segments of rod photoreceptors (36).

All three Na+/Ca2+ exchangers are modeled to have 11 transmembrane segments with a large intracellular loop between the fifth and sixth transmembrane segments. At the functional level, only NCX1 has been well characterized. For example, biophysical analyses of the transport cycle have been reported in detail (8, 11, 15). Mutagenesis experiments indicate that specific regions of the transmembrane segments known as the alpha  repeats are critical in the ion transport process (29). The large intracellular loop of NCX1 is primarily involved in various regulatory properties. For example, mutations within the loop region affect two processes referred to as intracellular Na+-dependent inactivation (7, 10, 26) and Ca2+-dependent regulation (7, 21, 27). That is, the exchanger transports Na+ and Ca2+ but is also separately modulated by both ions. Also, a peptide (XIP) with the same sequence as a region of the exchanger loop (the endogenous XIP region) is a relatively potent inhibitor of NCX1 activity (23). The endogenous XIP region is apparently involved in the Na+-dependent inactivation process (26). NCX1 activity is potently modulated by the level of membrane phosphatidylinositol 4,5-bisphosphate (PIP2) (9), although no information is available on the modulation of other exchangers.

Each of the NCX exchangers has distinctive putative phosphorylation sites, although roles for each of these sites have not been elucidated. NCX1 of rat smooth and cardiac muscle is phosphorylated by protein kinase C, with a resultant modest stimulation of transport activity (12, 13). NCX1, NCX2, and NCX3 have similar concensus sites for tyrosine phosphorylation, and in a preliminary report (2) NCX2 was found to be inhibited by genistein, a tyrosine kinase inhibitor. The Na+/Ca2+ exchangers in squid axon (4) and frog myocardium (5) are modulated by phosphorylation reactions.

In this study we compare, for the first time, the functional properties of NCX1, NCX2, and NCX3 in stably transfected BHK cells. We examined transport and regulatory properties of the three exchangers. 45Ca2+ fluxes were assessed in whole cells and in vesicles prepared from the cells. In addition, giant patches excised from transfected cells were used for electrophysiological analysis of the exchangers.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Cells. As described previously (31), BHK cells were stably transfected with pNUT transfer vectors containing cDNA inserts encoding for canine cardiac NCX1 (30), rat brain NCX2 (22), or rat brain NCX3 (31).

Membrane vesicle preparation. Transfected BHK cells were grown overnight on 150-mm petri dishes in the presence of 60 µM ZnCl2. The cells were scraped from the plates and homogenized with 20 strokes in a Teflon-glass homogenizer. Soluble proteins were removed by centrifuging the homogenate for 5 min at 10,000 rpm at 4°C and washing the pellet twice with 140 mM NaCl and 10 mM 3-(N-morpholino)propanesulfonic acid-tris(hydroxymethyl)aminomethane, pH 7.4. The resuspended pellet was centrifuged at 2,000 rpm for 5 min to remove aggregated particles. The supernatant was collected and centrifuged for 5 min at 10,000 rpm at 4°C to pellet the membrane vesicles. Vesicles were resuspended in the same solution to 0.5-1.5 mg protein/ml. All operations were carried out on ice. Aliquots of membrane vesicles were frozen and stored at -70°C until use. Membranes were solubilized and reconstituted as described previously (38).

45Ca2+ uptake assay in cells. The Ca2+ uptake assays in nontransfected BHK cells and cells transfected with NCX1, NCX2, or NCX3 were performed as described previously (24). Briefly, Na+-loaded cells are diluted into media containing 45Ca2+ and either K+ (to measure Na+ gradient-dependent Ca2+ uptake) or Na+ (to measure the small Na+ gradient-independent Ca2+ uptake). These values were subtracted as a measure of Na+/Ca2+ exchange.

45Ca2+ uptake assay in membrane vesicles. Intracellular Na+-dependent Ca2+ uptake was measured by diluting 10 µl (0.5-1.5 mg/ml) of membrane vesicles into 240 µl of Ca2+ uptake medium containing 140 mM KCl or NaCl, 10 mM 3-(N-morpholino)propanesulfonic acid-tris(hydroxymethyl)aminomethane, pH 7.4, 10 µM CaCl2, 0.3 µCi of 45CaCl2, and 0.36 µM valinomycin (37°C). Ca2+ uptake was terminated after 1 s by addition of 30 µl of 140 mM KCl and 10 mM ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and subsequent addition of 1 ml of 140 mM KCl and 1 mM EGTA at 4°C. One milliliter of the reaction mixture was applied to a Millipore nitrocellulose filter (0.45 µm). The filter was washed twice with 3 ml of 140 mM KCl and 1 mM EGTA at 4°C and then subjected to scintillation counting. Data are presented as the Na+ gradient-dependent uptake of 45Ca2+ (uptake in the NaCl-containing medium is subtracted from uptake in the KCl-containing medium). Ca2+ concentrations have been corrected by 2.5 µM Ca2+, the approximate level of contamination in solutions. We have used these techniques extensively in previous studies (38).

Assay of Na+/Ca2+ exchange activity in giant membrane patches. Outward Na+/Ca2+ exchange currents were measured using the giant excised patch technique (7). In all cases, Ca2+ was present within the patch pipette at the extracellular surface. Outward currents were initiated by the application of Na+ to the bathing medium at the cytoplasmic surface. Solutions were the same as those used to record exchange currents in patches from cardiomyocytes (10). Unless indicated otherwise, cytoplasmic free Ca2+ was 0.5 µM. Mild trypsin treatment (0.5 mg/ml for 1 min) was used to remove BHK cells from dishes and to facilitate seal formation. Pipettes were prepared with inner tip diameters slightly smaller than the diameter of the free BHK cells (~12 µm). Pipettes were coated with a light mineral oil-Parafilm mixture. Cells were briskly sucked into the pipette orifice, and gigohm seals formed around the entire cell (>50% success). The outer half of the cell was removed with a smaller pipette with use of suction and lateral manipulations to tear it away.

Materials. Forskolin, phorbol 12-myristate 13-acetate (PMA), genistein, phenylarsine oxide (PAO), cantharidin, calphostin C, dibutyryl cAMP, isoproterenol, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), rotenone, oligomycin, alpha -chymotrypsin, cytochalasin D, phosphatidylcholine (PC), phosphatidylserine (PS), and cholesterol were obtained from Sigma Chemical, asolectin from Associated Concentrates, and 2-{2-[4-(4-nitrobenzyloxy)phenyl]ethyl}isothiourea methanesulfonate (KB-R7943) from Kanebo (Osaka, Japan). Peptides XIP1 (RRLLFYKYVYKRYRAGKQRG), XIP2 (RRLLFYKYVYKRYRTDPRSG), and XIP3 (KRLLFYKYMHKRYRTDKHRG) were synthesized by Bayer (Wuppertal, Germany). Peptide FRCRCFa was a kind gift from Dr. D. Khananshvilli.

Data analysis. Data were compared using Student's t-test. Differences were considered statistically significant if P < 0.05. Values are means ± SE.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Na+/Ca2+ exchange activity in stably transfected BHK cells. Intracellular Na+-dependent Ca2+ uptake was measured in BHK cells stably transfected with NCX1, NCX2, or NCX3 (Fig. 1). Substantial Na+/Ca2+ exchange activity was seen with each of the three exchangers. Little background Ca2+ uptake was observed with nontransfected BHK cells. Maximal Ca2+ uptake occurred after 5-10 min. At 50 µM Ca2+ (Fig. 1), cells transfected with NCX1 had the largest Ca2+ uptake, followed by NCX3 and NCX2 transfected cells. Fluorescent labeling of cells with antibodies against the corresponding NCX showed intense staining of the membrane in transfected cells but no detectable signal in the nontransfected cells (not shown). ZnCl2 (60 µM) was added to the medium 16 h before the uptake assay to induce the expression of the NCX proteins under the control of the metallothionein promoter. The preincubation with ZnCl2 increased Na+/Ca2+ exchange activity by only ~20-30% for NCX1, NCX2, and NCX3 (n = 1-2). Presumably, the level of contaminating heavy metals in the culture medium was sufficient to activate the metallothionein promoter, even in the absence of exogenous Zn2+.


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Fig. 1.   Time course of intracellular Na+-dependent 45Ca2+ uptake by nontransfected BHK cells (open circle ) or BHK cells expressing NCX1 (bullet ), NCX2 (black-square), or NCX3 (black-triangle). Ca2+ uptake occurred in presence of 50 µM Ca2+ (n = 2-4). All data are corrected for Ca2+ uptake that occurred in absence of an outwardly directed Na+ gradient (i.e., Ca2+ uptake from an Na+ medium rather than a K+ medium). This Na+ gradient-independent Ca2+ uptake was <10% of uptake in presence of an Na+ gradient. This small correction has also been made for data presented in subsequent figures. Some error bars are omitted for clarity.

Influence of Ca2+, Na+, and pH on intracellular Na+-dependent Ca2+ uptake in cells. Figure 2 shows the dependencies on extracellular Ca2+ for the three exchangers. The K1/2(Ca2+) was similar for NCX1, NCX2, and NCX3, with values of 140, 100, and 130 µM, respectively. Maximum reaction velocities (Vmax) were ~14, 7, and 13 nmol · mg protein-1 · min-1 for NCX1, NCX2, and NCX3, respectively. Thus the maximal activities of NCX1 and NCX3 were comparable and were about twofold higher than those of NCX2. Interpretation of relative Vmax values is not possible, since we cannot determine whether equal amounts of the different exchangers are being expressed in the BHK cells.


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Fig. 2.   Effect of extracellular Ca2+ concentration ([Ca2+]) on intracellular Na+-dependent Ca2+ uptake in nontransfected BHK cells (open circle ) or BHK cells expressing NCX1 (bullet ), NCX2 (black-square), or NCX3 (black-triangle). Ca2+ uptake was measured for 1 min. Calculated K1/2 values are 140 µM for NCX1 (n = 4-5), 100 µM for NCX2 (n = 4-6), and 130 µM for NCX3 (n = 4-6).

It is difficult to directly measure apparent Na+ affinities using flux studies. Thus interaction of extracellular Na+ with the Na+/Ca2+ exchangers was assessed indirectly by measuring the ability of extracellular Na+ to inhibit extracellular Ca2+ uptake. This inhibition may be due to the ability of extracellular Na+ to compete with extracellular Ca2+ for binding sites on the exchanger but may also indicate a kinetic influence of Na+ on different modes of operation of the exchanger. We measured intracellular Na+-dependent 45Ca2+ uptake into the cells at various extracellular Na+ concentrations (Fig. 3). Extracellular Na+ inhibited Ca2+ uptake with K1/2 values of 14 mM for NCX1, 11 mM for NCX2, and 15 mM for NCX3. Overall, the results indicate that NCX1, NCX2, and NCX3 have similar binding affinities for extracellular Ca2+ and Na+.


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Fig. 3.   Effect of extracellular Na+ concentration ([Na+], 0-140 mM) on intracellular Na+-dependent Ca2+ uptake in nontransfected BHK cells (open circle ) or BHK cells expressing NCX1 (bullet ), NCX2 (black-square), or NCX3 (black-triangle). Ca2+ uptake was measured for 1 min in presence of 50 µM Ca2+. K1/2 values were ~14 mM for NCX1 (n = 3), 11 mM for NCX2 (n = 3), and 15 mM for NCX3 (n = 3).

We also investigated the response of the exchangers to alterations of external pH. As shown in Fig. 4, the activity of exchanger-expressing cells increased monotonically as pH was raised from 5 to 9. Activities were negligible at pH 5. The activity of NCX1 increased much more substantially than the activities of NCX2 and NCX3 as pH was raised from pH 5 to 6. 


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Fig. 4.   Effect of pH on intracellular Na+-dependent Ca2+ uptake in nontransfected BHK cells (open circle ) or BHK cells expressing NCX1 (bullet ), NCX2 (black-square), or NCX3 (black-triangle). Ca2+ uptake was measured for 1 min in presence of 50 µM Ca2+ (n = 2-3).

Inhibition of Na+/Ca2+ exchange activity in BHK cells. Cytoplasmic Ca2+ stimulates intracellular Na+-dependent Ca2+ uptake by binding to a regulatory site on the intracellular surface of the exchanger molecule (3, 7). Although this site on NCX1 has been analyzed in detail, only minimal evidence for the presence of a Ca2+ regulatory site on NCX2 and NCX3 is available. To detect secondary Ca2+ regulation of the three exchangers, we preincubated cells for 20 min in the presence of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-acetoxymethyl ester (20 µM) to chelate intracellular Ca2+. Table 1 shows diminished Ca2+ uptake in BAPTA-acetoxymethyl ester-pretreated cells for all three Na+/Ca2+ exchangers. Thus Ca2+ regulation of NCX1, NCX2, and NCX3 can be detected in intact cells.

We tested the effects of KB-R7943, a recently described inhibitor of Na+/Ca2+ exchange activity (14, 39), on the transfected BHK cells. KB-R7943 is an isothiourea derivative and inhibits the exchange activity of intact cells and membrane vesicles. Cells were pretreated with 30 µM KB-R7943 for 20 min and then used for Ca2+ uptake assays. Although the compound has been reported to be a potent inhibitor (50% inhibitory concentration = 1.2-2.4 µM) in cardiomyocytes and NCX1-transfected fibroblasts, we found only weak inhibition of NCX1, NCX2, and NCX3 exchange activities in BHK cells (Table 1).

                              
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Table 1.   Effects of various compounds on Na+/Ca2+ exchange activity in BHK cells

Effect of ATP depletion and cytochalasin D on Na+/Ca2+ exchange activity in BHK cells. ATP depletion and cytochalasin D inhibit the activity of NCX1 expressed in Chinese hamster ovary (CHO) cells (1). Both interventions were also observed to disrupt actin microfilaments, and it was suggested that exchanger inhibition was secondary to changes in the actin cytoskeleton (1). We compared the effects of ATP depletion and cytochalasin D on NCX1, NCX2, and NCX3 in BHK cells. Cells were depleted of ATP by preincubation with the mitochondrial inhibitors rotenone (2 µM) and oligomycin (2.5 µg/ml) for 20 min and then assayed for exchange activity. As shown in Table 1, ATP depletion induced a reduction of Na+/Ca2+ exchange activity for NCX1 and NCX2, whereas the activity for NCX3 remained unaffected. In separate experiments the cytoskeleton was disrupted by 3 µM cytochalasin D for 30 min before Ca2+ uptake assays. Cytochalasin D did not influence Na+/Ca2+ exchange activity (Table 1). Levels of ATP depletion and cytoskeletal disruption were not assessed.

Possible regulation of NCX1, NCX2, and NCX3 by phosphorylation. We examined the effects on NCX1, NCX2, and NCX3 activities of various agents that influence different protein kinases and phosphatases (Table 2). Intracellular Na+-dependent Ca2+ uptake was measured for 1 min in the presence of 50 µM Ca2+ after preincubation with each agent for 30 min. Forskolin (100 µM), an activator of adenylyl cyclase, enhanced the activity of BHK cells expressing NCX3 by 42%. This effect was antagonized by H-7 (50 µM), an inhibitor of nucleotide-dependent protein kinases (not shown). Moreover, isoproterenol (100 µM) and dibutyryl cAMP (50 µM), which also stimulate adenylyl cylase, increased Na+/Ca2+ exchange activity of NCX3-expressing cells by ~20% (data not shown). The results suggest that a protein kinase A-dependent pathway can modulate NCX3 activity. Modest effects of forskolin on NCX1 and NCX2 were also observed. Possible influences of protein kinase C on Na+/Ca2+ exchange activities were determined by pretreatment with 0.3 µM PMA. Modest enhancements of activity for NCX1 and NCX3 were observed (P > 0.05). Protein kinase C is downregulated by prolonged exposure to PMA. Therefore, cells were incubated with 0.1 µM PMA overnight. Intracellular Na+-dependent Ca2+ uptake was inhibited in cells expressing NCX1 and NCX3. Stimulation after short-term incubation and inhibition after long-term incubation with PMA support a possible protein kinase C-dependent activation for NCX1 and NCX3, although the effect was not blocked by calphostin C, an inhibitor of protein kinase C (not shown). Genistein (a tyrosine kinase inhibitor) and cantharidin (an inhibitor of protein phosphatases type 1 and 2A) had no effects on Na+/Ca2+ exchange activity. In preliminary experiments the tyrosine phosphatase inhibitor PAO also had no effect on Na+/Ca2+ exchange activities.

                              
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Table 2.   Effect of phosphorylation on Na+/Ca2+ exchange activity in BHK cells

Influence of Ca2+ and Na+ on Na+/Ca2+ exchange in membrane vesicles. Flux measurements using intact cells (above) provide ready access to the extracellular surface of the Na+/Ca2+ exchanger. To investigate the effects of ions at the intracellular surface of the exchanger, we prepared membrane vesicles from BHK cells transfected with NCX1, NCX2, or NCX3. The Na+/Ca2+ exchange of membrane vesicles primarily reflects transport by inside-out vesicles (23). The dependence of exchange on extravesicular Ca2+ is shown in Fig. 5A. All three exchangers had K1/2(Ca2+) values of ~12 µM, similar to the apparent Ca2+ affinity of cardiac sarcolemmal vesicles. The membrane vesicles exhibited Vmax values of ~16, 6, and 6 nmol · mg protein-1 · s-1 for NCX1, NCX2, and NCX3, respectively. Figure 5B shows vesicular Ca2+ uptake as a function of time in the presence of 10 µM Ca2+. The time course was similar for NCX1, NCX2, and NCX3. It is somewhat striking that Ca2+ uptake plateaus more rapidly (1-2 s) than previously observed with use of cardiac sarcolemmal vesicles (33, 38). The reason for this discrepancy is unknown. The effects of extravesicular Na+ on Ca2+ uptake are presented in Fig. 6. Na+ inhibited Ca2+ uptake with K1/2 of ~8 mM for NCX1, NCX2, and NCX3. Thus the interactions of NCX1, NCX2, and NCX3 with extravesicular Na+ and Ca2+ were similar.


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Fig. 5.   Effect of extravesicular Ca2+ on intracellular Na+-dependent Ca2+ uptake in membrane vesicles isolated from nontransfected BHK cells (open circle ) or BHK cells expressing NCX1 (bullet ), NCX2 (black-square), or NCX3 (black-triangle). A: Ca2+ dependency was determined as a function of extravesicular Ca2+ (0-200 µM). Ca2+ uptake was measured for 1 s and revealed K1/2 of ~12 µM for NCX1, NCX2, and NCX3 (n = 3-4). B: Ca2+ uptake was measured as a function of time (1-60 s) in presence of 10 µM Ca2+ (n = 4).


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Fig. 6.   Effect of extravesicular Na+ (0-140 mM) on intracellular Na+-dependent Ca2+ uptake in membrane vesicles isolated from cells expressing NCX1 (bullet ), NCX2 (black-square), or NCX3 (black-triangle). Ca2+ uptake was measured in presence of 10 µM Ca2+. K1/2 was ~8 mM for NCX1, NCX2, and NCX3 (n = 2-5).

Inhibition and stimulation of Na+/Ca2+ exchange activity in membrane vesicles. We tested the effects of the Na+/Ca2+ exchange inhibitor KB-R7943 (14, 39). At 10 µM, KB-R7943 inhibited the activities of the three exchangers by 30-45% (Table 3). This potency is lower than that described by Iwamoto et al. (14) (inhibitory constant = 5.4 µM) for the inhibition of intracellular Na+-dependent Ca2+ uptake by cardiac sarcolemmal vesicles. We observed this same reduced potency with canine cardiac sarcolemmal vesicles (not shown). FRCRCFa, a positively charged cyclic hexapeptide, has also been described to inhibit the Na+/Ca2+ exchange activity of cardiac sarcolemmal vesicles (16). We found that a high concentration of FRCRCFa (50 µM) had only modest inhibitory effects on NCX1, NCX2, and NCX3 (Table 3). We found a similar lack of potency using canine cardiac sarcolemmal vesicles. KB-R7943 and FRCRCFa were also tested at other concentrations, and consistently low inhibitory potencies were obtained (not shown).

                              
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Table 3.   Effects of various agents on Na+/Ca2+ exchange activity in membrane vesicles

Proteinase pretreatment stimulates Na+/Ca2+ exchange activity in membrane vesicles (34). The response to proteinase treatment of intracellular Na+-dependent Ca2+ uptake in membrane vesicles containing NCX1, NCX2, or NCX3 is shown in Table 3. Membrane vesicles were preincubated with 1 U of alpha -chymotrypsin for 20 min and immediately used for Na+/Ca2+ exchange assays. alpha -Chymotrypsin stimulated the activity of all three Na+/Ca2+ exchangers.

Inhibitory effect of different XIPs on NCX1 activity. A peptide XIP with the same amino acid sequence as an autoregulatory region of NCX1 is a relatively potent inhibitor of Na+/Ca2+ exchange (23). NCX2 and NCX3 have homologous XIP regions. We compared the relative potency of each of the three corresponding peptides (XIP1, XIP2, and XIP3) on NCX1, NCX2, and NCX3. The amino acid sequences of the three peptides (20-mers) are listed in METHODS. Data obtained using membrane vesicles prepared from NCX1-transfected BHK cells are shown in Fig. 7. All three XIPs were relatively poor inhibitors of Na+/Ca2+ exchange activity (~10% inhibition at 2 µM). The three XIP peptides were also only weak inhibitors of the exchange activity of membrane vesicles prepared from BHK cells expressing NCX2 and NCX3 (not shown). These data disagree with some of our previous results; XIP1 at this concentration inhibits NCX1 of cardiac sarcolemmal vesicles by ~50% (6, 23). Also, XIP2 had been found to be a poorer inhibitor of NCX1 than XIP1 (6). We repeated these experiments using canine cardiac sarcolemmal vesicles (Fig. 7) and confirmed our earlier results. The difference between the potencies of XIP peptides to inhibit the exchanger in BHK and those in sarcolemmal membranes is striking. To test the hypothesis that membrane lipid environment affects XIP potency, we solubilized and reconstituted NCX1 from BHK cells into liposomes. When NCX1 was reconstituted with asolectin as the source of lipid, the potency of XIP1 increased (Fig. 7) but was still less than that observed in sarcolemmal vesicles. However, when the liposomes were composed of 55% PC-25% PS-20% cholesterol, full XIP potency was restored.


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Fig. 7.   Effects of XIP1, XIP2, and XIP3 on NCX1 activity. Intracellular Na+-dependent 45Ca2+ uptake was assayed in membrane vesicles prepared from canine myocardium (SL) or from BHK cells expressing NCX1. Exchange activity of proteoliposomes reconstituted from solubilized BHK-NCX1 membranes was also assayed. Lipids used for reconstitution were asolectin (Aso) and 55% phosphatidylcholine-25% phosphatidylserine-20% cholesterol (PS). XIP peptide concentration was 2 µM. n = 3.

Giant patch recordings of exchange currents from transfected BHK cells. To further compare NCX1, NCX2, and NCX3, inside-out giant patches were formed from the transfected BHK cells for electrophysiological analysis. Relatively large BHK cells (15-20 µm diameter) were selected for giant patch experiments, and "half-cell" excised patches were prepared. Briefly, cells were sucked onto the pipette orifice, and then the outer half of the cell was removed by suction with a second smaller pipette (see METHODS). Na+/Ca2+ exchange currents recorded from patches from NCX1-expressing cells (Fig. 8A) were similar to those previously recorded from cardiac membranes. However, current densities (10-50 pA/pF) were about five times larger than those in guinea pig cardiac membrane patches. On application of bath (cytoplasmic) Na+, an outward exchange current is generated. The current partially decays to a new steady-state level (Na+-dependent inactivation). Addition of ATP to the bath stimulates exchange activity, as also occurs with patches from cardiac cells (9). Direct application of anionic phospholipids, such as PIP2, to the patch also elicited this stimulatory response (not shown). After ATP is removed, the stimulatory effects are reversed over a period of minutes. For the experiment in Fig. 8A, 0.5 µM cytoplasmic Ca2+ was present at all times. Exchange currents were abolished on removal of this bath Ca2+ (not shown). Thus NCX1 was fully regulated by cytoplasmic Ca2+.


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Fig. 8.   Giant patch recordings from BHK cells transfected with NCX1 (A), NCX2 (B), or NCX3 (C). Ca2+ was present at all times within patch pipette at extracellular surface. Outward Na+/Ca2+ exchange currents were initiated by application of Na+ to intracellular surface of excised patch. No exchange currents were elicited in nontransfected BHK cells. Cytoplasmic regulatory Ca2+ was 0.5 µM, except where otherwise noted. Ca2+i, intracellular Ca2+; Na+i, intracellular Na+.

Exchange currents recorded from NCX2-expressing cells (Fig. 8B) were not as well regulated as those previously reported from oocytes (22) and were smaller than those obtained from NCX1-expressing cells. Na+-dependent inactivation was small or absent, and stimulatory effects of ATP were also small. A large component of outward current was insensitive to removal of cytoplasmic Ca2+. This lack of regulation is characteristic of an exchanger that has been partially proteolyzed. Perhaps NCX2 is susceptible to a protease of BHK cells that becomes activated during patch formation. Exchange currents recorded from patches of NCX3-expressing cells were small and often close to the level of good resolution. NCX3 exchange currents showed Na+-dependent inactivation and were reduced 50-80% by removal of cytoplasmic regulatory Ca2+, but ATP effects were small or absent. It is notable that NCX3 expressed well, as assessed by isotope flux studies, but expressed poor activity in excised patches. At least 20 patches were analyzed for each exchanger isoform.

Overall, there is a striking discrepancy between functional results for NCX2 and NCX3 using Ca2+ flux techniques and those obtained using excised patches. The patches displayed small current densities for NCX2 and NCX3 relative to those for NCX1. We tested a number of possibilities to account for this discrepancy, assuming that exchangers might become inactivated after removal of cells from dishes or during patch formation. A variety of phosphatase inhibitors (fluoride, vanadate, pyrophosphate, and okadaic acid), protease inhibitors, and the microfilament stabilizer phalloidin were included in the incubation medium before experiments and in the solution into which patches were excised. The magnitudes of the NCX2 and NCX3 currents remained small. Tests for stimulation of exchange currents by G protein subunits were negative for all three isoforms.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

Although much insight has been gained on the workings of NCX1, little analysis has been reported on the more recently cloned NCX2 and NCX3. Previous functional analysis of NCX2 used only electrophysiological techniques (22). NCX3 expression has been observed using 45Ca2+ fluxes with intact oocytes or transfected BHK cells, but no kinetic characterization was reported (31). These three mammalian exchanger isoforms are ~70% identical to one another at the amino acid level and display unique patterns of tissue distribution (31, 35). Here we express NCX1, NCX2, and NCX3 at high levels in stably transfected BHK cells. Using these cells, we are able to assess exchange activities with three distinctive assays: 1) Na+ gradient-dependent 45Ca2+ uptake into transfected BHK cells, 2) Na+ gradient-dependent 45Ca2+ uptake into membrane vesicles prepared from the BHK cells, and 3) outward Na+/Ca2+ exchange currents in giant patches excised from the transfected cells. The assay using intact cells allows ready access to the extracellular surface, whereas the assay using vesicles allows access to the cytoplasmic surface (23). Thus interactions of ions and other substances at either surface of the exchanger can be evaluated. Use of giant patches also provides availability to the cytoplasmic surface but, importantly, also permits analysis of certain regulatory properties that cannot be studied by other techniques. It is striking that sizable exchange currents (especially for NCX1) could be measured with excised patches only ~12 µm in diameter. These currents were comparable in magnitude to those typically measured using giant patches 30 µm in diameter excised from cardiac myocytes or from oocytes expressing NCX1. The exchanger density in membranes from transfected BHK cells is about fivefold higher than in myocytes or oocytes; use of this system is highly promising for future studies.

We were unable to detect any striking differences in the interactions of Ca2+, Na+, or H+ with transport sites on the three different exchangers. Apparent affinities for Ca2+ at the extracellular and intracellular surfaces of each of the exchangers were ~120 and 12 µM, respectively. This asymmetry in apparent binding constants for Ca2+ on the two sides of the membrane has been observed previously (32). The ion transport sites on the three different exchanger proteins would appear to have similar molecular architecture.

Condrescu et al. (1) reported that ATP depletion inhibits the activity of NCX1 in stably transfected CHO cells. We were able to reproduce this finding for NCX1 in BHK cells and have a similar result for NCX2 (Table 1). In contrast, NCX3 activity appeared to be unaffected by ATP depletion. Hilgemann and Ball (9) (see below) found that ATP stimulates the NCX1 exchanger in giant patches by increasing the level of membrane PIP2. It is unclear whether this mechanism is relevant during cellular ATP depletion, however, inasmuch as ATP depletion would be expected to alter many cellular functions. Thus we are unable to speculate on the lack of effect of ATP depletion on NCX3. Condrescu et al. found that ATP depletion disrupted the actin cytoskeleton of CHO cells and that the functional effects of ATP depletion were mimicked by cytochalasin D. The authors suggested that the changes in the actin cytoskeleton affected exchanger activity. In contrast, however, we find no effects of cytochalasin D on exchange activity (Table 1). Cytoskeletal interactions in CHO cells may be different from those in BHK cells, but in any case, the inhibitory effects of ATP depletion on NCX1 and NCX2 do not appear to be linked to cytoskeletal disruption in BHK cells.

NCX1 of arterial myocytes and of cardiac myocytes has been reported to be modestly stimulated by phosphorylation mediated by protein kinase C (12, 13). The Na+/Ca2+ exchanger of frog myocardium is markedly inhibited after beta -adrenergic stimulation (5). Also, the exchanger of the squid giant axon is markedly stimulated by ATP, possibly because of a phosphorylation event (4). We tested the effects on exchanger activities of a variety of agents that modify protein phosphorylation state. Each of the exchangers contains multiple potential phosphorylation sites (22, 30, 31, 35). The most prominent effects we observed occurred with NCX3 (Table 2). Activity of NCX3 was increased by treatment of cells with agents that activate protein kinase A, although small effects were also seen with NCX1 and NCX2. NCX3 does possess unique sites for potential phosphorylation by protein kinase A (31), which may account for our results. Consistent with earlier work, we found that PMA activated NCX1 and NCX3 and that overnight treatment with PMA to downregulate protein kinase C decreased exchange activity, although the effect was statistically significant only for NCX3. Possibly, the effects of protein kinases A and C on Na+/Ca2+ exchange activity are indirect. For example, the kinases may affect the Na+ pump with consequent effects on the Na+ gradient. Each of the Na+/Ca2+ exchangers also contains putative tyrosine phosphorylation sites (35). However, no effects of the tyrosine kinase inhibitor genistein or the tyrosine phosphatase inhibitor PAO were observed. In contrast, Condrescu et al. (2) provided preliminary evidence that genistein treatment inhibited the exchange activity of CHO cells expressing NCX2. Possibly, this discrepancy is due to a difference in cell signaling pathways between CHO and BHK cells.

The peptide inhibitor XIP has the same amino acid sequence as a region of the Na+/Ca2+ exchanger suggested to have an autoregulatory role (23, 26). Homologous XIP regions are present in NCX1, NCX2, and NCX3, and we call the corresponding peptides XIP1, XIP2, and XIP3. The XIP peptides were not as potent as expected as inhibitors of NCX1 expressed in BHK cells. However, XIP1 gave the expected results when tested with sarcolemmal vesicles (Fig. 7). XIP has been found to bind to phospholipids (37), and perhaps the lipid environment of BHK cells is different from that in cardiac sarcolemma. We tested this hypothesis by reconstituting NCX1 from BHK cells into asolectin or PC-PS-cholesterol liposomes. These two different lipid mixtures were chosen, since liposomes containing asolectin or PS have been shown to support high levels of exchange activity (38). The presence of PS fully restored XIP1 sensitivity to NCX1 (Fig. 7). The results support the hypothesis that exogenous and endogenous XIP may interact with membrane lipids.

NCX1 is regulated by intracellular Ca2+, which activates the exchanger (7), and by intracellular Na+, which tends to inactivate the exchanger (10). Both of these regulatory properties involve the large intracellular loop of the exchanger (26, 27). In addition, anionic phospholipids (most notably PIP2) (9) also put the exchanger into an activated state. All forms of regulation are removed by chymotrypsin treatment, which puts the exchanger into a constitutively active state. We find that chymotrypsin stimulates the exchange activity of membrane vesicles isolated from BHK cells expressing NCX1, NCX2, or NCX3. Likewise, BAPTA loading of BHK cells to chelate intracellular Ca2+ removes Ca2+ activation and inhibits subsequent exchange activity for all three exchangers. Thus Ca2+ regulation and stimulation by chymotrypsin are common features of the three exchangers.

Electrophysiological analysis confirmed and extended these observations on regulation. The stimulation of NCX1 in excised patches from BHK cells by ATP (Fig. 8A) is a characteristic response of the native exchanger of cardiac myocytes and is due to increased synthesis of PIP2 (9). In contrast, when NCX1 is expressed in Xenopus oocytes, little response to ATP is observed. Thus the cloned NCX1 behaves like the native cardiac exchanger with respect to regulation by ATP when expressed in BHK cells but not when expressed in oocytes. This difference may be due to a high level of PIP2 phosphatase activity in Xenopus membranes (D. Hilgemann, unpublished observation).

In conclusion, the overall functional properties of NCX1, NCX2, and NCX3 are not fundamentally different. This statement regarding the similarity of the three exchangers, however, is subject to certain limitations. First, the NCX1 clone was derived from dog myocardium, whereas the NCX2 and NCX3 clones were derived from rat brain. Species differences in the amino acid sequences are minimal, however, and are unlikely to confound our analysis. For example, the canine and rat heart exchangers are 97% identical (25). Second, different splice variants of the three exchangers are expressed in a tissue-dependent manner. The splice variants differ in the use of six small exons coding for a portion of the large intracellular loop. In this study, we examined only one splice variant of each of the exchanger genes. By use of the terminology of Quednau et al. (35), the three splice variants used here are NCX1.1, NCX2.1, and NCX3.3. Perhaps some of the observed subtle differences are due to the presence or absence of specific exons. Third, some properties of the exchangers may be dependent on the BHK cell expression system. For example, the membrane lipid environment or the presence or absence of specific signaling molecules in the BHK cells may influence results. The need for three separate NCX genes is unclear but may allow for differential regulation and expression of different exchangers in various tissues according to cellular demands. Further analysis of the properties and regulation of the three exchangers is ongoing and should be fruitful.

    ACKNOWLEDGEMENTS

We thank Drs. D. A. Nicoll and B. D. Quednau for detailed comments on the manuscript.

    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grants HL-48509, HL-49101, and HL-51323, the American Heart Association, Greater Los Angeles Affiliate, and the Laubisch Foundation.

Present address of B. Linck: Institute for Pharmacology and Toxicology, Westfalische Wilhelms Universität, Domagkstr. 12, 48149 Münster, Germany.

Address for reprint requests: K. D. Philipson, Cardiovascular Research Laboratory, MRL 3-645, UCLA School of Medicine, Los Angeles CA 90095-1760.

Received 14 May 1997; accepted in final form 7 October 1997.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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