Determinants of cardiac Na+/Ca2+ exchanger temperature dependence: NH2-terminal transmembrane segments

Christian Marshall1,*, Chadwick Elias2,*, Xiao-Hua Xue1,*, Hoa Dinh Le2, Alexander Omelchenko2, Larry V. Hryshko2, and Glen F. Tibbits1,3

1 Cardiac Membrane Research Laboratory, Simon Fraser University, Burnaby, British Columbia V5A 1S6; 2 Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, University of Manitoba, Winnipeg, Manitoba R2H 2A6; and 3 Cardiovascular Sciences, British Columbia Research Institute for Children and Women's Health, Vancouver, British Columbia, Canada V5Z 4H4


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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The cardiac Na+/Ca2+ exchanger (NCX) in trout exhibits profoundly lower temperature sensitivity in comparison to the mammalian NCX. In this study, we attempt to characterize the regions of the NCX molecule that are responsible for its temperature sensitivity. Chimeric NCX molecules were constructed using wild-type trout and canine NCX cDNA and expressed in Xenopus oocytes. NCX-mediated currents were measured at 7, 14, and 30°C using the giant excised-patch technique. By using this approach, the differential temperature dependence of NCX was found to reside within the NH2-terminal region of the molecule. Specifically, we found that ~75% of the Na+/Ca2+ exchange differential energy of activation is attributable to sequence differences in the region that include the first four transmembrane segments, and the remainder is attributable to transmembrane segment five and the exchanger inhibitory peptide site.

myocardial contractility; excitation-contraction coupling; salmonid; calcium handling


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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THE NA+/CA2+ EXCHANGER (NCX) is an integral membrane protein that plays an important role in the regulation of Ca2+ concentration in the cytosol. Utilizing the Na+ electrochemical gradient, the NCX transports Ca2+ across the membrane with a stoichiometry of three Na+ ions to one Ca2+ ion. Although the NCX is present in many cell types, the cardiac-specific mammalian isoform (NCX1.1) has been the most extensively characterized, where it serves as the prime mechanism of Ca2+ extrusion from the cardiomyocyte (1, 2, 5). Active transporters such as NCX1.1, which are involved in ion translocation in the mammalian heart, are highly temperature dependent. For example, it has been demonstrated that the Q10 value for NCX1.1 is in the range of 2.2-4.0 (12, 16) in mammals. Cardiac function in active salmonid species such as rainbow trout (Oncorhynchus mykiss) is distinguished by the ability of the heart to maintain adequate contractility under hypothermic conditions that are cardioplegic to mammals. Studies of Na+/Ca2+ exchange in trout sarcolemmal vesicles have shown that >75% of NCX activity is maintained after reducing the temperature from 21 to 7°C, whereas in canines that value is diminished to <10% (30). This behavior of NCX was observed in both the native membranes and when exchangers were reconstituted into asolectin vesicles, which suggests that the differential temperature dependencies between isoforms are due to differences in primary structure. The recent cloning of the trout cardiac NCX (33) provides us with a molecular model for further investigation of the temperature dependence of the NCX molecule (7).

The wild-type trout exchanger NCX-TR1.0 has a predicted topology similar to that of the mammalian NCX1.1 based on hydropathy analysis and sequence identity. At the amino acid level, the NCX-TR1.0 shows ~75% overall identity to NCX1.1. However, sequence identity between these exchangers is significantly higher in regions of the molecule known to be functionally important such as the alpha -repeats (~92%), the exchanger inhibitory peptide (XIP) site (85%), and regulatory Ca2+ binding domains (~86%) (33). Based on these comparisons, the two isoforms appear similar from a molecular perspective despite exhibiting very different temperature dependencies. In a recent study (7), we characterized in detail the temperature dependencies of NCX1.1 and TR-NCX1.0 wild-type exchangers expressed in oocytes by measuring outward currents using the giant excised-patch technique. The peak outward current of NCX1.1 exhibited typical mammalian temperature sensitivities with a Q10 value of 2.4, whereas the NCX-TR1.0 peak current was relatively temperature insensitive with a Q10 value of 1.2 (7). Furthermore, it was found that the disparities in temperature dependence between these two exchanger isoforms are unlikely due to either differences in inactivation kinetics or NCX regulatory mechanisms (7).

The purpose of this study was to delineate the regions of the NCX molecule that are responsible for its temperature sensitivity. The strategy used involved the construction of chimeric NCX molecules using cDNA derived from canine and trout NCX wild-type cDNA. Outward currents for each chimeric construct were measured in Xenopus oocytes over a temperature range of 7-30°C using the giant excised-patch technique. By using this approach, the majority of the differential temperature dependence of the NCX isoforms was found to reside within the region of the molecule that includes the first four transmembrane (TM) segments.


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Construction of chimeras. The strategy used for the construction of the chimeras is shown in Fig. 1. Initially, three chimeras were constructed and tested. Wild-type dog and trout exchanger cDNA were cut twice at homologous places into three domains: an NH2-terminal domain that comprises the first five TM segments and the XIP site; the intracellular loop, which contains the Ca2+-binding domain and alternative splicing site; and a COOH terminal domain that includes the final four TM segments. For the trout NCX-TR1.0 (33), a silent mutation was made to introduce a second BclI site at nucleotide 2128 (relative to the start codon) by using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The mutations were made in a 548-bp cassette generated by AatII digestion.


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Fig. 1.   Strategy for chimera construction. Shown is a linear schematic of the cardiac Na+/Ca2+ exchanger (NCX) topology. Transmembrane (TM) segments are depicted as black bars and are numbered accordingly. Intracellular loop contains the exchanger inhibitory peptide (XIP) region, Ca2+ binding region, and alternative splicing region as shown. Arrows with relevant restriction enzymes (StuI and BclI) denote approximate areas where the NCX molecule was cut for chimera construction. Sequence alignment is also shown with the numbers indicating the exact amino acid number at which the cut was made. NCX1.1, cardiac-specific mammalian exchanger isoform; NCX-TR1.0, wild-type trout exchanger isoform.

NCX-TR1.0 was digested with BclI and the intracellular loop fragment (Tloop) from amino acid 280 to 710 was isolated, which does not include the XIP region but spans most of the intracellular loop (from amino acid 258 to 767). Dog NCX1.1 cDNA was digested with BclI to remove a fragment from amino acid 272 to 711. The construct DTD was generated by inserting the fragment Tloop into the digested dog NCX1.1. To generate the chimera DTT, wild-type NCX-TR1.0 cDNA (without the BclI site that was introduced) was digested with BclI and BglII (at nucleotide 49 after the stop codon). The resultant fragment, Tloop+TMC, was then inserted into NCX1.1 cDNA, which had the region of Dloop+TMC removed by digestion with BclI and EagI (at nucleotide 58 after the stop codon). To generate the chimera TDD, the cDNA of wild-type NCX-TR1.0 was digested with BclI and PstI (located at the beginning of the 5' untranslated region) and a fragment that included the NH2-terminal TM segments and the XIP site (TTMN+XIP) was isolated. The cDNA of NCX1.1 was digested with BclI and BamHI (located at the beginning of the 5' untranslated region) and the fragment spanning from the beginning of the NH2 terminus to the XIP site was removed. The remaining NCX1.1 fragment was ligated with the TTMN+XIP to form the construct TDD. A fourth construct called DTTT was produced to isolate the effect of sequence differences within the XIP site and TM segment 5 (TM5) regions on the differential temperature dependence. For this construct, a StuI site was introduced through silent mutation into the cDNA of NCX-TR1.0 at nucleotide 719 as well as the chimera DTT at nucleotide 695, relative to the start codon, respectively. The cDNA was then digested with StuI and BclI. A fragment from NCX-TR1.0 cDNA spanning the region from amino acid 240 to 279 was inserted into the corresponding region of treated DTT from amino acid 232 to 271 and generating chimera DTTT. All constructs were confirmed by sequencing.

Expression of chimeras in Xenopus oocytes. Expression of chimeric exchangers in Xenopus oocytes was carried out as described previously (7, 23). In brief, chimeric cDNA was prepared from XL1-Blue Escherichia coli (Stratagene) by using a QIAprep miniprep kit (Qiagen, Mississauga, ON) and was then linearized with HindIII. Chimera cRNA was synthesized by using the T3 mMessage mMachine in vitro transcription kit (Ambion, Austin, TX) and run on a 1% agarose gel to assess purity. Oocytes were prepared as described by Longoni et al. (18) and injected with 5 ng of cRNA. Exchange activity was measured 3-4 days after injection (see Assay of Na+/Ca2+ exchange activity).

Assay of Na+/Ca2+ exchange activity. Outward Na+/Ca2+ exchange currents were measured by using the giant excised-patch technique as described previously (7, 23). Briefly, oocytes were placed in a solution containing (in mM) 100 KOH, 100 2-(N-morpholino)ethanesulfonic acid (MES), 20 HEPES, 5 EGTA, and 5 MgCl2; pH 7.0 at room temperature with MES. Gigaohm seals were formed via suction by using borosilicate glass pipettes (inner diameter of approx 20-30 µm), and membrane patches were excised by movements of the pipette tip. The pipette solution contained (in mM) 100 N-methyl glucamine (NMG)-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 Li+- to 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 either 0 or 1 µM, respectively, with MAXC software (3). All experiments were conducted first at room temperature (22-23°C), and then exchange currents were measured at different temperatures (30, 14, and 7°C) by heating or refrigerating the bath solutions. Axon Instruments hardware and software were used for data acquisition and analysis.

Inactivation kinetics and temperature-dependence parameters. To explore further the differences in chimeric and wild-type exchangers, we examined the inactivation kinetics and temperature-dependence parameters for all constructs. For the inactivation kinetics, we calculated the inactivation rate constant (lambda ) and the ratio of steady-state current to peak current (Fss) for each trace. The lambda  values were obtained by fitting current-time traces to a single exponential (7, 12). The Fss was calculated as the ratio of steady-state current over the peak current measured for the same current-time trace as based on the one-step I1 inactivation model of Hilgemann (12). The energy of activation (Eact) and temperature coefficient (Q10), were calculated for each chimera as indices of temperature dependence. The Eact was estimated from the equation
ln<FENCE><FR><NU><IT>I</IT></NU><DE><IT>I</IT><SUB>303</SUB></DE></FR></FENCE>=−<FR><NU><IT>E</IT><SUB>act</SUB></NU><DE><IT>R</IT></DE></FR><FENCE><FR><NU>303<IT>−T<SUB>i</SUB></IT></NU><DE>303<IT>×T<SUB>i</SUB></IT></DE></FR></FENCE>
where I/I303 is the normalized current, R is the universal gas constant, and Ti is the experimental temperature (in K). To allow for statistical analysis of data obtained from different patches, exchange current was normalized to that obtained at 30°C (or ~303 K). The Q10 values for the chimeric exchangers were estimated for each patch by averaging results calculated for the three pairs of temperatures by using the equation
Q<SUB>10</SUB><IT>=</IT><FENCE><FR><NU><IT>I</IT><SUB><IT>T</IT><SUB>2</SUB></SUB></NU><DE><IT>I</IT><SUB><IT>T</IT><SUB>1</SUB></SUB></DE></FR></FENCE><SUP>10<IT>/T</IT><SUB>2</SUB>−<IT>T</IT><SUB>1</SUB></SUP>
where IT1 and IT2 are the currents at the corresponding experimental temperatures T1 or T2.

Data analysis and statistics. Statistical significance of the results was determined by unpaired Student's t-test and one-way ANOVA using Microcal Origin and GraphPad software. Unless indicated otherwise, a value of P < 0.05 was considered significantly different.


    RESULTS
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Temperature dependence of chimeric proteins. The goal of this study was to determine the domains of the NCX molecule responsible for the unique temperature dependencies observed between mammalian and salmonid NCX. To accomplish this, four different chimeras were constructed from the cDNA of the temperature-sensitive canine NCX1.1 and the relatively temperature-insensitive trout NCX-TR1.0. We examined the temperature dependence of NCX current for chimeric and wild-type exchangers expressed in Xenopus oocytes. Representative current traces are shown in Fig. 2. Canine NCX1.1 and trout NCX-TR1.0 currents were qualitatively similar at 30°C, and both exchangers exhibited decreased peak and steady-state currents with decreasing temperature (Fig. 2, A and B).


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Fig. 2.   Giant patch recordings from Xenopus oocytes expressing wild-type and chimeric exchangers. Representative current traces for wild-type dog (A), trout (B), and chimeric (C-F) Na+/Ca2+ exchangers obtained from inside-out giant membrane patches. Currents were induced by the rapid application of 100 mM Na+ to the cytoplasmic surface of the patch. Regulatory Ca2+ (1 µM) and transport Ca2+ (8 mM) were present in the bath and pipette solutions, respectively.

However, this decrease in current was much more pronounced in the canine NCX1.1, in which the exchanger current at 7°C was <10% of that measured at 30°C. In contrast, trout NCX-TR1.0 activity remained relatively high at ~60% of its activity at 7°C compared to that at 30°C (7).

To examine the mechanisms of the differential temperature dependence of the NCX molecule, the temperature dependence of four chimeric NCX exchangers was examined. Initially three chimeras, DTD, DTT, and TDD, were constructed and the outward currents were measured at 7, 14, and 30°C (Fig. 2, C-E). All three NCX chimeras displayed a typical outward NCX current with an initial peak current, which then decayed to a steady-state level. However, the temperature dependence of the chimeric exchangers varied, and based on the current traces at the different temperatures, each chimera can be qualitatively labeled as having either a predominately trout- or dog-temperature phenotype. In this regard, it is clear that DTD, which includes the canine NH2-terminal TM segments and trout loop, displays characteristics similar to that of canine NCX1.1 outward exchange-current temperature dependence (Fig. 2C). At 7°C, DTD maintained only ~10% of the peak and steady-state currents measured at 30°C. DTT, in which the canine portion includes only the five NH2-terminal TM segments and the XIP site, also displayed a temperature dependence similar to the canine NCX1.1 (Fig. 2E). In contrast, currents from TDD exhibited a temperature phenotype similar to wild-type trout NCX-TR1.0 (Fig. 2D). Specifically, at 7°C, TDD maintained ~50% of its peak and steady-state currents measured at 30°C. The TDD construct includes the five NH2-terminal TM segments and the XIP site with the rest of the exchanger cDNA being derived from dog. From these results, it appears that the region responsible for the differential temperature dependencies of the NCX molecule is localized within the NH2-terminal portion of the molecule, a region that includes the XIP site. To determine whether the XIP site had any effect on the temperature dependence of the NCX, a fourth chimera was constructed and designated DTTT. In this construct the NH2-terminal end including the first seven amino acids within TM5 are canine, and the rest of the exchanger was salmonid wild type. The temperature dependence of DTTT was clearly more similar to that of the dog wild-type exchanger (Fig. 2F). At 7°C, DTTT was attenuated to ~20% of the peak and steady-state current measured at 30°C. From these results, differences in the sequences within the TM5 and XIP regions appear to play a rather minor role in the temperature dependence of the NCX molecule.

Inactivation kinetics and temperature-dependence parameters. To investigate the temperature dependence of the NCX molecule in a more quantitative manner, the effect of temperature on the Na+-dependent inactivation of the chimeric exchangers was examined. The inactivation rate constant, lambda , and Fss values for each current trace were determined. The temperature dependence of lambda  for NCX currents from the chimeric exchangers is shown in Fig. 3. In general, the lambda  values decreased monotonically with temperature for all constructs. For the chimeras DTD and DTTT, there is a slight inflection point at 14°C as the mean lambda  value for this temperature is less than the lambda  value at 7°C; however, these differences are not statistically significant.


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Fig. 3.   Temperature dependence of the inactivation rate constant, lambda , for NCX currents from chimeric exchangers. For DTD, data points represent mean values from 6 patches and 6, 8, and 17 measurements obtained for 7, 14, and 30°C, respectively. For DTT, data points represent mean values from 7 patches and 9, 12, and 28 measurements obtained for 7, 14, and 30°C, respectively. For DTTT, data points represent mean values from 6 patches and 9, 11, and 32 measurements obtained for 7, 14, and 30°C, respectively. For TDD, data points represent mean values from 5 patches and 7, 5, and 17 measurements obtained for 7, 14, and 30°C, respectively. Values of lambda  were obtained by fitting current-time traces to a single exponential.

Based on the absolute value of lambda , constructs can be placed into two distinct groups depending on the isoform origin of the NH2-terminal portion of the exchanger. The lambda  values for the trout wild type (data not shown) and TDD are significantly (P < 0.01) lower at all temperatures compared with DTD, DTT, and DTTT and canine wild type. From the one-step I1 inactivation model (12), the Fss value characterizes the extent of I1 inactivation because 1-Fss is the fraction of inactivation. For a given exchanger construct, there were no statistically significant differences between Fss values determined at the three different temperatures (Table 1).

                              
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Table 1.   Temperature dependence of Fss values for chimeric NCX

The average values of Q10 for peak and steady-state currents for the chimeras are shown in Table 2. Again, the canine wild type, DTD, DTT, and DTTT can be grouped together as having Q10 values in the range of 2.0-2.7, whereas the Q10 values for TDD and the trout wild type are significantly different and fall within the range of 1.2-1.3.

                              
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Table 2.   Q10 values for chimeric NCX

Previously, it was reported that the Eact values for peak and steady-state currents were 53 ± 1 and 66 ± 9 kJ/mol for canine NCX1.1 and 7 ± 2 and 6 ± 0.1 kJ/mol for NCX-TR1.0, respectively (7). Arrhenius plots of Na+/Ca2+ exchange peak and steady-state currents for the chimeric exchangers are shown in Fig. 4.


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Fig. 4.   Arrhenius plots of NCX currents for chimeric exchangers. NCX peak (A) and steady-state (B) current values for wild-type dog (NCX1.1), trout (NCX-TR1.0), and various chimeric exchangers. Exchange currents were normalized to those obtained at 30°C (~303K). For the peak currents, data points are mean values from 3-4 measurements from 3-4 patches, 3-5 measurements from 3-5 patches, 5 measurements from 5 patches, 6 measurements from 6 patches, 6 measurements from 6 patches, and 3-4 measurements from 4 patches for NCX1.1, NCX-TR1.0, DTD, DTT, DTTT, and TDD, respectively. For the steady-state currents, data points are averaged from 3-4 measurements from 3-4 patches, 3-5 measurements from 3-5 patches, 4 measurements from 4 patches, 5-7 measurements from 7 patches, 6 measurements from 6 patches, and 3-4 measurements from 4 patches for NCX1.1, NCX-TR1.0, DTD, DTT, DTTT, and TDD, respectively. Solid lines represent linear least-squares fit to the experimental values of currents. Fitting of energy of activation (Eact) for each exchanger value is presented in the table and figure.

We calculated the Eact values for the four chimeras, and these values together with the Eact values of wild-type dog and trout are summarized in Fig. 5. From these Eact values, it is clear that DTD and DTT exhibited the canine phenotype, whereas TDD was troutlike with respect to temperature dependence. The chimera DTTT is clearly closer to the canine than trout phenotype in this regard, as the Eact calculated from either the peak or steady-state currents is ~75% of the canine wild type. Statistical significance of Eact values between all exchangers is reported in Table 3.


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Fig. 5.   Eact for wild-type (WT) and chimeric exchangers as a function of topology. For the representation of topology, trout NCX-TR1.0 is shown in black and dog NCX1.1 is shown in gray. Eact from peak currents are shown in shaded bars and open bars represent Eact from steady-state currents. +P < 0.05, significant difference vs. trout wild-type exchanger; * P < 0.05, significant difference vs. dog wild-type exchanger.


                              
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Table 3.   Statistical significance of differences in Eact values


    DISCUSSION
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In this study, we attempt to elucidate this temperature dependence discrepancy at the molecular level by using chimeric proteins derived from wild-type trout and dog exchangers. The use of chimeric proteins in studies involving NCX (6, 13, 20) and the thermostability of proteins (25, 27) has proven useful in comparing functional differences between isoforms. Qualitative examination of outward exchange currents for the chimeras DTD, DTT, and TDD over a range of temperatures placed the region responsible for NCX temperature dependence within the NH2-terminal TM segments and the XIP site. A fourth chimera, DTTT, revealed that the differences within TM5 and the XIP regions have an effect on the temperature dependence of the NCX molecule, but the contribution is relatively small compared with the first four TM segments. This is consistent with earlier findings that deregulation of both wild-type trout and dog exchangers with chymotrypsin treatment had no significant effect on the temperature dependencies of the NCX isoforms (7). However, with the use of chimeras it is possible to more closely examine the relative contribution to the temperature dependence of the NCX by different regions of the molecule. For instance, at 7°C, the chimeras DTD and DTT (in which the NH2-terminal end including TM5 and the XIP site are dog in origin) maintain only ~10% activity of the peak current measured at 30°C. The activity of the chimera DTTT at 7°C is relatively higher, retaining ~20% of peak current measured at 30°C. From this observation, it appears that differences in sequence in TM5 and XIP do contribute to the temperature dependence of the NCX molecule, albeit in a relatively minor capacity.

The effect of temperature on the inactivation kinetics of NCX currents was characterized using lambda  and Fss. As a general trend, lambda  decreased with temperature for all chimeras measured (see Fig. 3), which is consistent with what was found in wild-type trout and dog NCX values (7). However, it should be noted that the DTT chimera had abnormally high lambda  values with large SEs at all temperatures, a phenomenon that cannot currently be explained. The inactivation rates of DTD, DTT, and DTTT were consistently faster than that of TDD over the temperature range of 7-30°C and were not significantly different than canine wild type values. This supports further the conclusion that sequence differences in the NH2-terminal region of the NCX molecule are responsible for temperature-dependence disparities between isoforms and that the XIP site and TM5 have only a small effect on the temperature dependence of the NCX. Fss values were higher for TDD compared with the other chimeras, which again indicates that the Na+-dependent inactivation was slower for this construct. However, for each chimera there is no statistically significant difference between Fss values determined over the temperature range of 7-30°C. This indicates that an increase in the inactivation rate with temperature is accompanied by an increase in the rate of recovery from inactivation. The measures of lambda  and Fss to characterize the inactivation kinetics of these chimeras support the idea that the differential temperature dependencies of the NCX isoforms are not related to Na+-dependent inactivation. However, it is apparent that the NH2 terminal region of the NCX is a determining factor in the phenotype of the NCX inactivation kinetics.

In the present study, we calculated Q10 values (see Table 2) and Eact values (see Fig. 5 and Table 3) for the chimeric exchangers in an attempt to classify the temperature dependence as mammalian or troutlike. Consistent with earlier qualitative observations, the origin of the NH2-terminal region determines the temperature dependence phenotype exhibited by the chimeric exchangers. Similar to other mammalian species (15, 16), the exchanger activity of DTD and DTT is highly temperature dependent with Q10 values in the range of 2.3-2.7. With values in the range of 48-65 kJ/mol, the chimeras DTD and DTT exhibit Eact values typical of other mammalian NCX values such as dog (4, 7, 30), rabbit (4), and guinea pig (12). Conversely, the chimera TDD is relatively temperature insensitive and exhibits Q10 (1.2-1.3) and Eact values (14-15 kJ/mol) similar to those of the trout wild-type exchanger (7). By using these parameters, the chimera DTTT displays intermediate temperature dependencies. Even though not statistically significant, the Q10 values for DTTT are slightly lower than those for canine NCX and the chimeras DTD and DTT. The Eact values for DTTT are 39-40 kJ/mol, making them ~75% of the values for canine wild type (53-66 kJ/mol; Ref. 7) but much higher than the Eact values for the ectothermic species such as trout (6-7 kJ/mol; Ref. 7) and frog (21-25 kJ/mol; Ref. 4). These data indicate that the TM5 and XIP region play a relatively minor role in the temperature dependence of the NCX compared with the first four TM segments.

Figure 6 shows a sequence alignment comparing the NH2-terminal regions of canine NCX1.1 and trout NCX-TR1.0. For convenience, the sequence alignment is split where the restriction cuts were made to make the DTTT chimera. The section of TM5 and the XIP site that is responsible for ~25% of the temperature-dependence disparity between the trout and dog NCX contains minor sequence differences. The two nonconservative substitutions in the trout XIP site (F to V and Q to R at XIP positions 5 and 18, respectively) are possible causes for the minor role this region plays in the temperature disparity between isoforms. The structural effects of these substitutions are unknown but they are likely to make the trout XIP site less hydrophobic. The phenylalanine at position 5 (F5) of the XIP site has been found to be important in both the inactivation of the NCX (19) and inhibitory effects of the XIP peptide (10). Matsuoka et al. (19) found that making a F5E substitution increased the rate of inactivation sixfold and decreased Fss twofold. This substitution, in which the aromatic and hydrophobic amino acid phenylalanine is replaced by the negatively charged and hydrophilic amino acid glutamic acid, appears to induce the conformational changes involved in inactivation more rapidly (19). In the trout XIP site, the F5V substitution observed converts the highly hydrophobic amino acid to a nonaromatic and less-nonpolar amino acid. The data from Matsuoka et al. indicated the importance of the F5, but it is not clear what the impact of the valine substitution is at this point. Related to this is the observation that the potency of the exogenous XIP on NCX inhibition is sensitive to mutations of the phenylalanine at position 5 (10). Exogenously added peptide with the XIP sequence has been shown to inhibit exchanger function, which is consistent with the hypothesis that the endogenous XIP site has an autoregulatory function (17, 20). Substitution of F5 with a charged group (glutamate) decreases inhibition by a factor of five, whereas substitution with a conserved residue (tryptophan) has no effect on inhibition (10). Replacing the phenylalanine with an alanine or valine decreases the potency of XIP peptide inhibition by ~50 and 30%, respectively (10). Presumably, the XIP binds to the exchanger through hydrophobic interactions, and aromatics like F5 are required for maximal inhibition. The valine substitution at this position is the same substitution found in the trout NCX, and this may have an effect on the inactivation kinetics of the exchanger. However, further testing with site-directed mutagenesis is needed to confirm this theory. Interestingly, the frog (Xenopus laevis) NCX (14) shows complete identity to the canine NCX in the XIP region, even though the bullfrog (Rana catesbeian) NCX has a much lower Eact than the mammalian NCX isoform. Because the bullfrog NCX Eact values are higher than the trout NCX Eact values (21-25 vs. 6-7 kJ/mol, respectively), perhaps the amino acid substitutions in the XIP region may account for this difference. However, it must be pointed out that sequence differences in the XIP domain play a relatively minor role in the temperature dependence of the NCX molecule.


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Fig. 6.   Amino acid sequence alignment of dog NCX1.1 and trout NCX-TR1.0. Alignment is split into the first four TM segments (TM1, TM2, TM3, and TM4) and the TM5/XIP site according to the restriction enzyme used to construct the chimera DTTT. Arrows and the corresponding restriction enzyme indicate the site where the exchanger was cut to make the chimera. Identical amino acids are within black bars; conserved amino acids are within gray bars. Sequence alignment was constructed using CLUSTAL W (29).

Although fixed amounts of cRNA of each construct were injected into the oocytes, the absolute value of the peak currents varied substantially. Typically, peak currents for NCX-TR1.0 were lower than those observed for NCX 1.1. The reasons for this difference are not clear but could include disparities in unitary activity, stability of mRNA, and/or stability/longevity/trafficking of the protein in the membrane. These differences in current magnitude are unlikely to alter our conclusions for several reasons. The focus of the present study is on an intrinsic property, specifically, the temperature dependence of outward Na+/Ca2+ currents. The magnitude of these currents reflects both the number and unitary currents of exchangers within the patch. Although the number of NCX molecules within a patch is likely to vary considerably, possibly reflecting differences cited above and the heterogeneity of pipette and patch geometries, there is no evidence that differences of the number of exchangers within a patch alters its unitary properties. Second, the possibility of NCX dimerization has been raised (9, 31) but remains unproven and controversial. In our study, the putative potential of dimerization as an explanation of these results is extremely unlikely as it implies the following: 1) dimerization occurs, 2) the different constructs have varying propensities for dimerization, and 3) dimerization affects the unitary currents.

To date, most studies examining the molecular mechanisms of cold adaptation of proteins have used cytoplasmic enzymes from psychrotrophic bacteria (21, 28, 32) with some studies using enzymes from Antarctic fish (8). As of yet, there is no consensus as to which molecular mechanisms are responsible for low-temperature activity (26). General mechanisms by which proteins function at low temperatures include more polar and less hydrophobic residues; a decrease in or lack of salt bridges; fewer hydrogen bonds, aromatic interactions, and ion pairs; a decrease in the number of arginine and proline residues; and an increase in surface loops with increased polar residues (24, 26). No cold-adaptive protein displays all these features, which indicates that from an evolutionary perspective, there are multiple routes to cold adaptation (21, 32). However, these mechanisms help increase the conformational flexibility of proteins, which is a necessity for function at low temperatures. Even though the NCX is a membrane protein, some of these same principles may apply in its cold adaptation because it must be flexible for ion translocation across the membrane. It has been shown that as few as four amino acid substitutions are sufficient to generate an enzyme whose low-temperature activity is significantly greater than its parent enzyme (32). It is therefore not unreasonable to assume that only a few of the amino acid substitutions in the trout NCX-TR1.0 within the first four TM segments are responsible for its relatively high activity at low temperatures. An amino acid sequence comparison of this region between NCXs from mammalian species such as the dog (22) and from ectothermic species such frog (14), trout (33), and squid (11) revealed no general trends regarding the aforementioned adaptive mechanisms that proteins use to function at low temperature. The number of prolines (7 for dog and trout, 8 for frog, and 6 for squid) and arginines (5 for dog and 6 for trout, frog, and squid) in this region are fairly well conserved between species. In addition, substitutions in loop regions between isoforms did not show an overall increase or decrease in polarity (data not shown), nor were there large differences in the number of aromatic residues (21 for dog and 20 for trout, frog, and squid). Another feature that may promote structural flexibility are glycine residues, which are thought to be destabilizing in helices and stabilizing in loop regions (26). Within the first four TM segments, the number of glycine residues is conserved between these four isoforms except in the NH2-terminal loop (6 for dog, 5 for trout, 3 for frog, and 2 for squid). From sequence analysis, there is little to discern the specific mechanisms enabling the trout NCX to function at low temperatures. In fact, it is unlikely that there is one region or a few amino acid substitutions that can totally account for the cold activity of trout NCX-TR1.0. A more likely explanation is that a series of amino acid substitutions in different regions of the molecule all play a role in the activity of the trout NCX at low temperatures.

In summary, we have placed the region responsible for the temperature dependence of the NCX in the NH2-terminal region of the molecule. The majority of the differential temperature dependence between canine and trout isoforms seems to reside in the first four TM segments, with a minor role played by TM5 and the XIP site. Further mutational analysis is needed to determine the specific amino acids involved in the temperature dependence of the molecule, but we speculate that a series of amino acid substitutions within the first four TM segments are responsible for the activity of the trout NCX at low temperatures.


    ACKNOWLEDGEMENTS

We thank Drs. K. D. Philipson and D. A. Nicoll of University of California, Los Angeles for the kind gift of canine NCX 1.1 cDNA and for critical comments on the manuscript.


    FOOTNOTES

* C. Marshall, C. Elias, and X.-H. Xue contributed equally to this work.

The support of Natural Sciences and Engineering Research Council of Canada Grant no. OGP0002321 (to G. F. Tibbits) and the Canadian Institutes for Health Research (to L. V. Hryshko) is greatly appreciated.

Address for reprint requests and other correspondence: G. F. Tibbits, Cardiac Membrane Research Laboratory, Simon Fraser Univ., Burnaby, BC V5A 1S6 Canada (E-mail: tibbits{at}sfu.ca).

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. Section 1734 solely to indicate this fact.

March 20, 2002;10.1152/ajpcell.00558.2001

Received 20 November 2001; accepted in final form 14 March 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Cell Physiol 283(2):C512-C520
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