Differential inhibition of Na+/Ca2+ exchanger isoforms by divalent cations and isothiourea derivative

Takahiro Iwamoto and Munekazu Shigekawa

Department of Molecular Physiology, National Cardiovascular Center Research Institute, Suita, Osaka 565, Japan

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

We compared the properties of three mammalian Na+/Ca2+ exchanger isoforms, NCX1, NCX2, and NCX3, by analyzing the effects of Ni2+ and other cations as well as the recently identified inhibitor isothiourea derivatives on intracellular Na+-dependent 45Ca2+ uptake into CCL-39 (Dede) fibroblasts stably expressing each isoform. All these NCX isoforms had similar affinities for the extracellular transport substrates Ca2+ and Na+. Ni2+ inhibited 45Ca2+ uptake by competing with Ca2+ for the external transport site, with 10-fold less affinity in NCX3 than in NCX1 or NCX2. Ni2+ and Co2+ were most efficient in such discrimination of NCX isoforms, although their inhibitory potencies were less than those of La3+ and Cd2+. The monovalent cation Li+ stimulated 45Ca2+ uptake rate by all NCX isoforms similarly with low affinity, although the extent of stimulation was somewhat smaller in NCX1. On the other hand, the isothiourea derivative KB-R7943 was threefold more inhibitory to NCX3 than to NCX1 or NCX2. Thus distinct differences in the kinetic and pharmacological properties were detected between NCX3 and the other two isoforms.

ion transport; stable expression; sodium; calcium

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

THE SODIUM/CALCIUM exchanger of the plasma membrane is an electrogenic transporter that exchanges three Na+ for one Ca2+ (10). The physiological importance of the Na+/Ca2+ exchanger is best understood in cardiomyocytes. In this cell type, available evidence indicates that the exchanger plays a primary role in extrusion of Ca2+ from the cytoplasm during the excitation-contraction cycle (5, 33). In many other cell types, the exchanger is also considered to be important in Ca2+ extrusion, but its role still remains to be precisely defined (10).

Recent molecular cloning studies have shown that the Na+/Ca2+ exchanger forms a multigene family of homologous proteins comprising three isoforms, NCX1, NCX2, and NCX3 (16, 23, 24). These isoforms share ~70 and >80% amino acid identity in the overall sequences and sequences in the predicted membrane-spanning segments, respectively, and thus have similar molecular topology, consisting mainly of putative 11 membrane-spanning segments and a large central hydrophilic loop. NCX1 is highly expressed in cardiac muscle and to a lesser extent in many other tissues such as brain and kidney, whereas expression of NCX2 and NCX3 is significant only in brain and skeletal muscle (16, 23, 24). In addition, splice variants with variation in a small region near the carboxyl end of the large central loop are generated from genes of NCX1 and NCX3 in a tissue-specific manner (15, 22, 25). Quite recently, Linck et al. (17) have compared the functional properties of NCX1, NCX2, and NCX3 expressed stably in BHK cells. They found no fundamental difference in many properties of these isoforms. Thus little is known about the difference in the functions of these isoforms and their splice variants.

Specific inhibitors should be extremely useful for the study of the reaction mechanism of the Na+/Ca2+ exchanger and for the clarification of its physiological and pathological roles. Recently, the isothiourea derivative KB-R7943 has been reported to be a selective and potent inhibitor of the Ca2+ influx mode of Na+/Ca2+ exchange catalyzed by the NCX1 isoform (13, 32). In cardiac electrophysiology, on the other hand, Ni2+ has been known to inhibit the Na+/Ca2+ exchange current without a significant effect on background currents (6). This ion has been considered not to be transported by the Na+/Ca2+ exchanger. Using these two types of exchange inhibitors, we have explored in this study the difference in kinetic properties of NCX1, NCX2, and NCX3 expressed in CCL-39 fibroblasts. We show here that NCX3 responds to these inhibitors differently from NCX1 or NCX2.

    METHODS
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Materials. Chinese hamster lung fibroblasts (CCL-39, Dede) and human embryonic kidney cells (HEK293) were purchased from American Type Culture Collection. KB-R7943 (2-{2-[4-(4-nitrobenzyloxy)phenyl]ethyl}isothiourea methanesulfonate) and KB-R7898 (2-{2-[4-(3,4-dichlorobenzyloxy) phenyl]ethyl}isothiourea methanesulfonate) (Fig. 1) were synthesized by New Drug Research Laboratories, Kanebo, Japan. 45CaCl2 was purchased from DuPont NEN. All other chemicals were of the highest grade available.


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Fig. 1.   Structures of KB-R7943 and KB-R7898.

Cell culture. CCL-39, HEK293, and their NCX transfectants were maintained in DMEM supplemented with 7.5% heat-inactivated FCS, 50 U/ml penicillin, and 50 µg/ml streptomycin.

Cloning, construction, and stable expression of NCX1, NCX2, and NCX3 cDNAs. Isolation of NCX1 cDNA from dog heart was described previously (11). For isolation of NCX2 and NCX3 cDNAs, total RNA was extracted from rat brain with a TRIzol reagent (GIBCO BRL) and first-strand cDNAs were synthesized using an oligo(dT) primer and SuperScript preamplification system (GIBCO BRL). cDNAs were amplified by PCR using the following pairs of sense and anti-sense primers: for NCX2, 5'-CCCCCATGGCTCCCTTGGCTTTGG-3' and 5'-GTCCAAGAGCAAGTGCCAACAAGTCCC-3' [nucleotides -5-19 and 2792-2818 of NCX2 (16)]; for NCX3, 5'-AAGGGGAATCGGTCTCAGGCCTGT-3' and 5'-CTGGGAAG

GTTGGTGAGAAAGCGC-3' [nucleotides 762-785 and 3672-3695 of NCX3 (24)]. Amplified cDNA inserts were cloned into pCRII (Invitrogen) and sequenced using ABI PRISM dye terminator cycle sequencing kit (Perkin-Elmer). In the terminology of Quednau et al. (25), the splice variants used in this study are NCX1.1, NCX2.1, and NCX3.3. These cDNAs were inserted between Sac II and Hind III restriction sites of the mammalian expression vector pKCRH. CCL-39 and HEK293 cells were transfected with vectors with or without these inserts as described (11). Cell clones exhibiting high Na+/Ca2+ exchange activity were selected by treating colonies with 500 µg/ml G418 for 10 days and then with 10 µM ionomycin for 30 min. The ionomycin treatment ("Ca2+-killing") effectively eliminates cells with low exchange activity (12).

Assay of intracellular Na+-dependent 45Ca2+ uptake. Intracellular Na+ (Na+i)-dependent 45Ca2+ uptake was measured at 37°C as described previously (11, 31). Briefly, stable transfectants grown to confluence in 24-well dishes were loaded with Na+ by preincubation for 20 min in 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 simultaneously adding standard uptake medium [modified BSS containing 146 mM choline chloride, 0.1 mM 45CaCl2 (1.5 µCi/ml), and 1 mM ouabain] to each well through 24 temperature-controlled syringes equipped in a handmade holder. After an appropriate interval, 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. The salt concentrations in the uptake medium were modified as shown. When present, KB-R7943 and KB-R7898 were added to cells 30 s before uptake measurement. Na+i-dependent 45Ca2+ uptake was calculated by subtracting 45Ca2+ uptake in 146 mM Na+ from that obtained in medium devoid of Na+ or containing lower Na+. In this study, the rate of Na+i-dependent 45Ca2+ uptake was measured for the initial 30 s because uptake was linear for this period under the conditions used. Uptake rates were 10 ± 0.25, 4.1 ± 0.11, and 5.9 ± 0.28 nmol · mg-1 · 30 s-1 for NCX1, NCX2, and NCX3 transfectants, respectively, in standard uptake medium (see Fig. 4, A-C).

Statistical analysis. Data are expressed as means or means ± SE of three or four independent determinations. Differences for multiple comparisons were analyzed by unpaired t-test or one-way ANOVA followed by the Dunnett's test. Values of P < 0.05 were considered statistically significant.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

In the standard uptake medium of this study, containing 0.1 mM 45CaCl2, 146 mM choline chloride, 4 mM KCl, and 2 mM MgCl2, CCL-39 cells stably transfected with NCX1, NCX2, or NCX3 exhibited ~16-, 7-, or 12-fold greater 45Ca2+ uptake activity, respectively, compared with those measured in medium containing 146 mM NaCl in place of choline chloride. In nontransfected CCL-39 cells, however, 45Ca2+ uptake rates in the presence or absence of extracellular Na+ (Na+o) were low and not different, indicating that there is little endogenous Na+/Ca2+ exchange activity in these cells (see also Ref. 11). Immunoblot analyses with isoform-specific antibodies also revealed expression of very low (for NCX1) or undetectable (for NCX2 and NCX3) levels of NCX proteins in nontransfected cells but high levels of these proteins in the stable NCX transfectants (data not shown and see also Ref. 11).

Figure 2 shows the concentration-response profiles for inhibitors of Na+/Ca2+ exchange, i.e., isothiourea derivatives KB-R7943 and KB-R7898 (see Fig. 1) and Ni2+, measured under standard conditions. KB-R7943 dose dependently inhibited Na+i-dependent 45Ca2+ uptake by NCX1, NCX2, and NCX3 transfectants (Fig. 2A) with IC50 values of 4.9 ± 0.4, 4.1 ± 0.3, and 1.5 ± 0.1 µM, respectively (Table 1). NCX3 was thus about threefold more sensitive to inhibition by KB-R7943 than NCX1 or NCX2. In contrast, another isothiourea derivative, KB-R7898, had almost identical inhibition profiles for all these transfectants (IC50 = 1.4-1.9 µM) (Fig. 2B and Table 1). Intriguingly, a striking difference in the inhibition profile between NCX3 and the other two isoforms was noted with Ni2+ (Fig. 2C). Ni2+ inhibited Na+i-dependent 45Ca2+ uptake into NCX1 and NCX2 transfectants with similar IC50 values of 41 ± 4 and 30 ± 5 µM, respectively, whereas it inhibited the uptake by NCX3 transfectants with IC50 of 402 ± 45 µM (Table 1). Table 1 also shows the IC50 values for these inhibitors measured in medium containing 146 mM LiCl or 146 mM KCl in place of choline chloride. LiCl or KCl accelerated Na+i-dependent 45Ca2+ uptake into NCX transfectants 1.5- to 2.5-fold (see Fig. 5 for data with Li+ medium). Replacement of choline+ with Li+ or K+ did not cause significant changes in IC50 values for isothiourea derivatives (Table 1). Interestingly, however, the IC50 for Ni2+ measured in K+ medium was 2.3- to 3.5-fold larger than that obtained in choline+ medium in all three types of NCX transfectants, whereas the IC50 changed minimally when cells were placed in Li+ medium (Table 1). These effects of Ni2+ and isothiourea derivatives were observed similarly in HEK-293 cells stably expressing each NCX isoform (data not shown).


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Fig. 2.   Concentration-response profiles for inhibition by KB-R7943 (A), KB-R7898 (B), or Ni2+ (C) of intracellular Na+ (Na+i)-dependent 45Ca2+ uptake into Na+/Ca2+ exchanger isoform (NCX) transfectants. Rate of Na+i-dependent 45Ca2+ uptake into NCX1, NCX2, or NCX3 transfectants was measured in the presence or absence of inhibitor as described in METHODS. Data are presented as percentage of control values obtained in the absence of the inhibitor.

                              
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Table 1.   IC50 values for inhibitors obtained for different Na+/Ca2+ exchanger isoforms

We also analyzed the inhibitory effects of other divalent cations and La3+ on Na+i-dependent 45Ca2+ uptake by NCX transfectants in modified standard uptake medium containing no MgCl2; results are summarized in Table 2. Ni2+ and Co2+ caused the most pronounced difference in the inhibition profile, being ~10-fold more inhibitory to NCX1 and NCX2 than to NCX3, although they inhibited all three NCX isoforms less potently than did La3+ and Cd2+. Among the cations tested, La3+ and Cd2+ were most effective in inhibiting NCX isoforms, whereas Mg2+ was by far least effective (Table 2).

                              
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Table 2.   Inhibitory potencies of La3+ and divalent cations on Na+/Ca2+ exchanger isoforms

To obtain insight into the mechanism for the differential effects of KB-R7943 and Ni2+ on exchanger isoforms, rates of Na+i-dependent 45Ca2+ uptake into NCX1, NCX2, and NCX3 transfectants were measured under standard conditions as a function of extracellular Ca2+ concentration ([Ca2+]o) in the presence or absence of the inhibitor (Fig. 3). The double reciprocal plots of uptake rates vs. [Ca2+]o were all linear for each exchanger isoform (Fig. 3, A-C). The half-maximal Ca2+ concentration ( KCa) values for extracellular Ca2+ (Ca2+o) in NCX1, NCX2, and NCX3 were similar (0.17-0.21 mM) in the absence of the inhibitor, whereas the corresponding maximal velocity (Vmax) values were significantly different (Table 3), which probably reflects the expression level of each isoform. Thus there is essentially no difference in the affinity of the transport site for Ca2+o in NCX1, NCX2, and NCX3. In NCX1 and NCX2 transfectants, KB-R7943 at 5 µM decreased Vmax by 47 and 43%, respectively, and increased KCa by 76 and 29%, respectively (Fig. 3, A and B, and Table 3), suggesting a mixed type (competitive and noncompetitive) inhibition. In NCX3 transfectants, however, the same compound at 2 µM decreased Vmax by 59% without changing KCa (Fig. 3C and Table 3), indicating a noncompetitive type of inhibition. On the other hand, Ni2+, when added at 10 and 30 µM for NCX1 and NCX2 transfectants (Fig. 3, A and B) and at 100 and 300 µM for NCX3 transfectants (Fig. 3C), significantly increased the KCa values without influencing Vmax, suggesting that Ni2+ competes with Ca2+ at the extracellular transport site in each NCX isoform. We found that the intrinsic inhibition constant (Ki) for Ni2+ calculated from these data were ~7, 13, and 70 µM for NCX1, NCX2, and NCX3, respectively.


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Fig. 3.   Effects of Ni2+ and KB-R7943 on activation by extracellular Ca2+ of Na+i-dependent 45Ca2+ uptake into NCX1 (A), NCX2 (B), or NCX3 (C) transfectants. Rate of Na+i-dependent 45Ca2+ uptake was measured in modified standard medium containing 0.0625-2 mM CaCl2 in either absence of inhibitors (bullet ) or presence of inhibitors at the following concentrations: in A and B, 10 µM (open circle ) and 30 µM (triangle ) Ni2+ and 5 µM KB-R7943 (); in C, 100 µM (open circle ) and 300 µM (triangle ) Ni2+ and 2 µM KB-R7943 (). Data are presented in double reciprocal plots; straight lines were drawn using the least-squares method. V, velocity.

                              
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Table 3.   Kinetic parameters for extracellular Ca2+ concentration dependence of Na+/Ca2+ exchanger isoforms

Na+o has been shown to inhibit the reverse mode of Na+/Ca2+ exchange at high concentrations by competing with Ca2+o for the extracellular transport site (2, 18, 21, 26) but has been shown to accelerate it at low concentrations (3, 7) in cardiomyocytes, aortic myocytes, squid axons, and cardiac and synaptic membrane vesicles. Thus Na+o, depending on its concentration, could exert a complex effect on Na+/Ca2+ exchange. Figure 4, A-C, shows the effect of Na+o on Na+i-dependent 45Ca2+ uptake into NCX1, NCX2, and NCX3 transfectants in the presence or absence of the inhibitor. In the absence of the inhibitor, Na+o decreased rates of 45Ca2+ uptake by NCX transfectants with apparently similar IC50 values (42 ± 5, 35 ± 3, and 30 ± 3 mM for NCX1, NCX2, and NCX3, respectively). KB-R7943 at 2 µM (for NCX3) or 5 µM (for NCX1 and NCX2), although it decreased the rate of 45Ca2+ uptake roughly by 50% (Table 1), did not significantly affect the inhibition profiles of Na+o for all three NCX isoforms, giving similar IC50 values (Fig. 4, A-C). On the other hand, Ni2+ at 30 µM increased IC50 for Na+o slightly in NCX1 and NCX2 transfectants (by 14 ± 2 and 17 ± 4%, respectively) (Fig. 4, A and B), whereas the same cation at 300 µM increased the IC50 for Na+o twofold in NCX3 transfectants (Fig. 4C). In Fig. 4D, we further examined the effect of Ni2+ concentration on the rate of Na+i-dependent 45Ca2+ uptake by NCX transfectants in the presence of 60 mM Na+o. In NCX1 and NCX2 transfectants, Ni2+ monotonically inhibited 45Ca2+ uptake with IC50 values of 58 ± 2 and 42 ± 4 µM, respectively, which are only slightly larger than the values determined in the presence of 146 mM choline+ (see Table 1). In NCX3 transfectants, however, Ni2+ at low concentrations (0.1-1 mM) accelerated Na+idependent 45Ca2+ uptake considerably, although it inhibited the uptake at higher concentrations (Fig. 4D).


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Fig. 4.   Effects of Ni2+ and KB-R7943 on inhibition by extracellular Na+ of Na+i-dependent 45Ca2+ uptake into NCX transfectants. Rate of Na+i-dependent 45Ca2+ uptake into NCX1 (A), NCX2 (B), or NCX3 (C) transfectants was measured under standard conditions except that choline chloride was replaced by equimolar NaCl and inhibitor was included at the following concentrations: in A and B, no inhibitor (bullet ), 30 µM Ni2+ (triangle ), and 5 µM KB-R7943 (); in C, no inhibitor (bullet ), 300 µM Ni2+ (triangle ), and 2 µM KB-R7943 (). [Na+]o, extracellular Na+ concentration. In D, effect of Ni2+ concentration on Na+i-dependent 45Ca2+ uptake into NCX1, NCX2, and NCX3 transfectants was measured in the presence of 60 mM NaCl. Data are presented as percentage of control values obtained in the absence of Ni2+.

Extracellular monovalent cations have been shown to activate Na+i/Ca2+o or intracellular Ca2+/Ca2+o exchange in cardiac myocytes and squid axons (1, 3, 4, 9), although either stimulatory or inhibitory effects of these cations have been observed in membrane vesicles (27, 28). In Fig. 5, A-C, we examined the effect of extracellular Li+ (Li+o) concentration on the rate of Na+i-dependent 45Ca2+ uptake into NCX transfectants in the presence or absence of the inhibitor. In the absence of the inhibitor, increasing Li+o concentrations up to 146 mM increased 45Ca2+ uptake rates 1.5-, 2.2-, and 2.3-fold in NCX1, NCX2, and NCX3 transfectants, respectively. Li+o thus activated all three NCX isoforms with low affinity. We found that KB-R7943 at 2 µM (for NCX3) or 5 µM (for NCX1 and NCX2) or Ni2+ at 30 µM (for NCX1 and NCX2) or 300 µM (for NCX3) did not significantly affect the activation profiles of Li+o for all three NCX isoforms (Fig. 5, A-C). Conversely, Li+o, when present at 60 mM (Fig. 5D) or 146 mM (Table 1), minimally influenced the inhibition profiles of Ni2+ and isothiourea derivatives for all three NCX isoforms.


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Fig. 5.   Effects of Ni2+ and KB-R7943 on activation by extracellular Li+ of Na+i-dependent 45Ca2+ uptake into NCX transfectants. Rate of Na+i-dependent 45Ca2+ uptake into NCX1 (A), NCX2 (B), or NCX3 (C) transfectants was measured under standard conditions except that choline chloride was replaced by LiCl and inhibitor was included at the following concentrations: in A and B, no inhibitor (bullet ), 30 µM Ni2+ (triangle ), and 5 µM KB-R7943 (); in C, no inhibitor (bullet ), 300 µM Ni2+ (triangle ), and 2 µM KB-R7943 (). Data are presented as percentage of values obtained in the absence of extracellular Li+. [Li+]o, extracellular Li+ concentration. D: effect of Ni2+ concentration on Na+i-dependent 45Ca2+ uptake into NCX1, NCX2, and NCX3 transfectants was measured in the presence of 60 mM LiCl. Data are presented as percentage of control values obtained in the absence of Ni2+.

    DISCUSSION
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Abstract
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Methods
Results
Discussion
References

The objective of this work was to biochemically and pharmacologically compare functional properties of three mammalian Na+/Ca2+ exchanger isoforms. To characterize individual NCX isoforms in the same cellular context, we stably expressed each in CCL-39 fibroblasts that exhibited no detectable endogenous exchange activity (see RESULTS and Ref. 11). This study revealed that NCX3 is ~10-fold less sensitive to inhibition by Ni2+ or Co2+ but 3-fold more sensitive to inhibition by the isothiourea derivative KB-R7943 than NCX1 and NCX2 (Fig. 2, A-C, and Tables 1 and 2). Furthermore, acceleration of Na+i-dependent 45Ca2+ uptake by Ni2+, which has not previously been reported, was observed in a certain range of Na+o concentrations in NCX3 but not in NCX1 or NCX2 transfectants (Fig. 4D). Thus there are indeed some differences between NCX3 and the other two isoforms in their functional properties. In contrast, very similar KCa values were obtained for all three isoforms from the measurement of [Ca2+]o dependence on the rate of Na+i-dependent 45Ca2+ uptake under standard conditions (Fig. 3, A-C, and Table 3). The IC50 values for inhibitory Na+o in the presence of 0.1 mM 45Ca2+ were also similar in all three NCX isoforms (Fig. 4, A-C). These findings, consistent with similar findings by Linck et al. (17) who expressed NCX cDNAs in BHK cells, indicate that the exchanger isoforms bind Ca2+ or Na+ with similar affinities at their respective external sites. The binding site for the inhibitory Na+o is most likely to be the extracellular transport site because a competitive interaction between Ca2+o and relatively high concentrations of Na+o has been described in several previous studies (2, 18, 21, 26). In addition to these results, we observed that Li+ stimulates the rate of 45Ca2+ uptake by each isoform similarly with low affinity, although the extent of stimulation was somewhat smaller in NCX1 than in NCX2 or NCX3 (Fig. 5, A-C). Thus, despite the differences noted above, interactions of the external transport site with Ca2+ or Na+ and of the external monovalent cation site with Li+ are similar in all NCX isoforms.

Divalent and trivalent cations have been shown to inhibit Ca2+ transport by Na+/Ca2+ exchanger in a concentration-dependent manner (6, 14, 29, 30). Mg2+ inhibits Na+i-dependent 45Ca2+ uptake by competing with Ca2+ for the external exchanger site in cardiomyocytes and smooth muscle cells (14, 29). On the other hand, Ni2+ has been shown to be a mixed type inhibitor of the outward Na+/Ca2+ exchange current in cardiomyocytes (6). However, Ni2+ inhibited Na+i-dependent 45Ca2+ uptake into NCX transfectants competitively with Ca2+o under the standard conditions of this study (Fig. 3, A-C). The IC50 (and Ki) for Ni2+ was ~10-fold greater in NCX3 than in NCX1 or NCX2, as noted above (see also RESULTS). The IC50 values for Ni2+ in all NCX isoforms increased two- to threefold when cells were placed in medium containing 146 mM K+, although the same parameter changed minimally in medium containing 146 mM Li+ (Table 1). Thus it is likely that membrane depolarization increased the IC50 for Ni2+, suggesting that the affinity of Ni2+ for the inhibitory site is affected by membrane potential. Matsuoka and Hilgemann (20) have shown that the KCa for Ca2+o increases on depolarization when voltage dependence of the outward current was measured in giant membrane patches from cardiomyocytes. Furthermore, the inhibition profile of Ni2+ was not influenced by the presence or absence of Li+ (Figs. 2C and 5D), and Ni2+ did not significantly affect the activation profile of Li+ on 45Ca2+ uptake by all three NCX isoforms (Fig. 5, A-C). Ni2+ therefore does not influence occupancy of the activation site by the monovalent cation. All these results suggest that the remarkable difference in the inhibitory potency of Ni2+ between NCX3 and the other two isoforms arises from the difference in the intrinsic affinity for Ni2+ of the transport site or, more precisely, the binding site for these divalent cations within the ion transport pathway of the exchanger.

We have tested the effects of several other metal cations on Na+i-dependent 45Ca2+ uptake into NCX transfectants. Among the cations tested, La3+ and Cd2+ were the most effective inhibitors for all three NCX isoforms, whereas Mg2+ was the least effective (Table 2). The rank order of the inhibitory potency of La3+ >=  Cd2+ >=  Ni2+ > Co2+ approx  Mn2+ >>  Mg2+ for three NCX isoforms is similar to those obtained previously with cardiac sarcolemmal vesicles (30) or cultured smooth muscle cells (29). We found that Ni2+ was almost as effective as La3+ and Cd2+ in inhibiting Na+i-dependent 45Ca2+ uptake by NCX1 and NCX2 (Table 2). Because Ni2+, which has the effective ionic radius of 0.69 Å, is significantly smaller than Ca2+ (1.12 Å), La3+ (1.16 Å), or Cd2+ (0.95 Å) (19), ionic size does not appear to be a predominant factor in determining the inhibitory potency of these cations. On the other hand, Ni2+ and Co2+ were by far more efficient than other cations in discriminating NCX3 against NCX1 and NCX2. La3+, Cd2+, and Mg2+ discriminated the exchanger isoforms much less efficiently (Table 2). Because the ionic radius of Mg2+ (0.72 Å) is comparable to those of Ni2+ (0.69 Å) and Co2+ (0.74 Å) (19), ionic size again does not appear to be the primary factor in discriminating the exchanger isoforms. Currently, we know little about the mechanism by which the extracellular inhibitory site in the Na+/Ca2+ exchanger selects effective over noneffective cations.

Surprisingly, ~0.3 mM Ni2+ accelerated Na+idependent 45Ca2+ uptake by NCX3 in the presence of 60 mM Na+o, although Ni2+ monotonically inhibited the uptake by other NCX isoforms, with IC50 values similar to those obtained in the absence of Na+o (Fig. 4D, see also Figs. 2C and 5D). Consistent with this finding, the profile for the Na+o-dependent inhibition of 45Ca2+ uptake by NCX3 was complex in the presence of 300 µM Ni2+, giving a larger IC50 value for Na+o compared with those for NCX1 and NCX2 in the presence of 30 µM Ni2+ (Fig. 4, A-C). Intriguingly, such an activating effect of Ni2+ was not observed in choline+ medium (Fig. 2C), in which the monovalent cation site is unoccupied, as well as in medium containing 60 or 146 mM Li+ (Fig. 5, C and D), in which a significant portion of the monovalent cation site is occupied. The results suggest that Ni2+ neither directly activates NCX3 nor changes the extent of occupancy of the activation site by the monovalent cation. Furthermore, such a requirement of Na+o for the activation by Ni2+ cannot be explained by simple competition of Ni2+ with Na+o for the exchanger transport site. Thus it appears that a complex interaction exists between the effects of Na+o and Ni2+ in NCX3. At present, we have no explanation for the activation by Ni2+. In this context, it may be interesting to note that Na+o-dependent 45Ca2+ efflux from cultured aortic smooth muscle cells, which express NCX1, was accelerated by up to 60% by extracellular La3+ at concentrations between 10 and 300 µM in 146 mM Na+o (8). Low concentrations of trivalent cations have also been shown to stimulate Na+i-dependent 45Ca2+ uptake into cardiac sarcolemma vesicles in the absence of Na+o (30). However, the relationship between these previous and present findings is not clear.

We have recently shown that the isothiourea derivative KB-R7943 potently inhibits the Ca2+ influx mode of Na+/Ca2+ exchange catalyzed by NCX1 (13). As noted above, NCX3 was about threefold more sensitive to the inhibition by KB-R7943 than NCX1 or NCX2 (Fig. 2A). Interestingly, the new derivative KB-R7898 exerted a stronger inhibitory effect on NCX1 or NCX2 than did KB-R7943 and inhibited all the exchanger isoforms to a similar extent (Fig. 2B). In the structure-activity relationship of isothiourea derivatives, the isothiourea moiety, usually being protonated, and the benzyloxy moiety are essential for their inhibitory activity (13). Different inhibition profiles of KB-R7943 and KB-R7898 suggest that structural modification in the benzyloxy moiety is critically important for the isoform selectivity (Fig. 1).

For kinetic analysis of the drug-induced inhibition of Na+i-dependent 45Ca2+ uptake by NCX3 and the other two isoforms, we used 2 and 5 µM KB-R7943, respectively, which produced roughly 50% inhibition of each isoform in standard medium (Fig. 3, A-C, and Table 3). We found that inhibition of Na+i-dependent 45Ca2+ uptake by KB-R7943 was of a mixed type with respect to Ca2+o in NCX1 and NCX2, although the increases in KCa for Ca2+o were relatively small (Fig. 3, A and B, and Table 3). In NCX3, however, the inhibition was noncompetitive with respect to Ca2+o (Fig. 3C and Table 3). KB-R7943, on the other hand, did not appreciably change the IC50 values for inhibitory Na+o measured in the presence of 0.1 mM 45Ca2+ in all three NCX isoforms (Fig. 4, A-C). All these effects of KB-R7943 suggest that this agent inhibits NCX isoforms primarily by interacting with site(s) other than the transport site. It is unlikely that the inhibition by KB-R7943 involves the monovalent cation site because the extent of inhibition of Na+i-dependent 45Ca2+ uptake by this agent was not affected by the presence or absence of Li+o (Fig. 5, A-C) and Li+o did not change the inhibition profile of this agent (Table 1).

In summary, the present results suggest that Ni2+ inhibits Na+i-dependent 45Ca2+ uptake catalyzed by Na+/Ca2+ exchanger isoforms by apparently competing against Ca2+o for the external transport site, with 10-fold greater affinity in NCX1 and NCX2 than in NCX3. On the other hand, the isothiourea derivative KB-R7943, which is mainly a noncompetitive inhibitor with respect to Ca2+o, is threefold more inhibitory to NCX3 than to NCX1 or NCX2. Thus some distinct differences in the kinetic and pharmacological properties were detected between NCX3 and the other two exchanger isoforms, although they have high homology in the amino acid sequences (see the introduction) and similar affinities for transport substrates Ca2+o and Na+o or modulatory monovalent cation Li+o. These results, in particular those with inhibitory divalent cations, may provide an important basis for the structure-function study of the ion transport pathway in the exchanger molecule. Chimeras between the exchanger isoforms can be used to investigate this further.

    ACKNOWLEDGEMENTS

We thank Dr. A. Uehara of Fukuoka University School of Medicine and Drs. S. Wakabayashi and T. Nishitani of this department for fruitful discussion.

    FOOTNOTES

This work was supported by a Grant-in-Aid on Priority Areas and a Grant-in-Aid for Scientific Research (07457015) from the Ministry of Education, Science and Culture of Japan, and by Special Coordination Funds Promoting Science and Technology (Encouragement System of Center of Excellence) from the Science and Technology Agency of Japan.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: M. Shigekawa, Dept. of Molecular Physiology, National Cardiovascular Center Research Institute, Fujishiro-dai 5, Suita, Osaka 565, Japan.

Received 9 March 1998; accepted in final form 8 May 1998.

    REFERENCES
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Abstract
Introduction
Methods
Results
Discussion
References

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