Steady-state properties of calcium binding to parvalbumins from bullfrog skeletal muscle: effects of Mg2+, pH, ionic strength, and temperature

To improve our understanding of the physiological roles of parvalbumins, PA-1 (pI 4.78) and PA-2 (pI 4.97) parvalbumins were prepared from bullfrog skeletal muscle and their calcium binding properties were examined in a medium of constant ionic strength (I = 0.106, pH 6.80, at 20 degrees C) containing various concentrations of Mg2+ by using a metallo-indicator, tetramethylmurexide. Apparent binding constants for Ca2+ in the presence of Mg2+ changed in the manner expected if Ca2+ and Mg2+ compete for two independent homogeneous binding sites. The following values were obtained: for PA-1, KCa = 1 X 10(7) M-1, KMg = 900 M-1; for PA-2, KCa = 6 X 10(6) M-1, KMg = 830 M-1 (I = 0.106, pH 6.80, at 20 degrees C). The apparent binding constants are strongly dependent on temperature: at 10 degrees C for PA-1, KCa = 2 X 10(8) M-1, KMg = 10(4) M-1; for PA-2, KCa = 5 X 10(7) M-1, KMg = 5 X 10(3) M-1 (I = 0.106, pH 6.80). The dependence of the affinities for Ca2+ on ionic strength is similar to or less than that of GEDTA (EGTA). The affinities for Ca2+ and Mg2+ of parvalbumins are unchanged between pH 6.5 and 7.2.     

Mutation of the high affinity calcium binding sites in cardiac troponin C.

Fast skeletal and cardiac troponin C (TnC) contain two high affinity Ca2+/Mg2+ binding sites within the C-terminal domain that are thought to be important for association of TnC with the troponin complex of the thin filament. To test directly the function of these high affinity sites in cardiac TnC they were systematically altered by mutagenesis to generate proteins with a single inactive site III or IV (CBM-III and CBM-IV, respectively), or with both sites III and IV inactive (CBM-III-IV). Equilibrium dialysis indicated that the mutated sites did not bind Ca2+ at pCa 4. Both CBM-III and CBM-IV were similar to the wild type protein in their ability to regulate Ca(2+)-dependent contraction in slow skeletal muscle fibers, and Ca(2+)-dependent ATPase activity in fast skeletal and cardiac muscle myofibrils. The mutant CBM-III-IV is capable of regulating contraction in permeabilized slow muscle fibers but only if the fibers are maintained in a contraction solution containing a high concentration of the mutant protein. CBM-III-IV also regulates myofibril ATPase activity in fast skeletal and cardiac myofibrils but only at concentrations 10-100-fold greater than the normal protein. The pCa50 and Hill coefficient values for Ca(2+)-dependent activation of fast skeletal muscle myofibril ATPase activity by the normal protein and all three mutants are essentially the same. Competition between active and inactive forms of cardiac and slow TnC in a functional assay demonstrates that mutation of both sites III and IV greatly reduces the affinity of cardiac and slow TnC for its functionally relevant binding site in the myofibrils. The data indicate that although neither high affinity site is absolutely essential for regulation of muscle contraction in vitro, at least one active C-terminal site is required for tight association of cardiac troponin C with myofibrils. This requirement can be satisfied by either site III or IV.     

Influence of length on force and activation-dependent changes in troponin c structure in skinned cardiac and fast skeletal muscle

Linear dichroism of 5' tetramethyl-rhodamine (5'ATR) was measured to monitor the effect of sarcomere length (SL) on troponin C (TnC) structure during Ca2+ activation in single glycerinated rabbit psoas fibers and skinned right ventricular trabeculae from rats. Endogenous TnC was extracted, and the preparations were reconstituted with TnC fluorescently labeled with 5'ATR. In skinned psoas fibers reconstituted with sTnC labeled at Cys 98 with 5'ATR, dichroism was maximal during relaxation (pCa 9.2) and was minimal at pCa 4.0. In skinned cardiac trabeculae reconstituted with a mono-cysteine mutant cTnC (cTnC(C84)), dichroism of the 5'ATR probe attached to Cys 84 increased during Ca2+ activation of force. Force and dichroism-[Ca2+] relations were fit with the Hill equation to determine the pCa50 and slope (n). Increasing SL increased the Ca2+ sensitivity of force in both skinned psoas fibers and trabeculae. However, in skinned psoas fibers, neither SL changes or force inhibition had an effect on the Ca2+ sensitivity of dichroism. In contrast, increasing SL increased the Ca2+ sensitivity of both force and dichroism in skinned trabeculae. Furthermore, inhibition of force caused decreased Ca2+ sensitivity of dichroism, decreased dichroism at saturating [Ca2+], and loss of the influence of SL in cardiac muscle. The data indicate that in skeletal fibers SL-dependent shifts in the Ca2+ sensitivity of force are not caused by corresponding changes in Ca2+ binding to TnC and that strong cross-bridge binding has little effect on TnC structure at any SL or level of activation. On the other hand, in cardiac muscle, both force and activation-dependent changes in cTnC structure were influenced by SL. Additionally, the effect of SL on cardiac muscle activation was itself dependent on active, cycling cross-bridges.     

Function of the N-terminal calcium-binding sites in cardiac/slow troponin C assessed in fast skeletal muscle fibers

Fast skeletal troponin C (sTnC) has two low affinity Ca(2+)-binding sites (sites I and II), whereas in cardiac troponin C (cTnC) site I is inactive. By modifying the Ca2+ binding properties of sites I and II in cTnC it was demonstrated that binding of Ca2+ to an activated site I alone is not sufficient for triggering contraction in slow skeletal muscle fibers (Sweeney, H.L., Brito, R. M.M., Rosevear, P.R., and Putkey, J.A. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 9538-9542). However, a similar study using sTnC showed that Ca2+ binding to site I alone could partially activate force production in fast skeletal muscle fibers (Sheng, Z., Strauss, W.L., Francois, J.M., and Potter, J.D. (1990) J. Biol. Chem. 265, 21554-21560). The purpose of the current study was to examine the functional characteristics of modified cTnC derivatives in fast skeletal muscle fibers to assess whether or not either low affinity site can mediate force production when coupled to fast skeletal isoforms of troponin (Tn) I and TnT. Normal cTnC and sTnC were compared with engineered derivatives of cTnC having either both sites I and II active, or only site I active. In contrast to what is seen in slow muscle, binding of Ca2+ to site I alone recovered about 15-20% of the normal calcium-activated force and ATPase activity in skinned fast skeletal muscle fibers and myofibrils, respectively. This is most likely due to structural differences between TnI and/or TnT isoforms that allow for partial recognition and translation of the signal represented by binding Ca2+ to site I of TnC when associated with fast skeletal but not slow skeletal muscle.     

The calcium and magnesium binding sites on cardiac troponin and their role in the regulation of myofibrillar adenosine triphosphatase

The cardiac troponin (Tn) complex, consisting of a Ca2+-binding subunit (TnC), an inhibitory subunit (TnI), and a tropomyosin-binding subunit (TnT), has been reconstituted from purified troponin subunits isolated from bovine heart muscle. The Ca2+-binding properties of cardiac Tn were determined by equilibrium dialysis using either EGTA or EDTA to regulate the free Ca2+ concentration. Cardiac Tn binds 3 mol Ca2+/mol and contains two Ca2+-binding sites with a binding constant of 3 X 10(8) M-1 and one binding site with a binding constant of 2 X 10(6) M-1. In the presence of 4 mM MgC12, the binding constant of the sites of higher affinity is reduced to 3 X 10(7) M-1, while Ca2+ binding to the site at the lower affinity is unaffected. The two high affinity Ca2+-binding sites of cardiac Tn are analogous to the two Ca2+-Mg2+ sites of skeletal Tn, while the single low affinity site is similar to the two Ca2+-specific sites of skeletal Tn (Potter, J. D., and Gergely, J. (1975) J. Biol. Chem. 250, 4625-5633). The Ca2+-binding properties of the complex of TnC and TnI (1:1 molar ratio) were similar to those of Tn. Cardiac TnC also binds 3 mol of Ca2+/mol and contains two sites with a binding constant of 1 X 10(7) M-1 and a single site with a binding constant of 2 X 10(5) M-1. Assuming competition between Mg2+ and Ca2+ for the high affinity sites of TnC and Tn, the binding constants for Mg2+ were 0.7 and 3.0 X 10(3) M-1, respectively. The Ca2+ dependence of cardiac myofibrillar ATPase activity was similar to that of an actomyosin preparation regulated by the reconstituted troponin complex. Comparison by the Ca2+-binding properties of cardiac Tn and the cardiac myofibrillar ATPase activity as a function of [Ca2+] and at millimolar [Mg2+] suggests that activation of the ATPase occurs over the same range of [Ca2+] where the Ca2+-specific site of cardiac Tn binds Ca2+.     

Calcium-independent activation of skeletal muscle fibers by a modified form of cardiac troponin C.

A conformational change accompanying Ca2+ binding to troponin C (TnC) constitutes the initial event in contractile regulation of vertebrate striated muscle. We replaced endogenous TnC in single skinned fibers from rabbit psoas muscle with a modified form of cardiac TnC (cTnC) which, unlike native cTnC, probably contains an intramolecular disulfide bond. We found that such activating TnC (aTnC) enables force generation and shortening in the absence of calcium. With aTnC, both force and shortening velocity were the same at pCa 9.2 and pCa 4.0. aTnc could not be extracted under conditions which resulted in extraction of endogenous TnC. Thus, aTnC provides a stable model for structural studies of a calcium binding protein in the active conformation as well as a useful tool for physiological studies on the primary and secondary effects of Ca2+ on the molecular kinetics of muscle contraction.     

Altered Ca2+ dependence of tension development in skinned skeletal muscle fibers following modification of troponin by partial substitution with cardiac troponin C

Binding of Ca2+ to the troponin C (TnC) subunit of troponin is necessary for tension development in skeletal and cardiac muscles. Tension was measured in skinned fibers from rabbit skeletal muscle at various [Ca2+] before and after partial substitution of skeletal TnC with cardiac TnC. Following substitution, the tension-pCa relationship was altered in a manner consistent with the differences in the number of low-affinity Ca2+-binding sites on the two types of TnC and their affinities for Ca2+. The alterations in the tension-pCa relationship were for the most part reversed by reextraction of cardiac TnC and readdition of skeletal TnC into the fiber segments. These findings indicate that the type of TnC present plays an important role in determining the Ca2+ dependence of tension development in striated muscle.     

Resolution and calcium-binding properties of the two major isoforms of troponin C from crayfish

Crayfish tail muscle troponin C (TnC) has been fractionated into its five components and the Ca2+-binding properties of the two major isoforms (alpha and gamma) determined by equilibrium dialysis. alpha-TnC contains one Ca2+-binding site with a binding constant of 1 x 10(6) M-1 and one Ca2+ site with a binding constant of 1 x 10(4) M-1. In the complex of alpha-TnC with troponin I (TnI) or with TnI and troponin T (TnT), both sites bind Ca2+ with a single affinity constant of 2-4 x 10(6) M-1. gamma-TnC contains two Ca2+-binding sites with a binding constant of 2 x 10(4) M-1. In the gamma-TnC.TnI and gamma-TnC.TnI.TnT complexes, the binding constant of one of the sites is increased to 4-5 x 10(6) M-1, while Ca2+ binding to the second site is hardly affected (KCa = 4-7 x 10(4) M-1). In the presence of 10 mM MgCl2, the two Ca2+-binding sites of both TnC isoforms exhibit a 2-3-fold lower affinity. Assuming competition between Ca2+ and Mg2+ for these sites, their binding constants for Mg2+ were 120-230 M-1. In the absence of Ca2+, however, alpha-TnC and gamma-TnC bind 4-5 mol of Mg2+/mol with a binding constant of 1 x 10(3) M-1. These results suggest that the effect of Mg2+ on Ca2+ binding at the two Ca2+ sites is noncompetitive, i.e. Mg2+ does not bind directly to these sites (Ca2+-specific sites). Since the formation of the complex of crayfish TnI with alpha-TnC or gamma-TnC increases significantly the affinity of one of their two Ca2+-specific sites, I conclude that the binding of Ca2+ to only one site (regulatory Ca2+-specific site) controls the Ca2+-dependent interaction between crayfish TnCs and TnI.     

The effect of [Mg2+] on the Ca2+ dependence of ATPase and tension development of fast skeletal muscle. The role of the Ca2+-specific sites of troponin C

Conflicting reports have appeared concerning the effect of [Mg2+] on muscle activity. Several groups have found that increasing [Mg2+] produces a right-ward shift of the pCa-tension curve, while others have found no effect of [Mg2+] on myofibrillar ATPase activity. The present study is a careful evaluation of the effect of [Mg2+] on myofibrillar ATPase, skinned fiber tension development, TnCDANZ (troponin C (TnC)-labeled with 5-dimethylaminonaphthalene-1-sulfonyl aziridine) fluorescence, and simultaneous TnCDANZ fluorescence and tension development in the same fiber. A small effect of [Mg2+] on both ATPase and tension development was found with an apparent association constant of about 2 X 10(2) M-1. The Ca2+ dependence of TnCDANZ fluorescence was similarly effected by [Mg2+], either alone or when incorporated into TnC-depleted skinned fibers (K'Mg approximately equal to 2-3 X 10(2) M-1), suggesting that the effect of [Mg2+] on activity is due to an effect of [Mg2+] on Ca2+ binding to the Ca2+-specific sites of TnC. It is not yet clear whether this effect of [Mg2+] is through direct competition at the binding sites or through indirect effects. In either case, the calculated effect of physiological [Mg2+] is so small that the regulatory sites of TnC can still be considered "Ca2+-specific." In addition, a slightly greater effect of [Mg2+] on tension development (K'Mg = 4.62 X 10(2) M-1) was observed only for very low levels of [Mg2+], which might suggest an additional effect of Mg2+ on tension development which is saturated by millimolar Mg2+.     

Fast skeletal muscle skinned fibers and myofibrils reconstituted with N-terminal fluorescent analogues of troponin C

Glycerinated rabbit fast skeletal muscle fibers were chemically skinned with 1% Brij 35 and partially depleted of endogenous troponin C subunit (TnC) by exposure of the fibers to EDTA (Zot, H. G., and Potter, J. D. (1982) J. Biol. Chem. 257, 7678-7683). The TnC-depleted fibers exhibited a decrease in maximal tension that was mostly restored by readdition of TnC or by the addition of the fluorescent 5-dimethylaminonaphthalene-1-sulfonyl aziridine analogue, TnCDanz. TnCDanz is known to undergo an increase in fluorescence intensity when Ca2+ binds to the two low affinity Ca2+-specific regulatory sites of TnC. Steady-state fractional fluorescence and tension changes were measured simultaneously as a function of Ca2+. The Ca2+ sensitivity of the fluorescence curve was about 0.6 log unit greater than the tension curve. This difference in sensitivity could be explained if separate conformational states of TnC, brought about by Ca2+ binding to the Ca2+-specific sites, produce the fluorescence and tension changes. TnC-depleted fibers were also reconstituted with the fluorescent 2-[(4'-iodoacetamido)analino]naphthalene-6-sulfonic acid analogue, cardiac TnCIaans, which undergoes an increase in fluorescence intensity when Ca2+ binds to the single Ca2+- specific regulatory site. The steady-state fractional fluorescence and tension curves for fibers reconstituted with cardiac TnCIaans had nearly the same Ca2+ sensitivity. The steady-state fractional fluorescence of myofibrils reconstituted with TnCDanz was found to have a greater sensitivity to Ca2+ than the simultaneously measured ATPase. In all cases paired fractional fluorescence and activity curves tended to have parallel dependence on Ca2+. These procedures make it possible to study the Ca2+ binding properties of the Ca2+- specific sites in intact myofibrils and skinned fibers; the results presented suggest that the Ca2+ affinity of the Ca2+-specific sites of troponin are reduced in the thin filament compared to that of troponin in solution.     

Conformational change of skeletal muscle troponin

The fluorescence titration curve of skeletal muscle troponin containing TnI with 2-[4'-iodoacetamido)anilino)naphthalene-6-sulfonic acid-labeled Cys-48 and/or Cys-64 was composed of two transition curves. One transition occurred at the pCa region higher than 8.0, and the other between pCa 8.0 and 6.0. The transition at the lower pCa region had a midpoint of pCa 6.85, and the midpoint did not depend on Mg2+. The time course of the fluorescence change subsequent to the rapid pCa-jump of the solution was biphasic. The fast phase was due to the transition at the lower pCa region, and the rate constant of the process was characteristic of the conformational change of the protein induced by Ca2+ binding to the low affinity Ca2+-binding sites of TnC. The slow phase was from the transition at the higher pCa region, and its rate constant was characteristic of the conformational change of the protein induced by Ca2+ binding to the high affinity Ca2+-binding sites of TnC. Therefore we can conclude that the fluorescence probe bound to Cys-48 and/or Cys-64 of TnI detects the conformational change of the Tn complex induced by Ca2+ binding to both the low and high affinity Ca2+-binding sites of TnC. The fluorescence probe bound to Cys-133 of TnI or Met residues of TnT detected the conformational change of the Tn complex induced by Ca2+ binding to the low affinity Ca2+-binding sites of TnC.     

Effect of Ca2+ binding on troponin C. Changes in spin label mobility, extrinsic fluorescence, and sulfhydryl reactivity.

The Ca2+ binding component (TnC) of troponin has been selectively labeled with either a spin label, N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl) iodoacetamide, or with a fluorescent probe, S-mercuric-N-dansyl cysteine, presumably at its single cysteine residue (Cys-98) in order to probe the interactions of TnC with divalent metals and with other subunits of troponin. The modified protein has the same Ca2+ binding properties as native TnC (Potter, J. D., and Gergely, J. (1975) J. Biol. Chem. 250, 4628), viz. two Ca2+ binding sites at which Mg2+ appears to compete (Ca2+-Mg2+ sites, KCa = 2 X 10(7) M-1) and two sites at which Mg2+ does not compete (Ca2+-specific sites, KCa = 2 X 10(5) M-1). Either Ca2+ or Mg2+ alters the ESR spectrum of spin-labeled TnC in a manner that indicates a decrease in the mobility of the label, Ca2+ having a slightly greater effect. In systems containing both Ca2+ and Mg2+ the mobility of the spin label is identical with that in systems containing Ca2+ alone. The binding constants for Ca2+ and Mg2+ deduced from ESR spectral changes are 10(7) and 10(3) M-1, respectively, and the apparent affinity for Ca2+ decreases by about an order of magnitude on adding 2 mM Mg2+. Thus, the ESR spectral change is associated with binding of Ca2+ to one or both of the Ca2+-Mg2+ sites. Addition of Ca2+ to the binary complexes of spin-labeled TnC with either troponin T (TnT) or troponin I (TnI) produces greater reduction in the mobility of the spin label than in the case of spin-labeled TnC alone, and in the case of the complex with TnI the affinity for Ca2+ is increased by an order of magnitude. The fluorescence of dansyl (5-dimethylaminonaphthalene-1-sulfonyl)-labeled TnC is enhanced by Ca2+ binding to both high and low affinity sites with apparent binding constants of 2.6 X 10(7) M-1 and 2.9 X 10(5) M-1, respectively, calculated from the transition midpoints. The presence of 2 mM Mg2+, which produces no effect on dansyl fluorescence itself, in contrast to its effect on the spin label, shifts the high affinity constant to 2 X 10(6) M-1. Spectral changes produced by Ca2+ binding to the TnC-TnI complex furnish evidence that the affinity of TnC for Ca2+ is increased in the complex. The reactivity of Cys-98 to the labels and to 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2) is decreased by Ca2+ or Mg2+ both with native TnC and in 6 M urea. The reaction rate between Cys-98 and Nbs2 decreases to one-half the maximal value at a Ca2+ concentration that suggests binding to the Ca2+-Mg2+ sites. Formation of a binary complex between TnI and TnC reduces the rate of reaction, and there is a further reduction by Ca2+. The effect of Ca2+ takes place at concentrations that are 1 order of magnitude lower than in the case of TnC alone. These results suggest that the Ca2+ binding site adjacent to Cys-98 is one of the Ca2+-Mg2+ binding sites.     

Evidence that the Sr2+ activation properties of cardiac troponin C are altered when substituted into skinned skeletal muscle fibers

Troponin C (TnC) was extracted from skinned skeletal muscle fibers by a method similar to that used previously on myofibrils (Zot, H.G., and Potter, J.D. (1982) J. Biol. Chem. 257, 7678-7683) and replaced with either skeletal (fast-twitch) or cardiac TnC. The relationship between isometric tension and Sr2+ concentration remained essentially the same before removal and after replacement with skeletal or cardiac TnC. Therefore, the origin of the TnC made no difference in the Sr2+ activation properties of the skinned fiber. In contrast, the activation of skinned cardiac fibers is approximately an order of magnitude more sensitive to Sr2+ than skinned skeletal fibers. These results show that the affinity of cardiac TnC for Sr2+ is altered when substituted into skinned skeletal muscle fibers through protein-protein interactions.     

The control of myocardial contraction with skeletal fast muscle troponin C

The present study describes experiments on the myocardial trabeculae from the right ventricle of Syrian hamsters whose troponin C (TnC) moiety was exchanged with heterologous TnC from fast skeletal muscle of the rabbit. These experiments were designed to help define the role of the various classes of Ca2+-binding sites on TnC in setting the characteristic sensitivities for activations of cardiac and skeletal muscles. Thin trabeculae were skinned and about 75% of their troponin C extracted by chemical treatment. Tension development on activations by Ca2+ and Sr2+ was found to be nearly fully blocked in such TnC extracted preparations. Troponin C contents and the ability to develop tension on activations by Ca2+ and Sr2+ was permanently restored after incubation with 2-6 mg/ml purified TnC from either rabbit fast-twitch skeletal muscle (STnC) or the heart (CTnC, cardiac troponin C). The native (skinned) cardiac muscle is characteristically about 5 times more sensitive to activation by Sr2+ than fast muscle, but the STnC-loaded trabeculae gave response like fast muscle. Attempts were also made to exchange the TnC in psoas (fast-twitch muscle) fibers, but unlike cardiac muscle tension response of the maximally extracted psoas fibers could be restored only with homologous STnC. CTnC was effective in partially extracted fibers, even though the uptake of CTnC was complete in the maximally extracted fibers. The results in this study establish that troponin C subunit is the key in setting the characteristic sensitivity for tension control in the myocardium above that in the skeletal muscle. Since a major difference between skeletal and cardiac TnCs is that one of the trigger sites (site I, residues 28-40 from the N terminus) is modified in CTnC and has reduced affinity for Ca2+ binding, the possibility is raised that this site has a modulatory effect on activation in different tissues and limits the effectiveness of CTnC in skeletal fibers.     

Evidence that both Ca(2+)-specific sites of skeletal muscle TnC are required for full activity

To investigate the role of the Ca2(+)-specific (I and II) sites of fast skeletal muscle troponin C (TnC) in the regulation of contraction, we have produced two TnC mutants which have lost the ability to bind Ca2+ at either site I (VG1) or at site II (VG2). Both mutants were able to partially restore force to TnC-depleted skinned muscle fibers (approximately 25% for VG1 and approximately 50% for VG2). In contrast, bovine cardiac TnC (BCTnC), which like VG1 binds Ca2+ only at site II, could fully reactivate the contraction of TnC-depleted fibers. Higher concentrations of both mutants were required to restore force to the TnC-depleted fibers than with wild type TnC (WTnC) or BCTnC. VG1 and VG2 substituted fibers could not bind additional WTnC, indicating that all of the TnC-binding sites were saturated with the mutant TnC's. The Ca2+ concentration required for force activation was much higher for VG1 and VG2 substituted fibers than for WTnC or BCTnC substituted fibers. Also, the steepness of force activation was much less in VG1 and VG2 versus WTnC and BCTnC substituted fibers. These results suggest cooperative interactions between sites I and II in WTnC. In contrast, BCTnC has essentially the same apparent Ca2+ affinity and steepness of force activation as does WTnC. Thus, cardiac TnC must have structural differences from WTnC which compensate for the lack of site I, while in WTnC, both Ca2(+)-specific sites are probably crucial for full functional activity.     

Calmidazolium, a calmodulin antagonist, stimulates calcium-troponin C and calcium-calmodulin-dependent activation of striated muscle myofilaments

Although regulatory Ca2+-binding domains of calmodulin (CaM) and troponin C (TnC) are similar, it is interesting that agents that act as CaM antagonists appear to be TnC "agonists" in that they sensitize cardiac myofilaments to activation by Ca2+ (El-Saleh, S., and Solaro, R. J. (1987) Biophys. J. 51, 325 (abstr.). This indicates that the effects of agents that react with Ca2+-binding proteins may depend on protein-protein interactions involved in a particular Ca2+-dependent process. In experiments described here, we have explored this idea by testing effects of calmidazolium (CDZ), a potent calmodulin antagonist on striated muscle myofilaments regulated by cardiac TnC, skeletal TnC, and CaM. CDZ was shown to increase submaximal calcium activation of myofilament force and ATPase activity in both cardiac and skeletal muscle, but the effect was greater in the case of the cardiac preparations. In the presence of 10 microM CDZ, the free Ca2+ giving half-maximal activation was reduced to about 60% of the control value in the case of cardiac myofilaments. Analogous differential effects of CDZ were also seen in studies in which we measured direct effects of CDZ on Ca2+-dependent fluorescence changes of cardiac TnC and skeletal TnC labeled with probes reporting Ca2+ binding to the regulatory sites. Measurements were also done with myofibrillar preparations of psoas muscle in which the native skeletal TnC was removed and exchanged with cardiac TnC and CaM, both of which could substitute for skeletal TnC as a regulatory protein. CDZ was more effective in sensitizing Ca2+-dependent MgATPase activity of skeletal myofibrils containing CaM than in preparations containing the native TnC. However, CDZ was most effective in its Ca2+-sensitizing effect in the case of the preparations containing cardiac TnC. Our results indicate that effects of agents that bind to Ca2+-binding proteins depend not only on the particular variant, but also on the specific environment in which the Ca2+-binding proteins operate.     

Biological function and site II Ca2+-induced opening of the regulatory domain of skeletal troponin C are impaired by invariant site I or II Glu mutations

To investigate the roles of site I and II invariant Glu residues 41 and 77 in the functional properties and calcium-induced structural opening of skeletal muscle troponin C (TnC) regulatory domain, we have replaced them by Ala in intact F29W TnC and in wild-type and F29W N domains (TnC residues 1-90). Reconstitution of intact E41A/F29W and E77A/F29W mutants into TnC-depleted muscle skinned fibers showed that Ca(2+)-induced tension is greatly reduced compared with the F29W control. Circular dichroism measurements of wild-type N domain as a function of pCa (= -log[Ca(2+)]) demonstrated that approximately 90% of the total change in molar ellipticity at 222 nm ([theta](222 nm)) could be assigned to site II Ca(2+) binding. With E41A, E77A, and cardiac TnC N domains this [theta](222 nm) change attributable to site II was reduced to < or =40% of that seen with wild type, consistent with their structures remaining closed in +Ca(2+). Furthermore, the Ca(2+)-induced changes in fluorescence, near UV CD, and UV difference spectra observed with intact F29W are largely abolished with E41A/F29W and E77A/F29W TnCs. Taken together, the data indicate that the major structural change in N domain, including the closed to open transition, is triggered by site II Ca(2+) binding, an interpretation relevant to the energetics of the skeletal muscle TnC and cardiac TnC systems.     

Effects of mutations in the central helix of troponin C on its biological activity

To investigate the role of the central helix of skeletal muscle troponin C (TnC), five deletion mutants (Dobrowolski, Z., Xu, G.Q., and Hitchcock-DeGregori, S.E. (1991) J. Biol. Chem. 266, 5703-5710) of chicken TnC in the D/E linker region (K87EDAKGKSEEE97), dEDA, dKG, dKGK, dSEEE, and dKED-AKGK, were assayed for their ability to regulate muscle contraction by testing their effectiveness in restoring force and Ca2+ regulation to TnC-depleted rabbit skinned skeletal muscle fibers. By comparison with rabbit skeletal TnC, wild-type TnC, and chicken TnC, all mutants except dKG equally restored force development and Ca2+ regulation to TnC-depleted skinned muscle fibers. In contrast, approximately 4 times more dKG than rabbit skeletal TnC was required to reach 50% force restoration. Also, the pCa50 for dKG activation of force was significantly decreased. Thus, most of the TnC mutants that we studied did not have significantly altered biological activity in the skinned fiber assay. However, the 2-residue deletion in the central helix (dKG) significantly affected TnC activity. This deletion would be expected to produce a 160 degree rotation in the alpha-helix versus 60 degrees for dKGK and dEDA, 40 degrees in dSEEE, and 20 degrees in dKEDAKGK. Therefore, the change in orientation of the two Ca2(+)-binding domains appears to be a major parameter affecting TnC activity. The shift in the Ca2+ dependence in force activation may result from the inability of the Ca2(+)-specific domain to properly interact with its binding site on troponin I, an interaction which is known to increase the affinity of TnC for Ca2+ (Potter, J.D., and Gergely, J. (1975) J. Biol. Chem. 250, 4628-4633). In addition, the length of the central helix of TnC, Gly92, and the negatively charged cluster, EEE, appear not to be crucial for TnC activity.     

Site-directed mutation of the trigger calcium-binding sites in cardiac troponin C

The trigger Ca2+-binding sites in troponin C, those which initiate muscle contraction, are thought to be the first two of four potential sites (sites I-IV). In cardiac troponin C, the first Ca2+-binding site is inactive, and initiation of contraction in cardiac muscle appears to involve only the second site. To study this phenomenon and associated Ca2+-dependent protein conformational changes in cardiac troponin C, the cDNA for the chicken protein was incorporated into a bacterial expression plasmid to allow site-specific mutagenesis. Ca2+-binding site I was activated by deletion of Val-28 and conversion of amino acids 29-32 to those found at the first four positions in the active site I of fast skeletal troponin C. In a series of proteins, Ca2+-binding site II was inactivated by mutation of amino acids Asp-65, Asp-67, and Gly-70. All mutated proteins exhibited the predicted calcium-binding characteristics. The single mutation of converting Asp-65 to Ala was sufficient to inactivate site II. Ca2+-dependent conformational changes in the normal and mutated proteins were monitored by labeling with a sulfhydryl-specific fluorescent dye. Activation of Ca2+-binding site I or inactivation of site II, eliminated the large Ca2+-dependent increase in fluorescence seen in the wild type protein and there was, instead, a Ca2+-dependent decrease in fluorescence. All mutant proteins could associate with troponin I and troponin T to form a troponin complex. Activation of Ca2+-binding site I changed the characteristics of contraction in skinned slow skeletal muscle fibers such that the response to Ca2+ was more cooperative. Inactivation of Ca2+-binding site II abolished Ca2+-dependent contraction in skinned muscle fibers. The data provide a direct demonstration that Ca2+-binding site II in cardiac troponin C is essential for triggering muscle contraction and support the hypothesis that site I functions to modify the characteristics of contraction.