Temperature dependence of cloned mammalian and salmonid
cardiac Na+/Ca2+ exchanger isoforms
Chadwick L.
Elias1,*,
Xiao-Hua
Xue2,*,
Christian R.
Marshall2,
Alexander
Omelchenko1,
Larry V.
Hryshko1, and
Glen F.
Tibbits2
2 Cardiac Membrane Research Laboratory, Simon Fraser
University, Burnaby, British Columbia V5A 1S6; and
1 Institute of Cardiovascular Sciences, St. Boniface General
Hospital Research Centre, The University of Manitoba,
Winnipeg, Manitoba, Canada R2H 2A6
 |
ABSTRACT |
The cardiac
Na+/Ca2+ exchanger (NCX), an important
regulator of cytosolic Ca2+ concentration in contraction
and relaxation, has been shown in trout heart sarcolemmal vesicles to
have high activity at 7°C relative to its mammalian isoform. This
unique property is likely due to differences in protein structure. In
this study, outward NCX currents (INCX) of the
wild-type trout (NCX-TR1.0) and canine (NCX 1.1) exchangers expressed
in oocytes were measured to explore the potential contributions of
regulatory vs. transport mechanisms to this observation. cRNA was
transcribed in vitro from both wild-type cDNA and was injected into
Xenopus oocytes. INCX of NCX-TR1.0 and NCX1.1 were measured after 3-4 days over a temperature range of 7-30°C using the giant excised patch technique. The
INCX for both isoforms exhibited
Na+-dependent inactivation and Ca2+-dependent
positive regulation. The INCX of NCX1.1
exhibited typical mammalian temperature sensitivities with
Q10 values of 2.4 and 2.6 for peak and steady-state
currents, respectively. However, the INCX of
NCX-TR1.0 was relatively temperature insensitive with Q10
values of 1.2 and 1.1 for peak and steady-state currents, respectively.
INCX current decay was fit with a single
exponential, and the resultant rate constant of inactivation (
) was
determined as a function of temperature. As expected,
decreased
monotonically with temperature for both isoforms. Although
was
significantly greater in NCX1.1 compared with NCX-TR1.0 at all
temperatures, the effect of temperature on
was not different
between the two isoforms. These data suggest that the
disparities in INCX temperature dependence
between these two exchanger isoforms are unlikely due to differences in
their inactivation kinetics. In addition, similar differences in
temperature dependence were observed in both isoforms after
-chymotrypsin treatment that renders the exchanger in a deregulated
state. These data suggest that the differences in INCX temperature dependence between the two
isoforms are not due to potential disparities in either the
INCX regulatory mechanisms or structural
differences in the cytoplasmic loop but are likely predicated on
differences within the transmembrane segments.
teleosts; myocardium; contractility; calcium ions
 |
INTRODUCTION |
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 |
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 |
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).

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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).

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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.
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|
Figure 3 shows an Arrhenius plot of
Na+/Ca2+ exchange peak and steady-state
currents for the wild-type dog NCX1.1 and trout NCX-TR1.0. To allow
statistical treatment of data obtained in different patches, each
exchange current was normalized to that obtained at 30°C (~303°K). Therefore, the fitting function allowing estimation of
the energy of activation, Eact, is given by the
equation
where I/I303 is the normalized current,
R is the universal gas constant, and Ti is the
experimental value of temperature (°K). The values of
Eact for the peak and steady-state currents are
53 ± 1 and 66 ± 9 kJ/mol for NCX1.1 and 7 ± 2 and
6 ± 0.1 kJ/mol for NCX-TR1.0, respectively.

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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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|
The differences in the current inactivation rates for these two
exchanger isoforms are shown in Fig. 4.
The rate of inactivation was less for trout NCX-TR1.0 than for dog
NCX1.1 at both 30°C and 7°C. The inactivation rate constants,
,
of exchange current were obtained by fitting current-time traces to a
single exponential. The
values for NCX-TR1.0 and NCX1.1 were
0.16 ± 0.02 and 0.26 ± 0.03 s
1 at 30°C,
0.10 ± 0.01 and 0.16 ± 0.05 s
1 at 14°C, and
0.10 ± 0.01 and 0.14 ± 0.03 s
1 at 7°C,
respectively (Fig. 5).

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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.
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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.
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|
To investigate further whether possible differences in the exchanger
regulatory mechanisms are associated with the temperature dependence of
exchange activity, we measured current from proteolyzed patches. Figure
6 shows the exchange outward currents at
30°C, 14°C, and 7°C from
-chymotrypsin-treated patches (1 mg/ml for 1-2 min) expressing NCX. Similar to the result observed
in dog NCX1.1, proteolysis of the cytoplasmic side of patch for trout NCX-TR1.0 increased the peak current and dramatically reduced the
decay. Behaving like wild-type NCX, proteolyzed NCX1.1 yielded temperature-sensitive exchange current, while proteolyzed NCX-TR1.0 yielded relatively temperature-insensitive exchange current. Arrhenius plots of exchange peak and steady-state currents for proteolyzed NCX1.1
and NCX-TR1.0 are shown in Fig. 7. The
values of Eact were calculated to be 54 ± 6 and 72 ± 4 kJ/mol (NCX1.1) and 14 ± 2 and 17 ± 2 kJ/mol (NCX-TR1.0) for the peak and steady-state currents, respectively
(Table 1).

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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.
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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).
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|
The average values of Q10 for the wild-type and
-chymotrypsin-treated exchangers were calculated for each patch
using the equation
where IT1 and IT2
are the currents at the corresponding experimental temperatures
T1 or T2. The mean values of Q10
for each exchanger averaged from four to six patches are presented in
Table 2.
 |
DISCUSSION |
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 (Eact, expressed as
kJ/mol) values for both peak (53 ± 1) and steady-state (66 ± 9) INCX 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
Eact 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.
 |
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. L. Elias and X.-H. Xue contributed equally to this study.
The support of Natural Sciences and Engineering Research Council of
Canada (OGP0002321) to G. F. Tibbits and Medical Research Council
(GEC3) 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, Canada V5A 1S6 (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.
Received 15 February 2001; accepted in final form 13 April 2001.
 |
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