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
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ABSTRACT |
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
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INTRODUCTION |
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
-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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METHODS |
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.
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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
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
(
) and the ratio of steady-state current to peak current
(Fss) for each trace. The
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
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
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.
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RESULTS |
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.
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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,
, and Fss
values for each current trace were determined. The temperature
dependence of
for NCX currents from the chimeric exchangers is
shown in Fig. 3. In general, the
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
value for this temperature is less than the
value
at 7°C; however, these differences are not statistically significant.

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Fig. 3.
Temperature dependence of the
inactivation rate constant, , 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 were obtained by fitting current-time
traces to a single exponential.
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Based on the absolute value of
, constructs can be placed into
two distinct groups depending on the isoform origin of the NH2-terminal portion of the exchanger. The
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).
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.
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.
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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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DISCUSSION |
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
and Fss. As a general trend,
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
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
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.
 |
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