1 Department of Biological Sciences and 2 Cardiac Membrane Research Lab, Simon Fraser University, Burnaby, British Columbia V5A 1S6; 3 Department of Biology, Queens University, Kingston, Ontario K7L 3N6; and 4 Cardiovascular Sciences, British Columbia Research Institute for Children's and Women's Health, Vancouver, British Columbia, Canada V5Z 4H4
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ABSTRACT |
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Cardiac myofibrils isolated from trout heart have been demonstrated to have a higher sensitivity for Ca2+ than mammalian cardiac myofibrils. Using cardiac troponin C (cTnC) cloned from trout and mammalian hearts, we have previously demonstrated that this comparatively high Ca2+ sensitivity is due, in part, to trout cTnC (ScTnC) having twice the Ca2+ affinity of mammalian cTnC (McTnC) over a broad range of temperatures. The amino acid sequence of ScTnC is 92% identical to McTnC. To determine the residues responsible for the high Ca2+ affinity, the function of a number of ScTnC and McTnC mutants was characterized by monitoring an intrinsic fluorescent reporter that monitors Ca2+ binding to site II (F27W). The removal of the COOH terminus (amino acids 90-161) from ScTnC and McTnC maintained the difference in Ca2+ affinity between the truncated cTnC isoforms (ScNTnC and McNTnC). The replacement of Gln29 and Asp30 in ScNTnC with the corresponding residues from McNTnC, Leu and Gly, respectively, reduced Ca2+ affinity to that of McNTnC. These results demonstrate that Gln29 and Asp30 in ScTnC are required for the high Ca2+ affinity of site II.
myocyte contractility; Antarctic icefish; salmonid
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INTRODUCTION |
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MUSCLE CONTRACTION IS INITIATED when membrane depolarization leads to an increase in cytosolic Ca2+. The binding of Ca2+ to the low-affinity sites of troponin C (TnC) initiates a conformational change in the protein that is transmitted through the other components of the thin filament, allowing cross-bridge cycling between actin and myosin. Many of the characteristic responses of the contractile apparatus can be attributed to the Ca2+ binding properties of TnC. The differences between cardiac and skeletal myofibrils are due, in part, to the structural divergence in the TnC isoforms. While both fast skeletal muscle TnC (sTnC) and cardiac TnC (cTnC) are highly conserved within mammals and between mammals and the chicken, TnC from lower vertebrates exhibits less identity. Thus establishing the links between TnC structure and myofibrillar function should provide insight into both physiological responsiveness and evolutionary origins of contractile regulation.
TnC consists of two globular domains connected by an -helical
linker. Both the NH2- and COOH-terminal domains contain two EF-hand Ca2+-binding sites. The COOH-terminal domain
possesses two high-affinity sites (III and IV) that are always bound
with either Ca2+ or Mg2+. These sites are
considered to be structural sites, helping to anchor TnC into the
troponin complex. The two isoforms of TnC that exist in vertebrate
striated muscle differ in the number of active Ca2+-binding
sites in the NH2-terminal domain. In the isoform of TnC found in fast skeletal muscle (sTnC), both sites I and II are functional, low-affinity Ca2+-binding sites. In cTnC, which
is expressed in both cardiac and slow skeletal muscle, site I is
rendered nonfunctional by amino acid substitutions that disrupt its
ability to form the pentagonal bipyramid necessary to coordinate
Ca2+. In both isoforms, Ca2+ binding to the
NH2-terminal site(s) is responsible for triggering contraction (30, 31). NMR structural studies have
demonstrated that the Ca2+-induced conformational change is
greater in sTnC than in cTnC (35).
Many studies have attempted to assess the impact of the structural differences in TnC isoforms on the functional properties of striated muscle. Ca2+ affinity is very sensitive to pH in cardiac muscle but much less so in skeletal muscle. Studies using transgenic mice (24) and TnC replacement in skinned fibers (7, 25) have demonstrated that TnC isoforms influence the pH sensitivity of striated muscle. Cardiac muscle demonstrates important differences in the length-tension relationship, the basis of the Starling relationship, that have been attributed, at least in part, to cTnC (2, 13), though there is also compelling evidence to the contrary (22, 26). Differences in temperature dependence of cardiac and skeletal muscle contractility can also be attributed, in part, to the isoform of TnC that they contain. The Ca2+ affinity of skeletal myofilaments is relatively insensitive to temperature (11, 36), whereas cardiac muscle becomes desensitized to Ca2+ at low temperatures (5, 6, 14, 16, 37). Replacement of native cTnC in skinned cardiac muscle with sTnC attenuates this desensitizing effect of temperature (15). This loss of Ca2+ sensitivity at low temperature has been attributed to a reduction in Ca2+ affinity of cTnC site II (10).
The impact of temperature on myocardial Ca2+ sensitivity is particularly important in cold-blooded animals, such as fish, that experience low and fluctuating environmental temperatures. Functional comparison of cardiac myofibrils from mammals and rainbow trout suggests that trout cardiac muscle has a higher inherent Ca2+ affinity (6). It is thought that this higher affinity facilitates cardiac function at the low temperatures (0-20°C) normally experienced by this fish (6). We have demonstrated that the high Ca2+ sensitivity of trout myofibrils is due, in part, to the differences in the affinity of mammalian and trout cTnC for Ca2+ (10). Although the amino acid sequence of trout cTnC (ScTnC) is 91% identical to mammalian cTnC (McTnC) (27), and site II is completely conserved, ScTnC has 2.3-fold higher Ca2+ affinity than McTnC at 21°C (10). This difference in affinity was maintained when McTnC and ScTnC Ca2+ binding were compared at 7 and 37°C and over a range of pH values. The comparatively higher Ca2+ affinity of ScTnC would allow for force generation to be initiated at a lower intracellular [Ca2+] in the trout myocyte.
The purpose of the present study was to identify the residues responsible for interspecies differences in Ca2+ affinity of site II in cTnC. Although all known mammalian cTnC amino acid sequences exhibit 100% identity, there are differences between fish and mammals and within fish species. We created mutants to compare the impact of differences between salmonid (ScTnC) and McTnC. We also exploited naturally occurring differences between trout and Antarctic icefish (40). Ca2+ binding to site II was measured by titrating the F27W cTnC mutants with Ca2+ while measuring fluorescence. These results demonstrate that there is no difference in the Ca2+ affinity of ScTnC and icefish cTnC (IFcTnC), despite nine amino acid differences. The differences in Ca2+ affinity between mammals and fish can be attributed to amino acid substitutions in the NH2-terminal domain, because the removal of the COOH terminus (amino acids 90-161) through the creation of cNTnC mutants maintained the difference in Ca2+ affinity. Gln29 and Asp30 were also demonstrated as being required for the higher Ca2+ affinity of ScNTnC.
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METHODS |
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Construction of the IFcTnC F27W mutant. IFcTnC cDNA cloned into PCR2.1-TOPO was first subcloned into the pGex expression vector. To accomplish this, we engineered BamHI and EcoRI restriction sites onto the 5'- and 3'-ends, respectively, of the IFcTnC cDNA, using Accurase DNA polymerase (DNmp, Farnborough Hants, UK). The sequences of the 5' sense oligonucleotide primers used were as follows: BamHI, TCAGGATCCATCGAAGGTCGTATGAACGACATCTACAAAGCA; EcoRI, GAGTTCATGAAAGGAGTAGAATAAGAATTCGCA. After PCR, the product was purified by gel electrophoresis and use of the QIAquick gel extraction kit (Qiagen, Mississauga, ON, Canada) and then digested with BamHI and EcoRI (Pharmacia Biotech, Baie d'Urfé, QC, Canada). pGex plasmid was similarly digested and then ligated with the cassette using T4 DNA ligase (GIBCO BRL, Gaithersburg, MD), and the sequence of the insert was confirmed by sequencing at the Nucleic Acid/Protein Service Unit, University of British Columbia (UBC; Vancouver, BC, Canada) using AmpliTaq dye terminator cycle sequencing.
To substitute Trp for Phe at residue 27, we used the parental plasmid (pGex) containing the wild-type IFcTnC gene insert as a template for the extension of sense and antisense oligonucleotide primers containing a tryptophan point mutation as described in Gillis et al. (10). The sequence of the 5' sense oligonucleotide primer was GCCGCCTTTGACATCTGGGTACCAGATGCCGAG. A cassette containing the mutation was made from the mutated plasmid by using the restriction enzymes BamHI and StyI (New England Biolabs, Mississauga, ON, Canada) and was ligated into the similarly digested parental plasmid containing the wild-type IFcTnC gene. The nucleotide sequence of the newly mutated insert was confirmed by sequencing. From this point on, IFcTnC will refer to F27W IFcTnC.Construction of McNTnC and ScNTnC F27W mutants. To construct McNTnC and ScNTnC F27W mutants, we introduced a stop codon into McTnC and ScTnC cDNA after the Ser codon at residue 89 using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). Both of these gene constructs had been previously mutated to contain the F27W mutation and then cloned into the pGex expression plasmid from Pharmacia Biotech as described in Gillis et al. (10). These existing parental cDNA inserts were used as templates for the extension of sense and antisense oligonucleotide primers containing the stop codon TAA. The sequences of the 5' sense oligonucleotide primers were as follows: ScTnC, GGACGACAGCTAAGGGAAAACAGAGG; McTnC, GGTTCGGTGTATGAAAGATGACAGCTAAGGAAAAAC. Cassettes containing the mutation were then made using the restriction enzymes StyI and BsmI (New England Biolabs). Full-length F27W ScTnC and McTnC cDNA constructs subcloned into the pGex vector were similarly digested, and the cassette and plasmid were then purified as described above and ligated. The nucleotide sequences of the two newly mutated inserts were confirmed by sequencing. From this point on, McNTnC will refer to F27W McNTnC and ScNTnC will refer to F27W ScNTnC.
Construction of Q29L/D30G ScNTnC, Q29L ScNTnC, D30G ScNTnC, L29Q/G30D McNTnC, and D2N/L29Q/G30D McNTnC F27W mutants. The cDNA constructs in the pGex plasmid created to synthesize ScNTnC and McNTnC were used as templates for the extension of sense and antisense oligonucleotide primers containing codons to manipulate the residues at positions 29 and 30 using the QuickChange site-directed mutagenesis kit. The sequences of the 5' sense oligonucleotide primers used were as follows: Q29L/D30G ScNTnC, GCCTTTGACATCTGGATCCTGGGGGCGGAGGACGGC; Q29L ScNTnC, GCCTTTGACATCTGGATCCTGGATGCGGAGGACGGC; D30G ScNTnC, GCCTTTGACATCTGGATCCAGGGGGCGGAGGACGGC; L29Q/G30D McNTnC, GCCTTCGACATCTGGGTGCAGGATGCAGAGGATGGCTGC. Cassettes containing the mutations were made using SmaI (GIBCO BRL) and BsmI and were then ligated into the parental plasmids that had been similarly digested. The nucleotide sequences of the inserts were confirmed by sequencing. Once the sequence of L29Q/G30D McNTnC was confirmed, it was used as a template for the extension of sense and antisense oligonucleotide primers to make the D2N/L29Q/G30D McNTnC mutant. The sequence of the 5' sense oligonucleotide primer used to replace the Asp at residue 2 with an Asn was CCATCGAAGGTCGTATGAATGACATCTATAAGGCGGC. A cassette containing the mutation was made using BsmI and BamHI and was then ligated into the parental plasmid that had been similarly digested. The nucleotide sequence of the insert was confirmed by sequencing.
Expression and purification of cTnC mutants. The pGex plasmids containing the ScNTnC, McNTnC, IFcTnC, Q29L/D30G ScNTnC, Q29L ScNTnC, D30G ScNTnC, L29Q/G30D McNTnC, and D2N/L29Q/G30D McNTnC inserts were transformed into the Escherichia coli strain BL21 for protein expression. The cTnC mutants were expressed as glutathione S-transferase fusion proteins that were then digested and purified as described previously (10). The identities of all cTnC mutants were confirmed by NH2-terminal microsequencing and amino acid analysis completed at the Nucleic Acid/Protein Service Unit, UBC. The purities of the isolated proteins as well as their atomic masses were confirmed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry completed at the UBC Mass Spectrometry Center. Collectively, these tests confirmed the identity of all cTnC mutants.
Solutions used in fluorescence studies.
The solution used to measure fluorescence at 21°C, pH 7.0, was
identical to that described in Gillis et al. (10) (in mM: 1.0 EGTA, 0.03 CaCl2, 112.0 KCl, and 50 MOPS). A miniature
Ca2+ electrode, made according to Baudet et al.
(3), was used to confirm the purity and apparent
Ca2+ affinity constant
(K'Ca) of the EGTA used in the
solution under the experimental conditions. This electrode was also
used to test the initial pCa of the solution and the change in pCa
during Ca2+ titration. This electrode was calibrated by
using Ca2+ standards (Orion Research, Boston, MA) and was
sensitive to a pCa of 8.0. The EGTA had a calculated purity of 95.6%
and a K'Ca of 2.99 × 106 M1. It should be noted that
Mg2+ was not included in the reaction buffers despite its
physiological presence. The solution composition was carefully
considered for simplicity of interpretation of the in vitro experiments
as well as accuracy of pCa in the titrations, which were determined as described above.
Fluorescence studies. The fluorescence studies were carried out as previously described (10) using a Photon Technology International (PTI) model C-30 spectrofluorometer (London, ON, Canada) attached to a NesLab (Portsmouth, NJ) water bath to maintain the cuvette at 21.0 ± 0.1°C. Fluorescence was measured during Ca2+ titration of the cTnC mutants by using an excitation wavelength of 276 nm and an emission wavelength of 330 nm. The slit widths for both the excitation and fluorescence light pathways were adjusted to setting 4 on the PTI spectrofluorometer. The relative fluorescence was calculated as the ratio of the fluorescence of the protein and an internal rhodamine standard. The relative fluorescence was calculated as the ratio of the fluorescence of the protein and an internal rhodamine standard. Before the beginning of fluorescence measurements, the current gain setting of each photomultiplier tube (PMT) of the spectrofluorometer was adjusted so that the relative fluorescence of the Ca2+-saturated cTnC mutants was equal to unity for each mutant.
Data manipulation and statistical analysis.
The Ca2+-dependent component of the fluorescence
measurements from each titration was determined by subtracting the
fluorescence at basal [Ca2+] from all measurements and
then expressing the resultant values as percentages of the maximum
fluorescence. Each data set was fitted by using the Hill equation with
Origin 6.0. (Microcal Software, Northhampton, MA) as previously
described (10). The 2, which was used as a
goodness-of-fit index of the Hill equation to our data, ranged from
0.0017 ± 0.0006 (D30G ScNTnC) to 0.0003 ± 0.00008 (IFcTnC).
The effect of the mutations on the K1/2 values ([Ca2+] at half-maximal fluorescence) determined
by the Hill equation curve fitting were analyzed statistically using a
one-way repeated measures ANOVA followed by Bonferroni post hoc tests
using the statistical software package SigmaStat. The values reported
for K1/2 are expressed as means ± SE in pCa
units. Two means were considered to be significantly different when the
P value was <0.05.
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RESULTS |
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Ca2+ affinity of IFcTnC at 21°C, pH
7.0.
Because of its location in the cTnC molecule, the fluorescent reporter
monitors Ca2+ binding to site II. The
K1/2 values were used as a measure of the
Ca2+ binding characteristics of site II. Visual comparison
of the titration curves of IFcTnC and full-length ScTnC reveals that these curves are superimposable and are shifted to the left of McTnC
(Fig. 1A). The
K1/2 of IFcTnC is not significantly different from that previously reported for full-length ScTnC (10) and is 2.4 times less than that of McTnC under the
same conditions (Table 1). These results
indicate that site II of IFcTnC has Ca2+ binding abilities
similar to those of ScTnC.
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Comparison of the Ca2+ affinities of ScNTnC and McNTnC at 21°C, pH 7.0. Comparison of the Ca2+ titration curves of ScNTnC and McNTnC reveals that the curve for ScNTnC is shifted to the left of McNTnC, indicating that less Ca2+ is required to saturate site II of ScNTnC (Fig. 1B). The K1/2 of ScNTnC is ~0.30 pCa units greater than that of McNTnC, demonstrating that it has a higher Ca2+ affinity (Table 1). The removal of the COOH-terminal domain caused the K1/2 values of both ScNTnC and McNTnC to be ~0.44 pCa units higher at 21°C, pH 7.0, than the respective full-length isoform (Table 1).
Ca2+ affinities of Q29L/D30G
ScNTnC, Q29L ScNTnC, D30G ScNTnC, L29Q/G30D McNTnC, and
D2N/L29Q/G30D McNTnC at 21°C, pH 7.0.
The replacement of Gln29 and Asp30 in ScNTnC
with Leu and Gly, respectively, caused site II of Q29L/D30G ScNTnC to
have Ca2+ binding characteristics similar to those of
McNTnC. The titration curves of Q29L/D30G ScNTnC and McNTnC are
superimposable (Fig. 2A), and
there were no differences in the K1/2 values of the two proteins (Table 1). The Hill coefficient is typically used to
describe the cooperativity of Ca2+ binding to a single
molecule with multiple binding sites (sTnC) or of
Ca2+-activated tension in a functioning myofibril.
However, this study determined that in the in vitro Ca2+
binding of cTnC, which has a single activation site, cooperativity is
highly unlikely and is reflected in the fact the Hill coefficients were
approximately unity for all cTnCs. In these experiments, the Hill
coefficient is used only as a parameter of curve fitting, and a
physiological interpretation of these relatively small differences has
been avoided. The Hill coefficients for the Ca2+ binding
curves of McNTnC and Q29L/D30G ScNTnC do not differ, reflecting the
similarities in the shape of the Ca2+ binding curves (Table
2).
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DISCUSSION |
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Heart rate is determined by many factors, but with each contraction cycle, Ca2+ binds and releases from TnC. If the Ca2+ affinity of TnC is too high, the rate of relaxation will be limited. Thus the cTnC-Ca2+ on- and off-rate constants must be compatible with cardiac performance. Despite the difference of nearly three orders of magnitude in heart rate across birds and mammals, there is very little variation in cTnC primary sequence. Within mammals and between mammals and the chicken, cTnC variation does not seem to be an important determinant of interspecies differences in myocardial contractility. When cTnC structure is compared more widely across vertebrates, a greater degree of variability is seen. Among the differences between fish and mammals and within fishes (40), it is not clear which structural variants have functional consequences.
Of the 13 sequence differences between McTnC and ScTnC, 5 occur in the
NH2-terminal domain (40). To determine the
residues responsible for the higher Ca2+ affinity of ScTnC,
we focused on this region of the protein. Previous studies have
established that sequence manipulations within the
NH2-terminal domain can have significant effect on the
ability of TnC to bind Ca2+ (12, 31).
Examination of the cTnC isoform cloned from another fish, the Antarctic
icefish, reveals that there are nine differences in sequence between
icefish cTnC and ScTnC. However, of the five sequence differences
between ScTnC and McTnC in the NH2-terminal domain, three
also occur between icefish cTnC and McTnC. These are Asn2,
Gln29, and Asp30 (Fig.
3) (40). In McTnC, the
residues at these positions are Asp, Leu, and Gly, respectively (Fig.
3). The titration curves of IFcTnC and ScTnC are superimposable, and
the K1/2 values of these curves are
similar, demonstrating that the differences in sequence between IFcTnC
and ScTnC do not appear to affect Ca2+ affinity. IFcTnC
also contains Asn2, Gln29, and
Asp30, suggesting that one or a combination of these amino
acids is responsible for the high Ca2+ affinity of ScTnC
and IFcTnC.
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To confirm that sequence differences in the NH2-terminal domain of ScTnC are responsible for its higher Ca2+ affinity, we removed the COOH terminus from both ScTnC and McTnC, creating cNTnC mutants. cNTnC mutants have been used in previous studies to examine the structure and Ca2+ activation of the NH2-terminal domain (20, 34, 35). Spyracopoulos et al. (35) have demonstrated that the Ca2+-saturated NMR solution structure of McNTnC is similar to that of the Ca2+-saturated NH2 domain of the full-length mutant (C35S/C84S) chicken cTnC (33). In the present study, ScNTnC and McNTnC had a higher Ca2+ affinity than the respective full-length isoforms. The fact that this difference in affinity was similar for both McNTnC and ScNTnC suggests that any alteration in the ability of site II to bind Ca2+ caused by removal of the COOH-terminal domain was similar for both isoforms.
The difference in affinity between ScNTnC and McNTnC is consistent with that between the full-length ScTnC and McTnC (10) (Table 1). These data demonstrate that the removal of the COOH terminus of ScTnC and McTnC maintains the difference in affinity between isoforms while removing 8 of the 13 differences in amino acid sequence (Fig. 3). This result corroborates the IFcTnC data that suggested that NH2-terminal domain amino substitutions are responsible for the differences in Ca2+ affinity.
To identify the specific residues responsible for the high Ca2+ affinity of ScTnC, we next focused on Gln29 and Asp30 because these nonconservative substitutions occur in series in a region of the protein, site I, that while unable to bind Ca2+ directly has significant influence on the Ca2+ binding characteristics of cTnC (12, 31). Replacement of Gln29 and Asp30 with the corresponding residues from McTnC, Leu and Gly, caused the new construct, Q29L/D30G ScNTnC, to behave almost identically to McNTnC. The K1/2 of the mutant was not different from that of McNTnC, and the Ca2+ titration curves are virtually superimposed. Together, these results demonstrate that Gln29 and/or Asp30 is required for the higher affinity of ScNTnC.
The Ca2+ affinities of Q29L ScNTnC and D30G ScNTnC were measured to determine whether both Gln29 and Asp30 are necessary for the high Ca2+ affinity of ScNTnC. Both mutants demonstrated lower Ca2+ affinity than did ScNTnC. The replacement of Asp with Gly appears to make the Ca2+ affinity of the mutant intermediate between ScNTnC and McNTnC. The inability of D30G ScNTnC to become saturated at high [Ca2+], however, suggests that the protein, as a result of this mutation, has lost some functional integrity. We have previously observed a similar loss of functionality when the ability of ScTnC to bind Ca2+ was measured at 37°C (10). Because 37°C is well above the physiological temperature of the protein and lethal to the trout, this result was interpreted as a loss of thermal stability (10).
The specific mechanism by which the presence of Gln and Asp at residues
29 and 30, respectively, increases the affinity of site II in ScTnC is
not known; however, we suggest that it is due to an allosteric effect
on the ability of site II to bind Ca2+. Additionally,
through interpretation of 3JHNH coupling
constants and data from 15N relaxation measurements
obtained from NMR studies of apo- and Ca2+-saturated
McNTnC, Spyracopoulos et al. (35) have suggested that
sites I and II are structurally linked. It is not unreasonable, therefore, to propose that the manipulation of the sequence in this
area of the protein could allosterically affect the ability of site II
to bind Ca2+. The replacement of a hydrophobic residue
(Leu) with Gln and the addition of a negative charge through the
insertion of Asp are likely to have an effect on protein tertiary
structure, consistent with NMR solution observations (4).
Additionally, titration of 15N-labeled ScNTnC while
monitoring two-dimensional
1H,15N-heteronuclear single quantum
correlation NMR spectra has demonstrated that site I behaves
differently in ScNTnC than in McNTnC as it continues to remain
responsive to Ca2+ following the saturation of site II
(9). Together, these results demonstrate that site I in
ScNTnC differs both structurally and functionally from McNTnC.
To determine whether only Gln29 and Asp30 are
responsible for the high Ca2+ affinity of ScNTnC, we
mutated the sequence of McNTnC to insert these residues at positions 29 and 30 in place of Leu and Gly, respectively. The Ca2+
affinity of this new protein was significantly less than that of both
ScTnC and McTnC. This result suggests that one or more residues in
combination with Gln29 and Asp30 is/are
responsible for the high Ca2+ affinity of ScTnC. The three
remaining possibilities are Asn2, Asp14, and
Val28, which are in place of Asp, Glu, and Ile,
respectively, in McNTnC. The D14E and V28I substitutions are fairly
conservative. Additionally, IFcTnC, which has the same Ca2+
affinity as ScTnC, also contains Asn at position 2 (Fig. 3). The
sequence of cTnC cloned from the frog, Xenopus laevis,
another ectothermic species, contains Asp at position 2 as in McTnC
(41) (Fig. 3). This suggests that the presence of Asn at
residue 2 may be unique only to cTnC cloned from fish and is not a
common genotype of ectothermic hearts. To determine whether
Asn2 is required, in addition to Gln29 and
Asp30, for the high Ca2+ affinity of ScNTnC, we
replaced the Asp2 in L29Q/G30D McNTnC with an Asn. The
Ca2+ affinity of the resultant mutant was lower than that
of L29Q/G30D McNTnC. This suggests that either Asn2 is not
involved in the high Ca2+ affinity of site II in ScTnC or
that other residues in addition to Asn2, Gln29,
and Asp30 are required. This result also demonstrates that
the manipulation of the amino acid sequence far outside of site II
affects Ca2+ affinity. This finding is supported by earlier
work by Gulati et al. (12), who demonstrated that the
NH2-terminal -helical overhang of cTnC, where residue 2 is located, plays a role in regulating the Ca2+ binding
characteristics of site II in cTnC.
Fluorescent probes engineered into TnC through Phe to Trp mutation in
site I have been used previously to study the Ca2+ binding
dynamics of this molecule (8, 19, 21, 27, 29, 32, 38). We
have demonstrated the effectiveness of Trp at residue 27 in reporting
Ca2+ binding to site II in McTnC and ScTnC without
significantly affecting the -helical content of the protein, which
reflects the general structure of the molecule, using far UV circular
dichroism spectra (27). Additionally, the
K1/2 values of Ca2+ binding to the
F27W cNTnC mutants and full-length IFcTnC in the present study are
within the range previously reported for that of
Ca2+-triggered tension generation in cardiac myocytes under
similar conditions (16, 18, 23, 24, 28, 39). We therefore maintain that the F27W mutation effectively reports on the
Ca2+-induced conformational response of the cTnC isoforms
without significantly altering the functional characteristics of the proteins.
We do not feel that the absence of Mg2+ confounds the results, because Allen et al. (1) demonstrated that Mg2+ (1-8 mM) had no effect on the relationship between [Ca2+] and activated force in rat ventricular skinned fibers or on the relationship between pCa and ATPase activity in skinned cardiac cells. These results therefore indicate that Mg2+ has no measurable influence on the ability of Ca2+ to activate cTnC.
In summary, this study has demonstrated that it is the sequence differences between ScTnC and McTnC in the NH2-terminal domain that are responsible for the high Ca2+ affinity of ScTnC. Additionally, Gln29 and Asp30 were demonstrated as being required for the comparatively high Ca2+ affinity of ScTnC. Because the replacements of these two residues into McNTnC did not increase its Ca2+ affinity, it is clear that other residues in addition to Gln29 and Asp30 are required. The affinity of L29Q/G30D McNTnC was not increased through the replacement of Asp at residue 2 with an Asn, illustrating that other residues in place of, or in addition to, Asn2, along with Gln29 and Asp30, are required for the high Ca2+ affinity of ScTnC. The identification of these residues represents the focus of future experiments because they represent logical targets for pharmacological intervention to increase the Ca2+ affinity of McTnC to enhance the Ca2+ sensitivity of cardiac tissue. Such an agent would be useful in end-stage heart failure, allowing for increased inotropism and improved stroke volume.
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ACKNOWLEDGEMENTS |
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We thank Dr. J. S. Ballantyne for the use of the spectrofluorometer and C. R. Marshall for molecular biology assistance.
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FOOTNOTES |
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This work is supported by operating grants from the Heart and Stroke Foundation of British Columbia and Yukon and from the Natural Sciences and Engineering Research Council of Canada to G. F. Tibbits and by a Doctoral Fellowship from the Heart and Stroke Foundation of Canada to T. E. Gillis.
Address for reprint requests and other correspondence: G. F. Tibbits, Cardiac Membrane Research Lab, Simon Fraser Univ., 8888 Univ. Drive, 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.
First published January 8, 2003;10.1152/ajpcell.00339.2002
Received 23 July 2002; accepted in final form 11 December 2002.
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