©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
The Role of the Four Ca Binding Sites of Troponin C in the Regulation of Skeletal Muscle Contraction (*)

(Received for publication, October 27, 1995; and in revised form, January 11, 1996)

Danuta Szczesna Georgianna Guzman Todd Miller Jiaju Zhao Kamelia Farokhi Herman Ellemberger James D. Potter (§)

From the Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, Miami, Florida 33101

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In order to study the role of the Ca-specific sites (I and II) and the high affinity Ca-Mg sites (III and IV) of TnC in the regulation of muscle contraction, we have constructed four mutants and the wild type (WTnC) of chicken skeletal TnC, with inactivated Ca binding sites I and II (TnC1,2-), site III (TnC3-), site IV (TnC4-), and sites III and IV (TnC3,4C-). All Ca binding site mutations were generated by replacing the Asp at the X-coordinating position of the Ca binding loop with Ala. The binding of these mutated proteins to TnC-depleted skinned skeletal muscle fibers was investigated as well as the rate of their dissociation from these fibers. The proteins were also tested for their ability to restore steady state force to TnC-depleted fibers. We found that although the NH(2)-terminal mutant of TnC (TnC1,2-) bound to the TnC-depleted fibers (with a lower affinity than wild type TnC (WTnC)), it was unable to reactivate Ca-dependent force. This supports earlier findings that the low affinity Ca binding sites (I and II) in TnC are responsible for the Ca-dependent activation of skeletal muscle contraction. All three COOH-terminal mutants of TnC bound to the TnC-depleted fibers, had different rates of dissociation, and could restore steady state force to the level of unextracted fibers. Although both high affinity Ca binding sites (III and IV) are important for binding to the fibers, site III appears to be the primary determinant for maintaining the structural stability of TnC in the thin filament. Moreover, our results suggest an interaction between the NH(2)- and COOH-terminal domains of TnC, since alteration of sites I and II lowers the binding affinity of TnC to the fibers, and mutations in sites III and IV affect the Ca sensitivity of force development.


INTRODUCTION

Vertebrate skeletal muscle contraction is activated by the binding of Ca to the low affinity Ca binding sites of troponin-C (TnC), called the Ca-specific sites and designated as sites I and II(1) . These sites are located in the NH(2)-terminal domain of TnC, which is separated from the COOH-terminal domain of TnC by a 31-residue single helix (D/E)(2, 3) . The COOH-terminal region of TnC contains two high affinity Ca binding sites designated as sites III and IV and referred to as the Ca-Mg sites(1) . Sites I and II bind Ca specifically with K 3 times 10^5M, whereas sites III and IV bind Ca with K 2 times 10^7M and Mg with K 2 times 10^3M. Under physiological conditions, in relaxed muscle, sites III and IV of TnC are primarily occupied by Mg and can become partially saturated with Ca during contraction depending on the time course of the [Ca] transient(4) . While the process of Mg exchange for Ca is much too slow (k(d) 8 s) to trigger muscle contraction, the kinetics of Ca binding to the NH(2)-terminal domain sites I and II of TnC are coupled with the rate of muscle activation, implying that these sites are directly involved in muscle regulation. Inactivation of either of them significantly decreases regulatory function of TnC(5) , indicating that sites I and II are both required for the full regulatory activity of TnC. In contrast, the role of the high affinity Ca-Mg binding sites, III and IV, is still not entirely clear. A number of studies have suggested a structural role for these sites in maintaining the stability of the whole troponin complex in the thin filament, presumably through TnC-TnI interactions (1, 6) . The interaction interface of the COOH-terminal domain of TnC containing the high affinity Ca-Mg sites, III and IV, has been shown to be located in the NH(2)-terminal region of TnI (residues 1-98) and also near the NH(2)-terminal end of the inhibitory region of TnI containing residues 96-116(7, 8, 9, 10) . This so-called antiparallel fashion of TnC-TnI interaction is also true for the Ca-dependent interaction of the NH(2)-terminal regulatory domain of TnC with the COOH-terminal region of TnI as well as with the COOH-terminal end of the TnI inhibitory peptide(10, 11, 12) .

Recently we have shown that thrombin fragments of the NH(2)-terminal domain of TnC, containing Ca binding sites I and II, maintain the regulatory properties of intact TnC, whereas fragments from the COOH-terminal domain are mostly involved in the Ca-Mg-dependent interactions of TnC with TnI(13) . In the present study we have examined the role of the NH(2)- and COOH-terminal Ca binding sites of TnC using mutants of TnC that have either inactivated Ca-specific sites I and II (TnC1,2-) or structural site III (TnC3-), IV (TnC4-), or III and IV (TnC3,4-). The effect of these mutations on their structure and function was investigated using the TnC-depleted skinned skeletal muscle fiber system(5, 6) , where incorporation of the TnC mutants and steady state isometric force measurements are readily performed. We found that the NH(2)-terminal mutant of TnC (TnC1,2-) was able to bind to the fibers but failed to develop steady state force. Although TnC1,2- bound to the fibers in the pCa 8 relaxing solution, it was easy to displace it with WTnC, indicating a low affinity of this mutant for its binding sites in the fibers. All three COOH-terminal mutants of TnC (TnC3-, TnC4-, and TnC3,4-) demonstrated weakened ability to bind to TnC-depleted fibers. They dissociated from the fibers with different rates, implying that Ca binding sites III and IV of TnC contribute differently to the maintenance of the structural integrity of the whole troponin complex. Consistent with our thrombin fragment studies(13) , site III appears to be the primary determinant of the affinity of this interaction between TnC and its binding site in the fiber in the presence of Mg. Once bound to the fibers, all three of these mutants were able to activate steady state force as in unextracted intact fibers.


EXPERIMENTAL PROCEDURES

Site-directed Mutagenesis, Expression, and Purification

Ca-Mg binding sites III and IV of chicken skeletal TnC (cDNA of WTnC kindly provided by Hitchcock(14) ) were inactivated by the replacement of the single aspartic acid residue in the X positions of the Ca binding loops with alanine. Two mutated proteins, TnC3- (Asp Ala) and TnC4- (Asp Ala) were generated using the oligonucleotide-mediated, site-directed mutagenesis procedure of Kunkel et al.(15) . The double mutant, TnC3,4- (Asp Ala, Asp Ala) was constructed by replacing the wild type Ca binding site IV in TnC3- with the mutated Ca binding site IV of TnC4-, using the unique ClaI and HindIII sites (which flank the site IV coding region). The double mutant of TnC1,2- was constructed by replacing the wild type Ca binding site II in TnC1- (Asp Ala) obtained as described above (15) with mutated Ca binding site II (Asp Ala) using the EcoRV and KpnI sites (which flank the site II coding region).

The amino acid sequence of the WTnC and all mutants used in this study contained glutamic acid and aspartic acid at positions 99 and 100, respectively, as originally incorporated in the synthesized cDNA of TnC (14) used in this study. Subsequently it was found that the correct sequence of chicken skeletal TnC (16, 17) at these positions consists of alanine (Ala) and asparagine (Asn), respectively. To test the significance of this two-amino acid difference, the sequence of WTnC was corrected to Ala and Asn, and the protein was tested in the fiber experiments as described below. No difference was found between the corrected and noncorrected WTnCs with respect to steady state force development of WTnC reconstituted fibers (data not shown).

The mutated proteins were expressed in Escherichia coli BL-21 and purified based on Sheng (5) with the following modifications. One ml of a log phase subculture of E. coli was inoculated into 2 liters of enriched medium. After 12 h of culture, protein expression was induced with 0.4 mM isopropyl 1-thiol-beta-D-galactopyranoside, and the cells were harvested 4 h later. Cells pelleted by centrifugation (5 min at 6400 times g) were resuspended in 1M NaCl, 50 mM Tris, pH 7.5, 0.1 mM CaCl(2), 1 mM dithiothreitol with 1 µM leupeptin and pepstatin and 10 µM phenylmethylsulfonyl fluoride. Alternatively, cells were stored at this point at -20 °C for later isolation of protein or sonicated (for 6 min at high intensity in 30-s pulses with 90-s rests) on wet ice with a sonicator (Heat Systems model XL2020). Sonicates were cleared by centrifugation, adjusted to 1 M ammonium sulfate, and if a precipitate formed, centrifuged again. Cleared supernatants were applied to a 2.5 times 20-cm phenyl-Sepharose column, equilibrated in the above buffer containing 1 M ammonium sulfate (to increase the affinity of the expressed protein to the hydrophobic matrix), washed with the same buffer, and then eluted with 50 mM Tris, pH 7.5, 2 mM EDTA, 1 mM dithiothreitol. Protein fractions were dialyzed against a solution containing 50 mM Tris, 1 mM CaCl(2), 6 M urea, pH 8.0, and applied to a 5 times 8-cm DE-52 column. The column was washed with the same buffer, and protein was eluted with a salt gradient of 0-500 mM KCl (2 times 300 ml). Fractions of >95% purity (judged by SDS-PAGE) (^1)were pooled and dialyzed against 5 mM ammonium bicarbonate, lyophilized, and stored at -70 °C.

Ca Binding Measurements

One-ml aliquots of purified protein were dialyzed 24 h against 1 liter of 100 mM KCl, 10 mM MOPS, and 0.1 mM CaCl(2), pH 7.0, per Sheng(5) . Dialysis bags were transferred to another solution containing the same buffer to which 40 µCi of Ca had been added. Control aliquots of buffer were dialyzed in parallel and sampled to confirm that equilibrium was reached (cpm inside = cpm outside). At this point 100-µl aliquots were transferred to scintillation medium, and cpm was determined. Mol of Ca/mol of protein were calculated using net cpm and specific activity of Ca in the 1-liter solution.

SDS-PAGE of Mutated TnCs

Purified WTnC, TnC1,2-, TnC3-, TnC4-, and TnC3,4- were run on 15% SDS-PAGE gels, according to Laemmli et al.(18) . The samples contained either 1 mM CaCl(2) or 2 mM EGTA.

Skinned Fiber Preparation

Skeletal muscle fibers isolated from rabbit psoas muscle were dissected into small bundles and chemically skinned with 50% glycerol, 1% Triton X-100 in the relaxing solution (pCa 8) containing 10M [Ca], 1 mM [Mg], 7 mM EGTA, 5 mM [MgATP], 20 mM imidazole, pH 7.0, 20 mM creatinine phosphate, and 15 units/ml of creatine phosphokinase, ionic strength = 150 mM, for 24 h at 4° C. Fibers were then stored at -20 °C in the same solution without Triton X-100 for 2-4 weeks.

Measurements of Steady State Force Development

The skinned fibers were mounted on a force transducer equipped with stainless steel clips(19) , and the initial steady state isometric force was recorded in the pCa 4 (contracting) solution. The composition of this solution is the same as for the pCa 8 relaxing buffer except [Ca] = 10M. The endogenous TnC was extracted from the fibers by incubation with a 5 mM EDTA solution, pH 7.8, for 30 min. After TnC extraction, the fibers developed about 20% of the initial force (residual force) in the pCa 4 solution. Reconstitution of the fibers with the TnCs (WTnC, TnC1,2-, TnC3-, TnC4-, and TnC3,4-) was performed by incubation, with 0.1-0.5 mg/ml protein dissolved in the pCa 8 solution, for 15-20 min followed by washing with the pCa 8 relaxing buffer to remove excess unbound TnCs. Maximal steady state force was then measured in the pCa 4 solution.

Both the unextracted and the reconstituted fibers were also tested for force development in solutions of increasing [Ca], from pCa 8 to pCa 4. Data were analyzed using the following equations: 1) percentage of force restored = 100 times (force restored after reconstitution with TnC - residual force), and 2) percentage of change in force = 100 times [Ca]^n/([Ca]^n + [Ca]^n), where ``[Ca]'' is the free Ca concentration that produces 50% force and n is the Hill coefficient.

The Binding of AC-TnC1,2- to TnC-depleted Fibers: Simultaneous Force and Fluorescence Measurements

In order to test the binding of the non-force-producing mutant TnC1,2- to the TnC-depleted fibers, the mutant was fluorescently labeled with acrylodan (AC-TnC1,2-). The labeling was performed in a solution of 1 mg of TnC1,2- dissolved in 10 mM imidazole, pH 7.0, 100 mM KCl, 2 mM EGTA with a 5-fold molar excess of AC (6-acrylolyl-2-dimethylaminonaphtalene), for 4 h at room temperature. The reaction was terminated with 5 mM dithiothreitol, and excess reagents were removed by 16 h of dialysis at 4 °C against the pCa 8 solution. AC-TnC1,2- was incorporated into the TnC-depleted fibers with a 20-min incubation in the pCa 8 solution. Unbound AC-TnC1,2- was removed by further incubation in pCa 8 solution. Steady state force measurements were performed simultaneously with the fluorescence measurements on the reconstituted fibers(19, 20) . Steady state force was also measured for nonlabeled TnC1,2- reconstituted in the fibers. No difference was found with respect to the labeled or nonlabeled mutant.


RESULTS

Binding of Ca to WTnC, TnC1,2-, TnC3-, TnC4-, and TnC3,4-

Equilibrium dialysis was used for the determination of Ca binding to the four mutants of TnC to confirm the stoichiometry of the binding expected after inactivation of Ca binding sites I and II (TnC1,2-) or sites III (TnC3-), IV (TnC4-), or III and IV (TnC3,4-) of TnC. As is shown in Fig. 1, WTnC binds 3.8 ± 0.17 mol of Ca/mol of TnC, whereas mutants, TnC1,2-, TnC3-, TnC4-, and TnC3,4- bind 2.27 ± 0.11, 2.93 ± 0.02, 2.87 ± 0.33, and 2.12 ± 0.27, respectively. Replacement of Asp at the X-coordination position of the Ca binding loop with Ala has been shown to specifically inactivate Ca binding (21) to a Ca binding site.


Figure 1: Binding of Ca to WTnC, TnC1,2-, TnC3-, TnC4-, and TnC3,4-. The binding study was performed using the equilibrium dialysis method (see ``Materials and Methods''). WTnC bound 3.8 ± 0.17 mol of Ca/mol of protein, whereas TnC1,2-, TnC3-, TnC4-, and TnC3,4- bound 2.27 ± 0.11, 2.93 ± 0.02, 2.87 ± 0.33, and 2.14 ± 0.27, respectively. Data are the average of three experiments.



Effect of Ca Binding to WTnC, TnC1,2-, TnC3-, TnC4-, and TnC3,4- on Their Migration on SDS-PAGE Gels

Fig. 2represents SDS-PAGE patterns of the mutated proteins in the presence or absence of Ca. The rate of migration of the Ca-bound form of WTnC is faster compared with the Ca-free form (in the presence of EGTA). The same Ca-dependent change in the rate of migration was also observed for TnC1,2-. Thus, inactivation of the Ca-specific regulatory sites (I and II) of TnC did not change the Ca-dependent electrophoretic mobility shift of this mutant compared with WTnC. In contrast, inactivation of the COOH-terminal sites (III and IV) of TnC dramatically decreased the Ca-induced difference between the Ca-bound and the Ca-free forms of the mutants. TnC4- exhibited a small Ca-induced change in migration, but TnC3- and TnC3,4- showed no change in mobility, suggesting that Ca binding to site III has a significant effect on the conformation of TnC.


Figure 2: SDS-PAGE of WTnC, TnC1,2-, TnC3-, TnC4-, and TnC3,4-. Samples of the purified mutants of TnC were run on 15% SDS-PAGE in the presence of 1 mM CaCl(2) (+) (lanes 2, 4, 6, 8, and 10) or 2 mM EGTA(-) (lanes 1, 3, 5, 7, and 9). WTnC (lanes 1 and 2), TnC1,2- (lanes 3 and 4), TnC3- (lanes 5 and 6), TnC4- (lanes 7 and 8), and TnC3,4- (lanes 9 and 10).



Reconstitution of Skinned Skeletal Muscle Fibers with AC-TnC1,2-

Fig. 3shows the simultaneous force and fluorescence measurements of AC-TnC1,2- reconstituted fibers. Incubation of TnC-depleted fibers with AC-TnC1,2- in pCa 8 solution resulted in the binding of the mutated protein to the fibers (Fig. 3B). The fluorescence intensity increased by 13% when the fibers were transferred from pCa 8 to the pCa 4 solution as a result of the Ca-induced fluorescence change in AC-TnC1,2-. At the same time, the force measurements (Fig. 3A) showed that there was no tension developed by AC-TnC1,2- reconstituted fibers when tested in the pCa 4 solution. Thus, as expected, inactivation of Ca-specific sites I and II in TnC resulted in the inability of TnC1,2- reconstituted fibers to activate muscle contraction. To test the ability of WTnC to compete with AC-TnC1,2- already bound to the fibers, they were incubated with WTnC in pCa 8. Steady state force was restored to 90% of the level of the unextracted fibers, while the fluorescence intensity decreased to the basal level as a result of AC-TnC1,2- displacement by WTnC. This demonstrates that the TnC1,2- mutant was capable of binding to the TnC-depleted fibers but could not develop steady state force. It also showed that its binding to the fibers was weaker than WTnC, since WTnC was able to easily replace TnC1,2- within the fibers. Nonlabeled TnC1,2- behaved in the same manner as labeled TnC1,2- (data not shown), with the obvious exception that fluorescence could not be measured.


Figure 3: Simultaneous force (A) and fluorescence (B) measurements of skinned skeletal muscle fibers reconstituted with AC-TnC1,2-. After initial steady state force measurement of the skinned fibers (A), endogenous TnC was extracted with 5 mM EDTA, pH 7.8 (see ``Materials and Methods''). The extracted fibers were then incubated with a fluorescently labeled mutant of TnC having inactivated Ca binding sites I and II (AC-TnC1, 2-). Reconstitution was performed in pCa 8, and incorporation of the protein was detected utilizing fluorescence measurements (B). Steady state force was not changed as compared with the residual force of the extracted fibers (A). Inactivation of the regulatory Ca binding sites (I and II) of TnC resulted in a loss of force restoration (A). There was a small change in fluorescence intensity upon Ca binding to AC-TnC1,2- (B). The fibers were then exposed to WTnC, and the force and fluorescence measurements were repeated. Restoration of steady state force was about 90% compared with the initial force. The fluorescence intensity decreased to 10% as a result of the replacement of AC-TnC1,2- by WTnC.



The Binding of the COOH-terminal TnC Mutants to TnC-depleted Skinned Muscle Fibers

As shown in Fig. 4, all COOH-terminal mutants of TnC were able to bind to the TnC-depleted skinned fibers and reconstitute steady state isometric force to the level of the unextracted, intact fibers. The concentration dependence curves demonstrate that complete saturation of the fibers was achieved at a protein concentration of 10 µM. Because of the fast dissociation of TnC3,4- from the fibers (see below), this protein had to be present during the force measurements. In Fig. 4we show the concentration dependence of TnC3,4- reconstitution done two different ways. In both cases, the initial reconstitution was carried out in the pCa 8 solution. In one case (bullet), the force developed was measured in pCa 4 after the removal of unbound protein. In the second case (circle), the fibers were transferred directly to the pCa 4 solution containing the protein. As can be seen, less force was developed in the former case due to the dissociation of TnC3,4- during the pCa 8 wash.


Figure 4: The concentration dependence of force restoration in TnC-depleted skinned fibers reconstituted with WTnC, TnC3-, TnC4-, and TnC3,4-. Skinned skeletal fibers were tested for initial steady state isometric force and then extracted with 5 mM EDTA, pH 7.8, for 30 min. After TnC extraction, the fibers were incubated with increasing concentrations (abscissa) of WTnC (), TnC3- (up triangle), TnC4- () or TnC3,4- (bullet) dissolved in the pCa 8 solution for 15 min. Individual fibers were used for each concentration tested. Excess unbound protein was removed by washing in the pCa 8 solution followed by the measurement of force development in the pCa 4 solution. For TnC3,4- two curves are presented. The extra curve (circle) represents measurements with the protein present in the pCa 4 contraction solution. Data points are the average of three to six experiments.



Fig. 5illustrates the binding properties of WTnC, TnC3-, TnC4-, and TnC3,4- to TnC-depleted fibers. The experimental protocol is presented for TnC3- in Fig. 5A. The fibers, initially tested for force development in the pCa 4 solution, were extracted with 5 mM EDTA for 30 min. The residual force approximated the amount of endogenous TnC remaining in the fibers. TnC-depleted fibers were then reconstituted with TnC3- with a 20-min incubation, followed by force measurements to confirm that the maximal level of reconstitution had been achieved. Then steady state isometric force was measured at different time intervals to determine the rate of TnC3- dissociation from the fibers. Between the measurements the fibers were relaxed in the pCa 8 solution. The same protocol was used for WTnC and all TnC mutants. Fig. 5B demonstrates the time course of the dissociation of all of the mutated proteins and WTnC from the reconstituted fibers. As illustrated, WTnC and TnC4- exhibited similar slow dissociation from the fibers, while the dissociation of TnC3- was much faster. After 30 min of exposure of the TnC3- reconstituted fibers to the pCa 8 solution, 50% of the restored force was lost. The double mutant, TnC3,4- was the fastest dissociating protein, showing very low affinity for the fibers when incubated in the absence of Ca (presence of Mg). Isometric force dropped to the residual level after a 25-min incubation of the TnC3,4- reconstituted fibers in the pCa 8 solution (Fig. 5, B and C). After a 1- h exposure of the reconstituted fibers to the relaxing solution, they were tested to determine their ability to regain force when reconstituted with WTnC. These fibers were able to bind WTnC, and force was restored to 80-90% of the value of unextracted fibers (Fig. 5C). Thus the observed drop in force was due to TnC dissociation and not due to the deterioration of the fibers with time.


Figure 5: Dissociation rates of WTnC, TnC3-, TnC4-, and TnC3,4- from TnC-depleted skinned muscle fibers reconstituted with the mutated TnCs. A, the protocol for the dissociation rate measurements of the TnC3- reconstituted fibers. A-C, skeletal muscle fibers were tested for force development in the pCa 4 solution, and then the endogenous TnC was extracted with 5 mM EDTA, pH 7.8, for 30 min. Incorporation of WTnC, TnC3-, TnC4-, and TnC3,4- into the fibers was achieved with a 20-min incubation. B, steady state isometric force was measured at different time intervals to determine the rate of dissociation of the incorporated mutants from the fibers. circle, WTnC; bullet, TnC4-; down triangle, TnC3-; , TnC3,4-. C, after 1 h of dissociation of the reconstituted mutants from the fibers, the fibers were incubated with WTnC dissolved in pCa 8 solution. Force restoration was then tested in the pCa 4 solution. , WTnC; bullet, TnC4-; down triangle, TnC3-; box, TnC3,4-.



Effect of Inactivation of the COOH-terminal Ca Binding Sites in TnC on the Ca Dependence of Force Development

The force-pCa relationships were determined for the fibers reconstituted with WTnC, TnC3-, TnC4-, and TnC3,4-. As is shown in Fig. 6A, no change in the force-pCa relationship was observed for the fibers reconstituted with WTnC compared with unextracted fibers. For the mutants containing inactive site III (TnC3-), site IV (TnC4-), or sites III and IV (TnC3,4-), a leftward shift toward lower [Ca] of the force-pCa relationships was observed (Fig. 6, B, C, and D), indicating an increase in the Ca sensitivity of force development. This may indicate an interaction between the two domains of TnC where inactivation of the high affinity Ca binding sites (III and IV) affects the Ca sensitivity of force development maintained by the low affinity Ca binding sites (I and II).


Figure 6: The force-pCa relationship for fibers reconstituted with WTnC, TnC3-, TnC4-, and TnC3,4-. Solid curves, the unextracted fibers (bullet) were tested for force development in solutions of increasing Ca concentrations. Dotted curves, after extraction of endogenous TnC, the fibers () were reconstituted with WTnC (A), TnC3- (B), TnC4- (C), and TnC3,4- (D) and tested for force development in the different pCa solutions, from pCa 8 to pCa 4. The solid and dashed lines represent the best fit of the data to the Hill equation (see ``Materials and Methods''). A, control, pCa = 5.46, n = 2.99; after reconstitution with WTnC, pCa = 5.48, n = 2.60; B, control, pCa = 5.41, n = 3.32; after reconstitution with TnC3-, pCa = 5.57, n = 3.67; C, control, pCa = 5.46, n = 2.90; after reconstitution with TnC4-, pCa = 5.66, n = 3.15; D, control, pCa = 5.47, n = 4.28; after reconstitution with TnC3,4-, pCa = 5.72, n = 4.19.




DISCUSSION

We studied the role of Ca specific sites (I and II) and the high affinity Ca binding sites (III and IV) of TnC in the regulation of skeletal muscle contraction. Under physiological conditions the structural sites (III and IV) are saturated with Mg, whereas sites I and II are not occupied by metal. The initiation of muscle contraction occurs when Ca binds to the low affinity sites (I and II) in TnC since exchange of Mg for Ca at sites III and IV is too slow to account for muscle activation(4) . Therefore, sites I and II of TnC are the regulatory sites, whereas the high affinity Ca binding sites (III and IV) are thought to maintain the structural integrity of the whole troponin complex in the thin filament(6, 25) .

Our results demonstrate that NH(2)-terminal sites I and II of TnC indeed play an important role in the Ca-triggered regulation of muscle contraction. Skeletal muscle fibers reconstituted with the mutated TnC, having inactivated sites I and II (TnC1,2-), were not able to develop steady state force, although the mutant protein bound to the TnC depleted fibers even in the absence of Ca (presence of Mg). This is in accord with our previous study (5) where two separate mutants containing either inactive site I or II could only partially restore force to TnC-depleted skeletal fibers. Consistently, inactivation of the only regulatory site in cardiac TnC resulted in the loss of regulation of the Ca-dependent ATPase activity in TnC-extracted myofibrils(26) .

Interestingly, inactivation of the regulatory sites in TnC also affected the binding of TnC1,2- to the TnC-depleted fibers. Its binding affinity was lower than WTnC, since WTnC was able to displace TnC1,2- from the fibers. This suggests an interaction between two domains in TnC where altered NH(2)-terminal sites I and II have affected the structure and function of the COOH-terminal sites III and IV. Since the binding of TnC to the fibers is affected by the binding of Mg or Ca to the Ca-Mg sites (see below and (6) ), it is possible that inactivation of sites I and II lowers the metal binding affinity of sites III and IV and consequently the affinity of TnC1,2- for the fibers.

On the other hand, inactivation of either of the COOH-terminal high affinity Ca binding sites (III or IV) affected the association of the mutated TnCs from the thin filament. All three mutated proteins, TnC3-, TnC4-, and TnC3,4-, dissociated from the reconstituted fibers faster than WTnC, with the TnC3,4- dissociating the fastest. Interestingly, sites III and IV appeared not to be equal in terms of maintaining the structural stability of TnC in the fibers. The mutant of TnC that contained inactivated Ca binding site IV (TnC4-) bound to the TnC-depleted fibers and restored force almost as well as the wild type TnC. Its dissociation rate from the reconstituted fibers was also similar to the rate of dissociation of WTnC. The active Ca binding site III in this protein was therefore sufficient to maintain its Mg-dependent binding to the TnC-depleted fibers. In contrast to that, the mutant containing inactivated Ca binding site III (TnC3-) dissociated from the reconstituted fibers much faster than TnC4-. After 30 min exposure of the TnC3- reconstituted fibers to the pCa 8 solution, only half of the reincorporated protein remained bound to the fibers. The observed difference in the ability of these two sites (III and IV) to maintain the structural integrity of TnC in the fibers does not appear to be due to the different Ca binding stoichiometry of the mutated proteins. Both, TnC4- and TnC3- bound essentially 3 mol of Ca/mol of protein, respectively, as compared with the 4 mol of Ca bound by WTnC. They also bound to the TnC-depleted skinned fibers in a Mg-dependent manner over the same concentration range as WTnC. The observed difference in the rate of their dissociation from the fibers is in accord with our previous investigation on the thrombin fragments of TnC, demonstrating the functional differences between the Ca binding sites III and IV(13) , with the site III being more crucial for maintaining the structural integrity of the troponin complex.

The distinction between sites III and IV of TnC was also observed by Brito (22) in cardiac TnC (CTnC) mutants having either inactive Ca binding site III or IV. CTnC mutants containing inactivated site III produced greater instability in the C-terminal domain of CTnC than mutants containing inactivated site IV, as was judged by NMR spectroscopy. Our mutant with inactivated Ca binding site III (TnC3-) was also less stable in terms of its association with the thin filament in the fiber and dissociated from the reconstituted fibers faster than the mutant with inactivated site IV (TnC4-). Moreover, the Ca-induced difference of the electrophoretic migration of the mutated proteins in the presence of SDS was lost following inactivation of Ca binding site III (TnC3-) or III and IV (TnC3,4-). In contrast, the mutant TnC4- with site III still active demonstrated a small Ca-sensitive change in the electrophoretic migration. The greatest Ca-dependent mobility difference was observed for WTnC and TnC1,2-. Apparently, inactivation of the NH(2)-terminal sites I and II in TnC does not affect the structural change needed to produce this effect of Ca binding to sites III and IV. These results suggest that in the presence of Ca the NH(2)-terminal region of TnC is more stable than its COOH terminus, and inactivation of two high affinity Ca binding sites (III and IV) significantly decreased the structural stability of TnC, preventing the change in migration of TnC3,4- on SDS-PAGE due to Ca.

In another study, no functional difference between sites III and IV in CTnC was found with respect to the affinity of the mutated proteins for the thin filament(23) . This might be due to functional differences that have been found between skeletal and cardiac muscle preparations (23, 26) , differentiating the affinity of the TnC isoforms to the thin filament and possibly altering their interaction with the native TnI. However, consistent with our observation, the double mutant with both Ca binding sites III and IV inactivated was unable to restore force unless it was present in the contraction solution(23) . No difference between the dissociation rate of chicken skeletal TnC mutants containing either inactivated site III or IV and reconstituted to rabbit TnC-depleted fibers was observed by Sorenson et al.(24) . This may be due to the very fast dissociation rate of both mutated TnCs found by these authors in contrast to the rates detected in our study for TnC3- or TnC4-. After a 10-min exposure of the reconstituted fibers to the Mg-containing relaxing solution, they observed as much as 70% of the tension decrease for both mutated TnCs(24) . In contrast, comparable rates of dissociation were only observed by us for the double TnC mutant containing inactivated sites III and IV (TnC3,4-). This mutant demonstrated a significant inability to remain bound to the fibers in the presence of Mg and could restore steady state force only when it was present in the contraction (pCa 4) solution. The reason for this difference between our work and that of Sorenson et al.(24) remains unknown but could be due to their use of trifluoroperazine in their TnC extraction solution, which may interfere with subsequent TnC rebinding or to the fact that their preparation likely has a lower amount of endogenous TnC than ours. Interestingly, we observed that the force-pCa relationship was shifted toward lower Ca concentrations for all of our mutated TnCs, whereas in Sorenson et al.(24) only their equivalent TnC3- mutant showed an altered tension-pCa relationship, with a shift toward higher Ca concentrations. The increased Ca sensitivity of force development produced by our mutants suggests that the COOH-terminal high affinity Ca binding sites III and IV may affect the Ca binding and regulatory properties of the NH(2)-terminal low affinity Ca binding sites I and II. This was not observed by Negele et al.(23) where their wild types and mutants of cardiac TnCs, when reconstituted into TnC-depleted skeletal myofibrils showed no change in the Ca sensitivity of ATPase activity.

Our data clearly demonstrate that all four Ca binding sites of TnC are important for the physiological function of TnC in the thin filaments. Sites I and II are responsible for the Ca-dependent activation of muscle contraction, and inactivation of Ca binding to these sites completely abolishes the activation of muscle. Sites III and IV are important for the structural stability of TnC in the whole troponin complex, with site III being more critical for TnC association with the fibers. Site IV appears not to be essential for the structural stability of TnC within the thin filament, similar to our observations in our thrombin fragment studies(13) . The occupancy of site III by Mg appears to be sufficient to maintain the association of TnC4- with the other troponin subunits and prevent its dissociation from the fibers. Thus, Ca binding sites III and IV do not appear to contribute equally to metal-dependent TnC binding to the thin filament via its COOH-terminal domain. Additional studies will be necessary to distinguish between these sites in terms of the Ca/Mg-dependent interactions of TnC with TnI and other proteins in the thin filament. Moreover, the present results suggest an interaction between the two domains of TnC where the structural alterations within the NH(2)-terminal domain affect the function of its COOH-terminal region and vice versa. Inactivation of sites III and IV affected the Ca sensitivity of force development, whereas mutations of sites I and II lowered the binding affinity of TnC to the fibers.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants AR37701 and AR40727. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Molecular and Cellular Pharmacology, University of Miami School of Medicine, P.O. Box 016189, Miami, FL 33101.

(^1)
The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; MOPS, 4-morpholinepropanesulfonic acid; AC, acrylodan.


ACKNOWLEDGEMENTS

We thank Dr. Sarah E. Hitchcock-DeGregori for the cDNA of chicken skeletal TnC and Lois Rosenzweig for excellent help in preparing the manuscript.


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