©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Direct Regulatory Role for Troponin T and a Dual Role for Troponin C in the Ca Regulation of Muscle Contraction (*)

(Received for publication, July 27, 1994; and in revised form, November 18, 1994)

James D. Potter (§) Zelin Sheng (¶) Bo-Sheng Pan (**) Jiaju Zhao

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

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Troponin (Tn), containing three subunits: Ca binding (TnC), inhibitory (TnI), and tropomyosin binding (TnT), plays a crucial role in the Ca regulation of vertebrate striated muscle contraction. These three subunits function by interacting with each other and with the other thin filament proteins. Previous studies suggested that the primary role of TnT is to anchor the TnIbulletTnC complex to the thin filament, primarily through its interactions with TnI and tropomyosin. We propose here a new role for TnT. Our results indicate that, when TnT is combined with the TnIbulletTnC complex, there is an activation of actomyosin ATPase that is Ca-dependent. To determine whether the latter results from a direct effect of TnC on TnT or indirectly from an effect of TnC on TnI which is transmitted to TnT, we prepared a deletion mutant (deletion of residues 1-57) of TnI, TnI (Sheng et al. (1992) J. Biol. Chem. 267, 25407-25413), which interacts with TnC but not TnT. Both wild type (TnIbulletTnCbulletTnT) and mutant (TnIbulletTnCbulletTnT) Tn complexes demonstrated equivalent activity in the Ca regulation of actomyosin-S1 ATPase activity. Similarly, both TnI and TnI could equally reconstitute TnI-depleted skinned muscle fibers. Therefore, since TnI does not interact with TnT, these results suggest that TnT reconstitutes native Ca sensitivity via direct interaction with TnC. Thus Ca binding to TnC would have a dual role: 1) release of the ATPase inhibition by TnI and 2) activation of the ATPase through interaction with TnT.


INTRODUCTION

Much is known about the Ca regulation of striated muscle contraction by the thin filament proteins troponin (Tn) (^1)and tropomyosin (Tm). Tn consists of three subunits, the Ca binding (TnC), inhibitory (TnI), and Tm binding (TnT). In the Tn complex, TnT has been shown to interact primarily with TnI, and TnC has been shown to interact primarily with TnI. Ca binding to TnC initiates a chain of events which leads to changes in the interactions between the Tn subunits, Tm, and actin which results in the attachment of myosin cross-bridges and the resulting contraction (for review see Zot and Potter(1987) and Grabarek et al.(1992)). Previous studies have shown that one of the major steps in the regulatory process is a change in the interaction between TnI and TnC when Ca binds to the Ca-specific regulatory sites (Potter and Gergely, 1975) of TnC which results in the dissociation of TnI from actin (Potter and Gergely, 1974; Hitchcock, 1975).

Although there has been some evidence for Ca-dependent interaction between TnC and TnT (Pearlstone and Smillie, 1978, 1982; Heeley et al., 1987; Zot and Potter, 1987), little functional significance has been ascribed to it until recently (Pan and Potter, 1992). Previously it was thought that the role of TnT was primarily to anchor the TnIbulletTnC complex to the thin filament through the interaction of TnT with both Tm and TnI. However, Pan et al.(1992) showed that two different COOH-terminal isoform fragments (alpha and beta) of TnT affected the affinity of the Ca-specific sites of TnC differently, implying a possible physiological significance to this interaction.

To explore this further, we have studied the role of TnT in the regulation of actomyosin-S1 ATPase activity. It is known that although TnI can inhibit actomyosin ATPase activity, and TnC can neutralize this inhibition, TnT is required to reconstitute native Ca-dependent regulation (Zot and Potter, 1987). We demonstrate here that there is a Ca-dependent activation of the actomyosin-S1 ATPase that only occurs in the presence of TnT. We also present evidence that this TnT-dependent ATPase activation, utilizing a deletion mutant of TnI (TnI) which interacts with TnC but not TnT, results from a TnC Ca-specific site-dependent interaction between TnC and TnT. This result suggests that TnT probably plays a direct role in the Ca regulation of the actomyosin ATPase activity. Furthermore, these results combined with previous studies, argue strongly for a dual role for TnC in muscle regulation. Ca binding to the Ca-specific sites of TnC would result in TnC interacting with both TnI (dissociating TnI from actin, thereby relieving inhibition) and with TnT (resulting in ATPase activation), with Ca dissociation reversing both processes.


MATERIALS AND METHODS

Expression and Purification of WTnI and TnI

Wild type skeletal muscle TnI (WTnI) and TnI (a deletion mutant lacking residues 1-57 of WTnI) were expressed and purified as described previously (Sheng et al., 1992a, 1992b).

Actomyosin-S1-ATPase Assay

Myosin-S1, F-actin, Tm, rabbit skeletal muscle TnC (RTnC), TnI (RTnI), and TnT (RTnT) were prepared from rabbit skeletal muscle as described previously (Margossian and Lowey, 1982; Parder and Spudich, 1982; Potter, 1982). The ATPase assay was performed in a 2.0-ml reaction mixture as specified in the legend to Fig. 1. The ATPase reaction was initiated with the addition of ATP and stopped after 6 min with 5% trichloroacetic acid. The ATPase rate was linear at this time point. After sedimenting the precipitate, the inorganic phosphate concentration in the supernatant was determined according to the method of Carter and Kail(1982). The free Ca concentration was set as described previously (Robertson and Potter, 1984).


Figure 1: The effects of WTn, RTn, and Tn on actomyosin-S1 ATPase activity: Tn concentration dependence (A) and Ca dependence (B). A, three Tn complexes were formed, as described in the legend to Fig. 4, containing RTnT and RTnC and either RTnI(RTn) or WTnI(WTn) or TnI(Tn). The concentration dependence of these three complexes was tested on actomyosin-S1 ATPase in the presence of Tm. Conditions: 23 °C, 20 mM KCl, 4 mM MgCl(2), 2 mM ATP, 25 mM MOPS, pH 7.0, 1 mM EGTA, 1 µM myosin-S1, 7 µM actin, and 1 µM Tm. The concentration of Tn added is indicated on the abscissa. The dotted line indicates the level of inhibition of the actomyosin-S1bulletTm ATPase activity in the presence of 3 µM RTnI. The neutralization of this inhibition by 2 µM RTnC at pCa = 5.0 is shown by the star in a circle symbol. The dashed line represents basal activity. The solid symbols indicate the presence of Ca (pCa = 5.0), and the open symbols indicate a low concentration of Ca (pCa = 8.0); circles = RTn, triangles = WTn, and the squares = Tn. The data represent the average of three experiments. B, the Ca dependence was carried out under the same conditions as in A except that the Ca concentration was varied as described previously. The three Tn complexes, RTn (open triangles), WTn (open circles), and Tn (closed circles) were present at a concentration of 1.5 µM. The data were fitted to the Hill equation: relative ATPase activity (%) = [Ca]/([Ca] + pK); where pK is the midpoint (pCa) and n is the Hill coefficient. The pCa was 6.56 for RTn, 6.55 for WTn, and 6.50 for Tn. The n were 1.10, 1.06, and 1.01, respectively. The ATPase activity is expressed as a percentage of the difference between the velocity at pCa 5.0 and pCa 8.0. Each point is an average of six determinations.




Figure 4: Size exclusion chromatography of the ternary complexes of WTnI or TnI and TnC and TnT. WTnI (A) or TnI (B) were mixed with RTnT and RTnC (1:1:1, molar ratio) in 6 M urea, 1 M KCl, 25 mM MOPS, pH 7.0, 1 mM CaCl(2), and 1 mM DTT on ice for 60 min. The complexes were then dialyzed consecutively against KCl solutions of 1 M, 0.75 M, 0.5 M, 0.3 M, and 0.1 M, each containing 25 mM MOPS, pH 7.0, 1 mM CaCl(2), and 1 mM DTT prior to loading on a Sephacryl S-200 column equilibrated with the same solution. The insets are 4-20% gradient SDS-PAGE gels of the fractions indicated. Closed circles, OD.



TnT Affinity Chromatography

RTnT was immobilized on cyanogen bromide-activated Sepharose 4B (Sigma) according to the procedure suggested by the manufacturer. In brief, the protein was dissolved in coupling buffer (0.1 M NaHCO(3), pH 8.3, 0.5 M NaCl) and then mixed with a suitable amount (100 mg protein/5 g) of CNBr-activated-Sepharose 4B equilibrated with 1 mM HCl. After 2 h of incubation at room temperature, the gel was transferred to a blocking solution (0.2 M glycine, pH 8.0) and incubated for 2 h at room temperature. The gel was then washed with coupling buffer followed by a solution containing 0.1 M sodium acetate, pH 4.0, and 0.5 M NaCl and finally by coupling buffer again. The RTnT-Sepharose was packed into a column (0.5 times 25 cm) and equilibrated with the desired starting buffer (as described in the legend to Fig. 2).


Figure 2: TnT affinity chromatography. WTnI and TnI were equilibrated with the starting solution (starting solution = 50 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 0.5 mM CaCl(2), and 1 mM DTT), and after loading, the RTnT affinity column (see ``Materials and Methods'') was washed with this solution (A, tubes 1-12). This was followed by a linear gradient (0.15-1 M NaCl in SS: B, tubes 13-63) and then with a solution containing 1 M NaCl and 6 M urea in starting solution (C, tubes 63-80). Circles, OD; closed squares, conductivity. The peaks indicated to contain WTnI (tubes 69-75) and TnI (tubes 5-10) were demonstrated by SDS-PAGE and Western blotting.



Skinned Porcine Cardiac Muscle Preparation and Force/pCa Measurements

Skinned porcine cardiac muscle preparations (CSM) were prepared and treated with vanadate essentially as described by Strauss et al.(1992). Briefly, the fibers were isolated from porcine left ventricle and chemically skinned by incubation in a relaxing (pCa 8.0) solution (5 mM [Mg], 4 mM EGTA, 5 mM ATP, 20 mM creatine phosphate, and 10 mM imidazole, pH = 7.0, ionic strength = 150 mM, [Ca] = 10M) containing 50% glycerol and 1% Triton X-100 at 4 °C for 24 h. The CSM were then stored in the same pCa 8 solution without Triton X-100 at -20 °C until needed. Force measurements were made using a Guth force transducer setup as described previously (Sheng et al., 1990, 1991). The method of Strauss et al.(1992) was used to remove TnI and TnC from the CSM. In this method the CSM, after an initial test contraction, was treated for 10 min with 10 mM sodium orthovanadate in pCa 8 relaxing solution, followed by incubation in pCa 8 solution. Reconstitution of Ca sensitivity to the TnIbulletTnC-depleted CSM was improved over that previously reported by Strauss et al.(1992), as described under ``Results.'' The Ca dependence of force development of the CSM was measured as described previously (Sheng et al., 1991) and the [Ca] concentration was calculated as previously described (Robertson and Potter, 1984).


RESULTS

The Effect of TnI and Various Tn Complexes on Actomyosin-S1 ATPase

Fig. 1A illustrates the well known inhibitory effect of TnI on the ATPase activity (dotted line). This inhibition can be reversed by the addition of TnC (dashed line) and has been referred to as the ``neutralization'' (Weeks and Perry, 1978; Perry et al., 1972) of the TnI inhibitory activity. This reversal of the inhibition returns the ATPase activity to the basal level seen with actomyosin-S1 and Tm alone. The ATPase activity never exceeds this basal level even when higher concentrations of TnC are added to neutralize the inhibition (data not shown). In contrast, higher levels of ATPase activity are seen when TnT is included to form the Tn complex in the presence of Ca (solid circles = RTn + Ca). This activation is maximal at a ratio of 1.5 Tn/Tm and represents an 170% increase over the basal ATPase activity. In the absence of Ca (open circles = RTn - Ca) the ATPase is maximally inhibited at the same ratio of Tn/Tm and represents an 60% inhibition of the basal activity. Thus, although inhibition could be brought about by TnI and neutralized by TnC, the only time that activation was observed was in the presence of TnT and Ca, therefore suggesting that activation is a property of TnT that requires the intact Tn complex and the presence of Ca. This result further suggested that the activation brought about by TnT might involve either a Ca-dependent interaction between TnC and TnT or a Ca-dependent change in the interaction of TnC and TnI that is transmitted through TnI to TnT. To test these two possibilities, we attempted to produce a large fragment of TnI that retained the ability to inhibit ATPase activity and the ability to bind TnC but not TnT. Since previous studies had suggested that TnT interacts with the NH(2) terminus of TnI (Hitchcock-DeGregori, 1982; Chong and Hodges, 1982), we studied the ability of a deletion mutant (Sheng et al., 1992), TnI, in which residues 1-57 of wild type rabbit skeletal TnI (WTnI) are deleted, to interact with TnT.

TnT Affinity and Size Exclusion Chromatography

The interaction of WTnI and TnI with TnT was studied with the use of a TnT affinity column (prepared as described under ``Materials and Methods'') and is illustrated in Fig. 2. A mixture of WTnI and TnI was simultaneously applied to the TnT affinity column and eluted as indicated in the figure legend. It can be seen that TnI eluted in the wash solution and failed to bind to the column. In contrast, WTnI was not eluted with a NaCl gradient to 1 M, but was eluted with 1 M NaCl and 6 M urea. In a separate experiment, it was confirmed that TnI alone did not bind to the TnT affinity column (data not shown). The inability of TnI to bind to TnT was further shown with the use of size exclusion chromatography (Fig. 3). A high affinity interaction was observed between WTnI and RTnT (Fig. 3A), whereas no interaction was observed between TnI and RTnT (Fig. 3B). Thus, both methods failed to show an interaction between TnI and TnT but confirm the well known interaction between TnT and TnI. TnI was subsequently used in the following functional assays to determine if the activation seen with TnT in the WTn complex was retained in complexes containing TnI.


Figure 3: Size exclusion chromatography of WTnI or TnI and TnT. WTnI (A) or TnI (B) were mixed with RTnT (1:1 molar ratio) in a solution containing 6 M urea, 1 M NaCl, 25 mM MOPS, pH 7.0, 0.5 mM CaCl(2), and 1 mM DTT on ice for 60 min. These mixtures were then dialyzed consecutively against NaCl solutions of 1 M, 0.75 M, O.5 M, 0.3 M, and 0.2 M, each containing 25 mM MOPS, pH 7.0, 0.5 mM CaCl(2), and 1 mM DTT. These mixtures were then loaded on a TSK column (Phenomenex G2000 WS) equilibrated with 0.2 M NaCl, 25 mM MOPS, pH = 7.0, 0.5 mM CaCl(2), and 1 mM DTT. The insets on the figures are 15% SDS-PAGE gels representing the indicated fractions. Closed circles, OD.



Formation of the Ternary Complexes of WTnI or TnI with TnC and TnT

To test whether the Tn complex made with TnI (Tn) retained Tn function, RTnT, RTnC, and TnI or WTnI were mixed together (Fig. 4, A and B) in 6 M urea followed by dialysis against decreasing concentrations of KCl (Potter, 1982) in the presence of Ca to produce the Tn and WTn complexes. To ensure that complex formation had occurred in both cases, these complexes were chromatographed over Sephacryl S-200. Fig. 4, A and B, demonstrate that both complexes formed under the conditions used for the experiments. The WTn complex did not require the presence of Ca to form, but the Tn complex did. Once formed, the Tn complex appeared to be stable even in the presence of EGTA. This result is consistent with the ATPase results presented below, where the activity of Tn is comparable with that of WTn.

The Effects of WTn and Tn on Actomyosin-S1 ATPase Activity

Fig. 1A compares the effect of RTn with that of WTn and Tn. As can be seen, both WTn and Tn have essentially the same effect on the ATPase activity as RTn. All three have similar activation (pCa 5.0) and inhibitory (pCa 8.0) properties as well as the same concentration dependence, with maximal activation/inhibition occurring at a ratio of 1.5 Tn/Tm.

Fig. 1B shows the Ca dependence of actomyosin-S1 ATPase activity as a function of pCa. The measurements were made essentially the same as in Fig. 1A except the free Ca concentration was varied at a fixed ratio of Tn/Tm. All three Tn complexes showed essentially the same Ca dependence. These results suggest that, even in the absence of an interaction between TnI and TnT, that the activation properties of TnT are not lost, implying a Ca-dependent interaction between TnC and TnT in the Tn complex, as well as in the RTn and WTn complexes.

Effects of WTnIbulletRTnC and TnIbulletRTnC Complexes on TnIbulletTnC-depleted CSM

In addition to testing TnI in the actomyosin-S1 ATPase assay, we also tested its ability to restore force and regulate a TnIbulletTnC-depleted CSM preparation (see ``Materials and Methods''), and the results of these experiments are illustrated in Fig. 5, A and B. Basically, the CSM was treated with vanadate which has been shown previously to remove TnI and TnC (Strauss et al., 1992) and lead to the development Ca-independent force. Originally Strauss et al.(1992) restored Ca-dependent regulation to TnIbulletTnC-depleted CSM by first adding TnI to inhibit the Ca-independent force, followed by the addition of TnC to restore Ca regulation. We found, as they did, that only a small fraction of the original Ca sensitivity could be restored using this protocol. We have improved on their original method (Sheng et al., 1993) by restoring Ca sensitivity through the addition of preformed WTnIbulletRTnC complex (Fig. 5A). As can be seen, the addition of WTnIbulletRTnC (in pCa 8 relaxing solution) to the vanadate-treated CSM, which developed 90% of the original Ca-activated force, resulted in 85% inhibition of the original force. Presumably the added WTnIbulletRTnC complex bound to TnT remaining in the TnIbulletTnC-depleted CSM. Since this complex was rebound in the pCa 8 relaxing solution, the CSM relaxed as would be expected if the added TnIbulletTnC properly reconstituted the Tn complex. After washing the CSM free of unbound WTnIbulletRTnC, the CSM was returned to the pCa 4 contracting solution, force was regained and the CSM was once again sensitive to changes in free Ca concentration (Fig. 5A). With this new protocol for restoring Ca sensitivity, we formed and tested the TnIbulletRTnC complex in the same way (Fig. 5B). Interestingly, the vanadate-treated CSM was reconstituted almost the same with this complex as with WTnIbulletRTnC (Table 1), consistent with the ATPase measurements (Fig. 1). In both cases, 91% of the force present after vanadate treatment could be inhibited by the two complexes in relaxing solution (Table 1). After washout of the unbound complexes in pCa 8, 75% of the force could be restored in the pCa 4 contracting solution (Table 1).


Figure 5: Effects of WTnIbulletRTnC and TnIbulletRTnC complexes on vanadate-treated CSM. A and B, CSM were prepared as described under ``Materials and Methods.'' Two solutions were used: contracting solution (pCa 4) containing 10M [Ca], 5 mM [Mg], 4 mM EGTA, 5 mM ATP, 20 mM creatine phosphate, 20 units of CPK, and 10 mM imidazole, pH 7.0, ionic strength = 150 mM, and relaxing solution (pCa 8) which had the same composition except [Ca] = 10M. After an initial test contraction (A and B) the CSM was treated with 10 mM sodium orthovanadate in pCa 8 (see ``Materials and Methods'') for 10 min and then washed with the pCa 8 solution. After maximum force was obtained in pCa 8, the preparations were incubated with the WTnIbulletRTnC (A) or TnIbulletRTnC (B) complexes (10 µM) in pCa 8 solution. After maximal inhibition was achieved, the effect of the pCa 4 solution was tested. C, the Ca dependence (see ``Materials and Methods'') of these reconstituted CSM were tested (WTnIbulletRTnC, solid circles; TnIbulletRTnC, solid triangles) and compared with untreated fibers (solid circles). These data (average of three experiments) were fitted to the Hill equation (see Fig. 1) and the pCa and n for these are 5.74 (n = 2.46), 5.7 (n = 2.24), and 5.74 (n = 2.38), respectively.





Fig. 5C illustrates the Ca dependence of the CSM before vanadate treatment and the vanadate treated CSM reconstituted with either WTnIbulletRTnC or TnIbulletRTnC. The results for all three measurements were not significantly different and indicate that the original Ca sensitivity could be restored with either binary complex.


DISCUSSION

The results presented in this paper suggest that TnT plays an important role in the regulation of contraction that has not been considered previously. Basically, TnT appears to activate actomyosin-S1 ATPase activity when it is present in Tn complexes. TnI alone fully inhibits the actomyosin-S1 ATPase activity in the presence of Tm as has been shown previously (Weeks and Perry, 1978; Perry et al., 1972; Greaser and Gergely, 1973; Greaser et al., 1972). Addition of TnC to this, fully reverses the ATPase activity to the basal ATPase level seen in the absence of TnI (Fig. 1), the so called neutralization of TnI's inhibitory activity (Perry et al., 1972). Interestingly, the ATPase activity could not be further stimulated by subsequent additions of TnC. However, in the presence of Ca, the ATPase activity with intact Tn was much higher than that seen with the TnIbulletTnC complex in the presence of Ca. This activation of the ATPase could only be observed in the intact Tn complex and suggested that this activation is a property of TnT that requires Ca binding to the regulatory Ca binding sites on TnC.

If the above is true, then there are at least two possibilities to account for this observation. Ca binding to TnC may cause a direct interaction to occur between TnC and TnT, thereby altering the conformation of TnT. This signal would be transduced to Tm and/or actin in some way leading to the activation of ATPase activity. Alternatively, Ca binding to TnC, which is known to change its interaction with TnI, could transmit information indirectly through a conformational change in TnI that would change the conformation of TnT, leading to ATPase activation. Since TnI interacts with TnT it is difficult to sort out these two possibilities. To overcome this, we reasoned that if we could eliminate the interaction between TnI and TnT without interfering with either TnI's inhibition or TnC neutralization capabilities, that we would be able to distinguish between these two possibilities directly.

As the results indicated, TnI retained both of these properties. Although the binding between TnI and TnT has been observed in previous studies (Zot and Potter, 1987; Hitchcock, 1982; Sheng et al., 1990; Horwitz et al., 1979; Chong and Hodges, 1982; Pearlstone and Smillie, 1982), the specific nature of this interaction has not been well understood. Previous cross-linking and lysine reactivity studies have suggested that the NH(2)-terminal region (residues 40-78) of TnI probably interacts with TnT (Hitchcock, 1982; Chong and Hodges, 1982). Consistent with this, we found that TnI did not bind to TnT (Potter et al., 1993).

Previous studies (Sheng et al., 1992a, 1992b) utilizing TnI, have revealed that there are at least three types of interaction which occur between TnC and TnI: 1) one which is dependent upon Ca binding to the Ca-specific sites of TnC; 2) one which is dependent upon Ca or Mg binding to the Ca-Mg sites of TnC; and 3) one which is metal-independent. TnI was shown to retain the Ca-specific site-dependent interaction but lost the Ca-Mg site-dependent interaction and exhibited a weaker metal independent interaction. Similar conclusions have been made by Farah et al.(1994). In spite of this difference from WTnI, TnI retained full actomyosin ATPase inhibitory activity that could be completely neutralized by TnC (Perry et al., 1972; Weeks and Perry, 1978; Sheng et al., 1992a, 1992b). Taken together, these results, and those presented in this paper, suggest that the NH(2) terminus of TnI may play a crucial role in stabilizing the interaction between not only TnC and TnI, but also between TnT and TnI and is probably of fundamental importance in maintaining the structural integrity of the Tn complex.

We reasoned that if the ATPase activation seen with intact Tn could still be observed in Tn complexes formed with TnI, that this would imply a direct effect of TnC on TnT since there would presumably be no interaction between TnI and TnT in the ternary complex. Interestingly, the TnI complex could only be formed in the presence of Ca, as might be expected, since TnT could no longer bind to TnI and unless complexed with TnC, TnT would precipitate under the conditions of this experiment. Although the latter interpretation seems plausible, it is not possible at present to completely exclude the possibility that the interaction between TnC and TnI may result in an interaction between TnI and TnT in the complex, that would not occur between TnI and TnT alone. We are currently carrying out additional experiments to unequivocally decide between these two possibilities.

Previous studies have shown that TnT binds to TnC in a Ca-dependent manner (Zot and Potter, 1987; Pearlstone and Smillie, 1978, 1982; Pan and Potter, 1992; Heeley et al., 1987). The COOH-terminal half of TnT probably contains the major binding site for TnC (Zot and Potter, 1987; Pearlstone and Smillie, 1978; Pan and Potter, 1992), whereas the NH(2)-terminal regulatory domain of TnC may contain the TnT interaction site (Grabarek et al., 1981; Leavis and Gergely, 1984). Recent cross-linking studies have further defined the interacting sites as including the residues 175-178 of TnT and a region in the vicinity of Cys-98 of TnC (Leszyk et al., 1988). Interestingly, this region of TnC also interacts with TnI in the presence of Ca (Leavis and Gergely, 1984; Leszyk et al., 1990). These observations suggest the possibility that both TnI and TnT participate in the Ca signal which is transmitted by TnC. It is also possible that both TnI and TnT interact in the same region or in close proximity on TnC.

The ability of the Tn complex to regulate actomyosin ATPase activity and contraction suggests that the sensitivity and cooperativity of Ca regulation were not affected either by a lack of an interaction between TnI and TnT or by the loss of the Ca-Mg-dependent interaction between TnI and TnC shown previously (Sheng et al., 1992a, 1992b).

The actomyosin and CSM results strongly suggest an important role for TnT in the regulation of contraction. Our results indicate that the Ca-dependent activation of actomyosin ATPase activity only occurs in the presence of TnT, even in the absence of an interaction between TnI (TnI) and TnT. Increasing evidence indicates that TnT plays an important modulatory role in the Ca regulation of striated muscle contraction. Several studies have established correlations between TnT isoform composition and the Ca sensitivity of force production by skinned skeletal and cardiac muscle fibers (Schachat et al., 1987; Reiser et al., 1992). Tobacman and Lee(1987) observed a difference in Ca sensitivity between reconstituted actomyosin systems regulated by two isoforms of bovine cardiac TnT. We recently found that bacterially expressed rabbit skeletal TnT fragments representing two carboxylterminal isoforms of TnT exhibited different affinities for TnC and that there was a significant difference in the Ca affinity of the regulatory sites of TnC in the binary complexes formed between the two TnT isoform fragments and TnC (Pan and Potter, 1992). These results are consistent with our hypothesis in this paper, that in addition to interacting with TnI, TnC interacts directly with TnT in the Ca sensitive activation of contraction.

The way in which TnC interacts with TnT and activates contraction is not well understood at this time. As mentioned above, Ca-dependent interactions between TnC and TnT are thought to occur between the NH(2) terminus of TnC and the carboxyl terminus of TnT. The carboxyl terminus of TnT (residues 159-259) has also been shown to interact with Tm in the vicinity of Cys-190. In the presence of Ca, the interaction between the T2 fragment of TnT (residues 159-259) is strengthened with TnC and weakened with Tm (Pearlstone and Smillie, 1983). In the absence of Ca the reverse is true. The interaction of the NH(2) terminus of TnT with Tm in the vicinity of the Tm overlap region has been shown to be insensitive to Ca (Pearlstone and Smillie, 1982). Thus Ca binding to the Ca-specific sites of TnC may produce an interaction between the NH(2) terminus of TnC and the carboxyl terminus of TnT that causes a dissociation or weakening of the interaction between the carboxyl terminus of TnT and Tm, resulting in the activation of the actomyosin ATPase, perhaps through a derepression of the ATPase activity when TnT changes its interaction with Tm, thereby altering the conformation of actin and its interaction with myosin.

Our previous results (Pan and Potter, 1992) suggest that the region coded by the variable carboxyl-terminal exon (amino acid residues 221-233) in TnT may be important in the interactions mentioned above, since the two carboxyl-terminal TnT fragments containing either the alpha or beta exon exhibited different affinities for Tm and also affected the Ca affinity of the Ca-specific sites of TnC differently. Work is in progress in our laboratory to further define these possibilities.

In summary, results presented here have suggested a new role to TnT in the Ca regulation of contraction and a dual role for TnC. In the absence of Ca, TnI would be bound to actin, inhibiting actomyosin ATPase activation and the carboxyl terminus of TnT would be bound to Tm. Upon muscle activation and Ca binding to the Ca-specific sites of TnC, TnC would interact with TnI at the Ca-specific site-dependent interaction site on TnI, thereby dissociating TnI from actin, relieving the actomyosin ATPase inhibition. Simultaneously, TnC would interact with the carboxyl terminus of TnT weakening the interaction of this region of TnT with Tm and thereby derepress the ATPase activity resulting in the activation ascribed here to TnT.


FOOTNOTES

*
This work was supported by Grants AR37701 and AR40727 from the National Institutes of Health. A preliminary report of this work was presented at the 1993 Biophysical Society meeting (Potter et al., 1993). 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.

Present address: University of California, San Diego, Dept. of Medicine, La Jolla, CA 92093.

**
Present address: Dept. of Pharmacology, MSD Research Laboratories, West Point, PA 19486.

(^1)
The abbreviations used are: Tn, the troponin complex; Tm, tropomyosin; RTn, rabbit skeletal muscle Tn; WTnI, wild type TnI; WTn, Tn made from RTnT, WTnI, and RTnC; RTnC, RTnI, and RTnT, Tn subunits from rabbit skeletal muscle; TnI, deletion mutant of WTnI; CSM, skinned porcine cardiac muscle preparation; Tn, Tn made from RTnT, TnI, and RTnC; PAGE, polyacrylamide gel electrophoresis; DTT, dithiothreitol; MOPS, 4-morpholinepropanesulfonic acid.


REFERENCES

  1. Carter, S. G., and Kail, D. W. (1982) J. Biochem. Biophys. Methods 7, 7-13 [CrossRef][Medline] [Order article via Infotrieve]
  2. Chong, P. C. S., and Hodges, R. S. (1982) J. Biol. Chem. 257, 11667-11672 [Abstract/Free Full Text]
  3. Farah, C. S., Miyamoto, C. A., Ramos, C. H., da Silva, A. C., Quaggio, R. B., Fujimori, K., Smillie, L. B., and Reinach, F. C. (1994) J. Biol. Chem. 269, 5230-5240 [Abstract/Free Full Text]
  4. Grabarek, Z., Drabikowski, W., Leavis, P. C., Rosenfeld, S. S., and Gergely, J. (1981) J. Biol. Chem. 256, 13121-13127 [Abstract/Free Full Text]
  5. Grabarek, Z., Tao, T., and Gergely, J. (1992) J. Muscle Res. 13, 383-393
  6. Greaser, M. L., and Gergely, J. (1973) J. Biol. Chem. 248, 2125-2133 [Abstract/Free Full Text]
  7. Greaser, M. L., Yamaguchi, M., Brekke, C., Potter, J., and Gergely, J. (1972) Cold Spring Harbor Symp. Quant. Biol. 37, 235-244
  8. Heeley, D. H., Golosinska, K., and Smillie, L. B. (1987) J. Biol. Chem. 262, 9971-9978 [Abstract/Free Full Text]
  9. Hitchcock, S. E. (1975) Eur. J. Biochem. 52, 255-263 [Abstract]
  10. Hitchcock-DeGregori, S. E. (1982) J. Biol. Chem. 257, 7372-7280 [Abstract/Free Full Text]
  11. Horwitz, J., Bullard, B., and Mercola, D. (1979) J. Biol. Chem. 254, 350-355 [Abstract]
  12. Leavis, P. C., and Gergely, J. (1984) CRC Crit. Rev. Biochem. 16, 235-305 [Medline] [Order article via Infotrieve]
  13. Leszyk, J., Collins, J. H., Leavis, P. C., and Tao, T. (1988) Biochemistry 27, 6983-6987 [Medline] [Order article via Infotrieve]
  14. Leszyk, J., Grabarek, Z., Gergely, J., and Collins, J. H. (1990) Biochemistry 29, 299-304 [Medline] [Order article via Infotrieve]
  15. Margossian, S. S., and Lowey, S. (1982) Methods Enzymol. 85, 55-71 [Medline] [Order article via Infotrieve]
  16. Pan, B. S., and Potter, J. D. (1992) J. Biol. Chem. 267, 23052-23056 [Abstract/Free Full Text]
  17. Parder, J. D., and Spudich, J. A. (1982) Methods Enzymol. 85, 234-240 [Medline] [Order article via Infotrieve]
  18. Pearlstone, J. R., and Smillie, L. B. (1978) Can. J. Biochem. 56, 521-527 [Medline] [Order article via Infotrieve]
  19. Pearlstone, J. R., and Smillie, L. B. (1982) J. Biol. Chem. 257, 10587-10592 [Abstract]
  20. Pearlstone, J. R., and Smillie, L. B. (1983) J. Biol. Chem. 258, 2534-2542 [Abstract/Free Full Text]
  21. Perry, S. V., Cole, H. A., Head, J. F., and Wilson, F. J. (1972) Cold Spring Harbor Symp. Quant. Biol. 37, 251-262
  22. Potter, J. D. (1982) Methods Enzymol. 85, 241-263 [Medline] [Order article via Infotrieve]
  23. Potter, J. D., and Gergely, J. (1974) Biochemistry 13, 2697-2703 [Medline] [Order article via Infotrieve]
  24. Potter, J. D., and Gergely, J. (1975) J. Biol. Chem. 250, 4628-4633 [Abstract]
  25. Potter, J. D., Sheng, Z., and Pan, B. S. (1993) Biophys. J. 64, A137 [Abstract]
  26. Reiser, P. J., Greaser, M. L. & Moss, R. L. (1992) J. Physiol. (Lond.) 449, 573-588 [Abstract]
  27. Robertson, S. P., and Potter, J. D. (1984) Methods Pharmacol. 5, 63-75
  28. Schachat, F. H., Diamond, M. S., and Brandt, P. W. (1987) J. Mol. Biol. 198, 551-554 [Medline] [Order article via Infotrieve]
  29. Sheng, Z., Strauss, W., Francois, J. M., and Potter, J. D. (1990) J. Biol. Chem. 265, 21554-21559 [Abstract/Free Full Text]
  30. Sheng, Z., Francois, J. M., Hitchcock-DeGregori, S. E., and Potter, J. D. (1991) J. Biol. Chem. 266, 5711-5715 [Abstract/Free Full Text]
  31. Sheng, Z., Pan, B.-S., Miller, T., and Potter, J. D. (1992a) J. Biol. Chem. 267, 25407-25413 [Abstract/Free Full Text]
  32. Sheng, Z., Pan, B.-S., Miller, T., and Potter, J. D. (1992b) J. Biol. Chem. 267,25407 [Abstract/Free Full Text] -25413; Correction (1993) J. Biol. Chem.268, 3016
  33. Sheng, Z., Kong, Y., Pan, B. S., Miller, T., and Potter, J. D. (1993) Biophys. J. 64, A136
  34. Strauss, J. D., Zeugner, C., Van Eyk, J. E., Bletz, C., Troschka, M., and Ruegg, J. C. (1992) FEBS Lett. 310, 229-234 [CrossRef][Medline] [Order article via Infotrieve]
  35. Tobacman, L. S., and Lee, R. (1987) J. Biol. Chem. 262, 4059-4064 [Abstract/Free Full Text]
  36. Weeks, R. A., and Perry, S. V. (1978) Biochem. J. 173, 449-457 [Medline] [Order article via Infotrieve]
  37. Zot, A. S., and Potter, J. D. (1987) Annu. Rev. Biophys. Biophys. Chem. 16, 535-559 [CrossRef][Medline] [Order article via Infotrieve]

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