INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

THE PLASMA MEMBRANE INTEGRAL protein Na⁺/Ca²⁺ exchanger (NCX) is crucial in cytosolic Ca²⁺ concentration regulation in a variety of cells. The cardiac-specific isoform of NCX in mammals (NCX1.1) is critical for mechanical relaxation because it serves as the prime mechanism of Ca²⁺ extrusion from the cardiomyocyte (1, 2, 5). Additionally, it has been postulated that, under certain physiological conditions, NCX1.1 can operate in reverse mode, in which it contributes to cardiomyocyte Ca²⁺ influx either through depolarization-induced Ca²⁺ influx (18) and/or Na⁺ current-induced Ca²⁺ influx (17). Thus the critical role that NCX1.1 plays in cardiac excitation-contraction (E-C) coupling is well documented.

Cardiac function in active salmonid species such as rainbow trout (Oncorhynchus mykiss) is distinguished by its ability to maintain adequate contractility under hypothermic conditions that are cardioplegic to mammals. Achieving this phenomenon poses interesting biological challenges, because all of the crucial proteins involved in Ca²⁺ regulation and E-C coupling in the mammalian heart are highly temperature dependent. For example, it has been demonstrated that the Q₁₀ (times change in activity for a 10°C change in temperature) for NCX1.1 is in the range of 2.2-4.0 (11, 16). Thus it has been proposed that at least some of the proteins involved in E-C coupling have evolved differently in these species (32) to maintain cardiac function under hypothermia. It has been demonstrated in atrial myocytes that the trout NCX plays an important role in E-C coupling (12). Studies of Na⁺/Ca²⁺ exchange in trout heart sarcolemmal vesicles have demonstrated properties of this protein that are both unique and common to the mammalian NCX1.1 (33). Similarities include antigenicity, electrogenicity, and stimulation by chymotrypsin treatment. Most obvious among the differences is that reducing the temperature from 21 to 7°C dramatically diminishes canine NCX1.1 activity to <10% of the initial level, whereas in trout the activity remains >75% (33). This behavior of NCX was observed in both the native membranes and when the exchangers were reconstituted into asolectin vesicles. These data strongly suggest that the differential temperature dependencies in the mammalian and teleost NCX isoforms are due to differences in their primary structures.

To understand the molecular mechanisms of these differences, we recently cloned trout cardiac NCX and designated it NCX-TR1.0 (36). The NCX-TR1.0 cDNA has an open reading frame that codes for a protein of 968 amino acids with a deduced molecular mass of 108 kDa. Based on the hydropathy analysis and sequence identity, the topology of NCX-TR1.0 is predicted to be similar to that of mammalian NCX1.1, which is now modeled to have nine transmembrane segments (13, 26). At the amino acid level, sequence comparison including the cleaved leader peptide showed ~75% identity to dog NCX1.1, 66% to rat NCX3, and 61% to rat NCX2. Like all NCXs, the sequence of NCX-TR1.0 shows the most divergence at the amino terminus (34). Sequence identity becomes very high (85%) within the putative transmembrane segments, consistent with their functional significance in ion translocation (25). Furthermore, the ₁ and ₂ repeats within the transmembrane segments, which play a critical role in ion translocation (25) and are modeled to face one another (30), exhibit ~92 and 94% amino acid identity, respectively, between these isoforms. Although the amino acid sequence of the intracellular loop of NCX-TR1.0 has only 73% identity overall with NCX1.1, those regions within the loop with known functional importance are well conserved. For example, the endogenous XIP site, consisting of 20 amino acids at the amino terminus of the loop, exhibits a high degree of conservation (17/20 identity with two conservative substitutions) and is critical for Na⁺-dependent inactivation (19). The regulatory Ca²⁺ binding domains are known to be highly conserved in a wide variety of species (7) including NCX-TR1.0 (~86-90%) (36), and the three consecutive aspartic acid residues characteristic of each of these domains are completely conserved in NCX-TR1.0.

In this study, we characterize in detail the temperature dependencies of the outward currents of trout NCX-TR1.0 and dog NCX1.1 expressed in Xenopus oocytes using the giant excised patch technique to elucidate the potential contributions to temperature sensitivity of the regulatory domains vs. transport mechanisms.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Expression of Na+/Ca²⁺ exchanger in Xenopus oocytes. Dog NCX 1.1 and trout NCX-TR1.0 cDNAs were subcloned into modified pBluescript as described previously (36) and then linearized with HindIII. cRNA was synthesized using T3 mMessage mMachine In Vitro Transcription Kit (Ambion, Austin, TX). Oocytes were prepared as described previously (20). Oocytes were injected with ~5 ng of cRNA, and exchange activity was measured 3-4 days after injection as exchanger current (see below).

Assay of Na+/Ca²⁺ exchange activity. Outward Na⁺/Ca²⁺ exchange currents were measured using the giant excised patch technique, as described previously (28), to investigate the potential contributions of regulatory vs. transport mechanisms to the differential temperature sensitivities in these two NCX isoforms. Borosilicate glass pipettes were pulled and polished to a final inner diameter of ~20-30 µm and coated with a Parafilm-mineral oil mixture. The vitellin layer was removed, and oocytes were placed in a solution containing (in mM) 100 KOH, 100 MES, 20 HEPES, 5 EGTA, and 5 MgCl₂ (pH 7.0 at room temperature with MES). Gigaohm seals were formed via suction, and membrane patches (inside-out configuration) were excised by movements of the pipette tip. A computer-controlled, 20-channel solution switcher was used for rapid solution changes. Axon Instruments hardware and software were used for data acquisition and analysis. The pipette solution contained (in mM) 100 N-methyl-D-glucamine-MES, 30 HEPES, 30 tetraethylammonium (TEA)-OH, 16 sulfamic acid, 8 CaCO₃, 6 KOH, 0.25 ouabain, 0.1 niflumic acid, and 0.1 flufenamic acid (pH 7.0 with MES). Outward Na⁺/Ca²⁺ exchange currents were activated by switching from intracellular Li⁺- to intracellular Na⁺-based bath solutions containing (in mM) 100 Na⁺- or Li⁺-aspartate, 20 MOPS, 20 TEA-OH, 20 CsOH, 10 EGTA, 0-7.3 CaCO₃, and 1.0-1.13 Mg(OH)₂ (pH 7.0 with MES or LiOH). Mg²⁺ and Ca²⁺ were adjusted to yield free concentrations of 1.0 mM and 0, 1, or 10 µM, respectively, using MAXC software (3). All experiments were conducted first at room temperature (22-23°C), and then exchange currents were measured at different temperatures (30°C, 14°C, and 7°C) by heating or cooling bath solutions.

Data analysis. All statistical data are shown as means ± SE. All comparisons between mammalian and trout Na⁺/Ca²⁺ exchangers were made using unpaired, two-tailed Student's t-test. P < 0.05 was considered as significantly different.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Exchange currents of NCX-TR1.0 and NCX 1.1. We measured the outward Na⁺/Ca²⁺ exchange currents in giant patches excised from Xenopus oocytes expressing NCX 1.1 and NCX-TR1.0 (Fig. 1). Currents were activated by the application of 100 mM Na⁺ to the cytoplasmic surface of an excised patch of oocyte membrane. As indicated on the overlapping current traces, records were obtained at different concentrations of regulatory Ca²⁺ (0, 1, and 10 µM) at the cytoplasmic surface. Outward Na⁺/Ca²⁺ exchange currents for both exchanger isoforms displayed similar characteristics. For both dog NCX1.1 and trout NCX-TR1.0, peak and steady-state outward currents were larger in the presence of regulatory Ca²⁺, demonstrating positive regulation of exchange current by intracellular Ca²⁺. Peak current at 10 µM Ca²⁺ was less than at 1 µM intracellular Ca²⁺ for both exchanger isoforms. In addition, both dog NCX1.1 and trout NCX-TR1.0 responded in a similar fashion to intracellular Na⁺ application. The current increased to a peak value and then slowly decayed in a time-dependent manner, indicative of intracellular Na⁺-dependent inactivation (9).

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Typical current traces demonstrating cytoplasmic Ca2+ regulation of outward Na+/Ca2+ exchange (NCX) currents obtained from inside-out giant membrane intact. Currents were induced by the rapid application of 100 mM Na+ to the cytoplasmic surface of the patch at the indicated regulatory intracellular (Ca) concentrations. Ca2+ within the pipette was constant at 8 mM and temperature was 30°C. NCX-TR1.0, wild-type trout exchanger; NCX 1.1, canine exchanger; and X, 0.1 or 10 µM Ca2+.

Temperature effect on exchange activity. We examined the temperature dependence of Na⁺/Ca²⁺ exchange current for the dog NCX1.1 and trout NCX-TR1.0 expressed in Xenopus oocytes. Figure 2A shows outward exchange currents activated by the rapid application of 100 mM Na⁺ to the cytoplasmic surface of an excised patch of oocyte membrane in the presence of 1 µM regulatory Ca²⁺ on the cytoplasmic side. At 30°C, the current properties of NCX1.1 and NCX-TR1.0 are similar. However, with decreasing temperature, both peak and steady-state currents of NCX1.1 decreased. The same trend was observed in NCX-TR1.0, but to a much lesser degree. At 7°C, NCX 1.1 maintained ~10% of its peak and steady-state currents measured at 30°C, while NCX-TR1.0 maintained ~60% of its activity (Fig. 2, B and C).

View larger version (21K): [in this window] [in a new window]   Fig. 2.   A: temperature dependence of Na+/Ca2+ exchange outward current induced by the rapid application of 100 mM Na+ to the cytoplasmic surface of intact patches. Regulatory Ca2+ (1 µM) and 8 mM transport Ca2+ were present in the bath and pipette solutions, respectively. B: representative traces demonstrating influence of temperature on Na+/Ca2+ exchange outward current for NCX1.1 and NCX-TR1.0. Currents at 7°C are expressed as a fraction of the respective peak currents at 30°C. C: influence of temperature on Na+/Ca2+ exchange outward current for NCX1.1 and NCX-TR1.0. Values of peak (1) and steady-state (2) currents at 7°C are presented as a fraction of the respective peak and steady-state currents at 30°C. Currents were activated by rapid application of 100 mM intracellular Na+ in the presence of 1 µM intracellular Ca2+. I, current.

View larger version (11K): [in this window] [in a new window]   Fig. 3.   Arrhenius plot of Na+/Ca2+ exchange peak (1, 3) and steady-state (2, 4) currents for the wild-type exchangers: dog (1, 2) and trout (3, 4) cardiac. For dog NCX1.1, data points represent mean values from 3-4 measurements obtained in 3-4 patches, and those for trout NCX1.0 represent mean values from 3-5 measurements obtained in 3-5 patches. Solid and dashed lines represent linear least-square fits to the experimental values of the peak and steady-state currents, respectively. To allow statistical treatment of data obtained in different patches, each exchange current was normalized to that obtained at 30°C (~303°K). Ti, temperature in °K.

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View larger version (16K): [in this window] [in a new window]   Fig. 4.   Representative traces demonstrating differences in the current inactivation rates for dog NCX1.1 and trout NCX-TR1.0 exchangers at 30°C (A) and 7°C (B). For each trace, currents were normalized to respective peak current.

View larger version (11K): [in this window] [in a new window]   Fig. 5.   Temperature dependence of the inactivation rate constant  of Na+/Ca2+ exchange current for NCX1.1 and NCX-TR1.0. For NCX1.1, data points represent mean values from 7 to 30 measurements obtained in 7-16 patches. For NCX-TR1.0, data points represent mean values from 8 to 27 measurements obtained in 4-8 patches. Inactivation rate constants were obtained by fitting current-time traces to a single exponential. The inactivation rate of NCX-TR1.0 was generally slower than that of NCX1.1 over the temperature range of 7-30°C, although this only achieved statistical significance at 30°C.

View larger version (12K): [in this window] [in a new window]   Fig. 6.   Temperature dependence of Na+/Ca2+ exchange outward current induced by the rapid application of 100 mM Na+ to the cytoplasmic surface of -chymotrypsin-treated patches. Regulatory Ca2+ (1 µM) and 8 mM transport Ca2+ were present in the bath and pipette solutions, respectively.

View larger version (12K): [in this window] [in a new window]   Fig. 7.   Arrhenius plot of Na+/Ca2+ exchange peak (1, 3) and steady-state (2, 4) currents for -chymotrypsin-treated exchangers: dog (1, 2) and trout (3, 4) cardiac. For dog NCX1.1, data points represent mean values from 3-4 measurements obtained in 3-4 patches, and those for trout NCX-TR1.0 represent mean values from 3-5 measurements obtained in 3-5 patches. Solid and dashed lines represent linear least-square fits to the experimental values of the peak and steady-state currents, respectively. To allow statistical treatment of data obtained in different patches, each exchange current was normalized to that obtained at 30°C (~303°K).

View this table: [in this window] [in a new window]   Table 1.   Mean Eact values for the control and -chymotrypsin-treated exchangers

View this table: [in this window] [in a new window]   Table 2.   Mean Q10 values for the control and -chymotrypsin-treated exchangers

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

We have confirmed that cloned trout heart Na⁺/Ca²⁺ exchanger, NCX-TR1.0, expressed in oocytes, displays similar regulatory properties as mammalian NCX. These properties include Na⁺-dependent inactivation, characterized by a slow, partial decline in outward exchange current on application of bath Na⁺ to initiate exchange and Ca²⁺-dependent positive regulation characterized by the requirement of the presence of micromolar levels of Ca²⁺ for full activation (9). It is known that these two forms of regulation are dependent on the presence of the XIP site and the Ca²⁺ binding sites of the cytoplasmic loop (22, 23). The similarity in regulation is consistent with the fact that the sequences of both the XIP site and the Ca²⁺ binding sites are highly conserved between trout NCX-TR1.0 and dog NCX1.1 (36). Peak currents for both isoforms declined at 10 µM cytoplasmic Ca²⁺, presumably reflecting competition between Na⁺ and Ca²⁺ for the intracellular transport sites (21).

Previous studies have demonstrated unequivocally that NCX activity of mammalian species is highly temperature dependent (11, 15, 16, 29), with Q₁₀ values in the range of 2.2-4.0. Using sarcolemmal vesicles, we (33) have demonstrated that the trout heart NCX activity is dramatically less temperature dependent than mammals with a Q₁₀ of ~1.2. Comparison of the temperature dependence of exchange activity in reconstituted proteoliposomes and native membrane indicates that the temperature sensitivity is likely an intrinsic property of the NCX protein rather than dependent on the lipid environment (4, 33). In the present study, the temperature effects on NCX activity were characterized further by measuring outward exchange currents of the cloned trout NCX-TR1.0 and dog NCX1.1 over a temperature range of 7-30°C. When the temperature was decreased from 30°C to 7°C, both the peak current and the steady-state current of dog NCX1.1 were greatly reduced (to ~10%), and the derived Q₁₀ values of 2.4 for peak current and 2.6 for steady-state current are consistent with previous measurements of mammalian NCX temperature dependence (11, 16, 33). However, when the temperature was reduced from 30°C to 7°C, the outward currents of cloned NCX-TR1.0 were largely maintained (~60%), with the derived Q₁₀ values being 1.2 for peak current and 1.1 for steady-state current. These values are strikingly similar to that determined for the Ca²⁺ uptake by native trout NCX (Q₁₀ ~ 1.2) in both native and asolectin-reconstituted sarcolemmal vesicles (33).

The energy of activation (E_act, expressed as kJ/mol) values for both peak (53 ± 1) and steady-state (66 ± 9) I_NCX for the expressed canine NCX determined in this study are in a range similar to those observed by others using various mammalian preparations. These results are summarized in Table 3, and a clear distinction can be made between the endothermic mammals in which E_act ranges from 48 to 67 kJ/mol and the ectothermic lower vertebrates such as frogs (21-25 kJ/mol) and trout (6-7 kJ/mol). This, we believe, is a reflection of the important role that NCX plays in the hearts of these two species under hypothermic conditions.

Characterization of the temperature effect on inactivation kinetics of Na⁺/Ca²⁺ exchange current was performed by fitting the current decay with a single exponential, and the inactivation rate constant  was determined as a function of temperature (Fig. 5). The  decreased monotonically with temperature for both isoforms, consistent with that observed in myocyte patches by some experimenters (10) but not others (27). The inactivation of NCX-TR1.0 was consistently slower than that of NCX1.1 over the temperature range of 30-7°C. However, the values of  for these two isoforms changed almost in parallel over this temperature range, as reflected in the fact that the inactivation rate constant Q₁₀ values were calculated to be 1.35 for NCX1.1 and 1.34 for NCX-TR1.0, over the range 7-30°C and were not significantly different. However, the derived Q₁₀ of the inactivation rate for the cloned canine NCX expressed in oocytes in this study of 1.35 is considerably lower than that of native NCX in excised patches from guinea pig cardiomyocytes (Q₁₀ 2.2) observed by Hilgemann et al. (11), and the reasons for this discrepancy are not clear. It is worth noting, however, that a recent study has shown that there are considerable differences in the rates of Na⁺-dependent inactivation for Na⁺/Ca²⁺ exchange, depending on the model system and technique employed for characterization (8).

To investigate further the relationship between the regulation and temperature dependency of NCX, we treated the cytoplasmic surface of the excised NCX patch with -chymotrypsin. Although it is not clear whether the 70-kDa fragment generated by chymotrypsin treatment represents either the carboxy terminus (14) or the amino terminus (31) of the molecule, it is well documented that this treatment eliminates all forms of regulation while maintaining the exchange activity (9). Comparable to mammalian NCX, proteolyzed NCX-TR1.0 was deregulated, with the exchange current no longer sensitive to changes of cytoplasmic Ca²⁺ concentration (data not shown), and with very little Na⁺-dependent inactivation (Fig. 6). The proteolyzed NCX isoforms from both species exhibited temperature dependencies similar to that of the wild-type exchangers, since the Q₁₀ values between wild-type and proteolyzed NCX were not significantly different, suggesting that the temperature dependence is not predicated on NCX regulatory mechanisms. Based on the observations that NCX loses its regulatory properties after treatment with -chymotrypsin (9) and that the large cytoplasmic loop is essential for regulation of NCX activity (24), it can be postulated that the substantial sequence differences in the loop are not associated with the disparate temperature dependencies of NCX isoforms. In support of this conclusion, we have preliminary evidence that replacing the cytoplasmic loop of the canine NCX with that of trout NCX-TR1.0 does not reduce the Q₁₀ of either the peak or steady-state currents of this chimera (35). Therefore, the difference in temperature dependence is likely to reside in the transmembrane segments in which NCX1.1 and NCX-TR1.0 exhibit ~85% identity at the amino acid level. The transmembrane segments, especially the  repeats, are known to be involved in ion binding and translocation. During ion translocation the protein undergoes conformational changes that are determined by the flexibility of the protein structure and that in turn are affected by temperature. Further experimentation is required to determine the molecular mechanisms involved in the different temperature sensitivities of these NCX isoforms.

It should be noted that in these experiments no ATP was included in either the bath or pipette solutions because it can activate confounding currents in the oocyte patch. ATP, however, is known to regulate the activity of NCX through the phosphatidylinositol 4,5-bisphosphate pathway (6). Furthermore, since most reactions involving phosphorylation and dephosphorylation are known to be temperature dependent, it remains to be seen whether this pathway also contributes to the differential temperature sensitivity between these NCX isoforms.

In summary, we have characterized the temperature differences between the dog and trout myocardial exchangers. These discrepancies are due to intrinsic differences in the NCX isoform structures and are likely related to sequence differences in the transmembrane segments of the protein.