The Role of the NH2- and COOH-terminal Domains of the Inhibitory Region of Troponin I in the Regulation of Skeletal Muscle Contraction
1999; Elsevier BV; Volume: 274; Issue: 41 Linguagem: Inglês
10.1074/jbc.274.41.29536
ISSN1083-351X
AutoresDanuta Szczȩsna, Ren Zhang, Jiaju Zhao, Michelle Jones, James D. Potter,
Tópico(s)Cardiovascular Effects of Exercise
ResumoThe role of the inhibitory region of troponin (Tn) I in the regulation of skeletal muscle contraction was studied with three deletion mutants of its inhibitory region: 1) complete (TnI-(Δ96–116)), 2) the COOH-terminal domain (TnI-(Δ105–115)), and 3) the NH2-terminal domain (TnI-(Δ95–106)). Measurements of Ca2+-regulated force and relaxation were performed in skinned skeletal muscle fibers whose endogenous TnI (along with TnT and TnC) was displaced with high concentrations of added troponin T. Reconstitution of the Tn-displaced fibers with a TnI·TnC complex restored the Ca2+ sensitivity of force; however, the levels of relaxation and force development varied. Relaxation of the fibers (pCa 8) was drastically impaired with two of the inhibitory region deletion mutants, TnI-(Δ96–116)·TnC and TnI-(Δ105–115)·TnC. The TnI-(Δ95–106)·TnC mutant retained ∼55% relaxation when reconstituted in the Tn-displaced fibers. Activation in skinned skeletal muscle fibers was enhanced with all TnI mutants compared with wild-type TnI. Interestingly, all three mutants of TnI increased the Ca2+ sensitivity of contraction. None of the TnI deletion mutants, when reconstituted into Tn, could inhibit actin-tropomyosin-activated myosin ATPase in the absence of Ca2+, and two of them (TnI-(Δ96–116) and TnI-(Δ105–115)) gave significant activation in the absence of Ca2+. These results suggest that the COOH terminus of the inhibitory region of TnI (residues 105–115) is much more critical for the biological activity of TnI than the NH2-terminal region, consisting of residues 95–106. Presumably, the COOH-terminal domain of the inhibitory region of TnI is a part of the Ca2+-sensitive molecular switch during muscle contraction. The role of the inhibitory region of troponin (Tn) I in the regulation of skeletal muscle contraction was studied with three deletion mutants of its inhibitory region: 1) complete (TnI-(Δ96–116)), 2) the COOH-terminal domain (TnI-(Δ105–115)), and 3) the NH2-terminal domain (TnI-(Δ95–106)). Measurements of Ca2+-regulated force and relaxation were performed in skinned skeletal muscle fibers whose endogenous TnI (along with TnT and TnC) was displaced with high concentrations of added troponin T. Reconstitution of the Tn-displaced fibers with a TnI·TnC complex restored the Ca2+ sensitivity of force; however, the levels of relaxation and force development varied. Relaxation of the fibers (pCa 8) was drastically impaired with two of the inhibitory region deletion mutants, TnI-(Δ96–116)·TnC and TnI-(Δ105–115)·TnC. The TnI-(Δ95–106)·TnC mutant retained ∼55% relaxation when reconstituted in the Tn-displaced fibers. Activation in skinned skeletal muscle fibers was enhanced with all TnI mutants compared with wild-type TnI. Interestingly, all three mutants of TnI increased the Ca2+ sensitivity of contraction. None of the TnI deletion mutants, when reconstituted into Tn, could inhibit actin-tropomyosin-activated myosin ATPase in the absence of Ca2+, and two of them (TnI-(Δ96–116) and TnI-(Δ105–115)) gave significant activation in the absence of Ca2+. These results suggest that the COOH terminus of the inhibitory region of TnI (residues 105–115) is much more critical for the biological activity of TnI than the NH2-terminal region, consisting of residues 95–106. Presumably, the COOH-terminal domain of the inhibitory region of TnI is a part of the Ca2+-sensitive molecular switch during muscle contraction. Contraction of skeletal muscle is initiated by the binding of Ca2+ to the regulatory sites of troponin (Tn) 1The abbreviations used are:TntroponinTmtropomyosinWTnIwild-type TnIRSTnTrabbit skeletal TnTPAGEpolyacrylamide gel electrophoresisMOPS4-morpholinepropanesulfonic acid1The abbreviations used are:TntroponinTmtropomyosinWTnIwild-type TnIRSTnTrabbit skeletal TnTPAGEpolyacrylamide gel electrophoresisMOPS4-morpholinepropanesulfonic acid C, the Ca2+-binding subunit of Tn, which causes an interaction with TnI, the inhibitory subunit of Tn, releasing its inhibition of actomyosin ATPase activity. For full biological function, Tn requires a third subunit, TnT, which anchors the TnI·TnC complex to the actin-tropomyosin (Tm) filaments and also plays a role in the Ca2+-mediated activation of actomyosin ATPase activity and/or contraction (1Potter J.D. Sheng Z. Pan B.-S. Zhao J. J. Biol. Chem. 1995; 270: 2557-2567Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 2Reinach F.C. Farah C.S. Monteiro P.B. Malnic B. Cell Struct. Funct. 1997; 22: 219-223Crossref PubMed Scopus (13) Google Scholar, 3Malnic B. Farah C.S. Reinach F.C. J. Biol. Chem. 1998; 273: 10594-10601Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 4Gergely J. Adv. Exp. Med. Biol. 1998; 453: 169-176Crossref PubMed Scopus (25) Google Scholar, 5Szczesna D. Potter J.D. Thomas D.D. dos Remedios C.G. Molecular Interactions of Actin. 2. Springer-Verlag, Heidelberg, Germany1999Google Scholar).The inhibitory function of Tn has been studied in many different ways, yet the structure-function of the inhibitory region of TnI, responsible for this, is still under investigation. The role of the putative inhibitory region of TnI, which consists of 21 amino acids (residues 96–116), has been studied previously utilizing synthetic peptides as well as proteolytic and recombinant fragments of TnI. The cyanogen bromide fragment (CN4) of TnI (residues 96–116) was originally found to possess all of the inhibitory properties of intact TnI (6Syska H. Wilkinson J.M. Grand R.J.A. Perry S.V. Biochem. J. 1976; 153: 375-387Crossref PubMed Scopus (192) Google Scholar). Studies with synthetic peptides have demonstrated that residues 105–114 represent the minimal sequence necessary to produce inhibition of actomyosin ATPase activity and to retain TnC binding (7Talbot J.A. Hodges R.S. J. Biol. Chem. 1979; 254: 3720-3723Abstract Full Text PDF PubMed Google Scholar, 8Talbot J.A. Hodges R.S. J. Biol. Chem. 1981; 256: 2798-2802Abstract Full Text PDF PubMed Google Scholar, 9Van Eyk J.E. Kay C.M. Hodges R.S. Biochemistry. 1991; 30: 9974-9981Crossref PubMed Scopus (27) Google Scholar, 10Campbell A.P Sykes B.D. J. Mol. Biol. 1991; 222: 405-421Crossref PubMed Scopus (73) Google Scholar); however, the CN4 fragment (TnI-(96–116)) has been shown to have an 8-fold higher affinity for TnC compared with TnI-(104–115) (11Chandra M. McCubbin W.D. Oikawa K. Kay C.M. Smillie L.B. Biochemistry. 1994; 33: 2961-2969Crossref PubMed Scopus (32) Google Scholar).Several regions of TnI, including its inhibitory region (residues 96–116), have been identified as interacting with actin-Tm and TnC (12Levine B.A. Moir A.J. Perry S.V. Eur. J. Biochem. 1988; 172: 389-397Crossref PubMed Scopus (60) Google Scholar, 13Van Eyk J.E. Hodges R.S. J. Biol. Chem. 1988; 263: 1726-1732Abstract Full Text PDF PubMed Google Scholar, 14Cachia P.J. Sykes B.D. Hodges R.S. Biochemistry. 1983; 22: 4145-4152Crossref PubMed Scopus (47) Google Scholar, 15Leszyk J. Collins J.H. Leavis P.C. Tao T. Biochemistry. 1987; 26: 7042-7047Crossref PubMed Scopus (48) Google Scholar, 16Leszyk J. Grabarek Z. Gergely J. Collins J. Biochemistry. 1990; 29: 299-304Crossref PubMed Scopus (65) Google Scholar). Sequence 104–115 of TnI has been shown to share both the actin-Tm- and TnC-binding sites. Studies of Tripet et al.(17Tripet B. Van Eyk J.E. Hodges R.S. J. Mol. Biol. 1997; 271: 728-750Crossref PubMed Scopus (184) Google Scholar) suggest that the region of TnI that follows the inhibitory sequence (residues 96–116) contains additional actin-Tm- and TnC-binding sites. A synthetic peptide consisting of residues 128–148 was able to bind specifically to the actin-Tm filament and could induce a weak inhibitory activity on its own. Truncation of residues 140–148 completely abolished the inhibitory effect of this region when compared with TnI-(96–115), suggesting that region 140–148 of TnI presumably contains the second actin-Tm-binding site (17Tripet B. Van Eyk J.E. Hodges R.S. J. Mol. Biol. 1997; 271: 728-750Crossref PubMed Scopus (184) Google Scholar). This is in agreement with Farah et al. (18Farah C.S. Miyamoto C.A. Ramos C.H.I. da Silva A.C.R. Quaggio R.B. Fujimori K. Smillie L.B. Reinach F.C. J. Biol. Chem. 1994; 269: 5230-5240Abstract Full Text PDF PubMed Google Scholar), who demonstrated that residues 116–156 are important for the Ca2+ regulation of actomyosin ATPase activity. The recombinant fragment TnI-(1–116) failed to inhibit ATPase activity in the absence of Ca2+compared with other fragments (TnI-(1–156) and TnI-(103–182)) that were able to regulate actomyosin ATPase activity in a Ca2+-dependent manner (18Farah C.S. Miyamoto C.A. Ramos C.H.I. da Silva A.C.R. Quaggio R.B. Fujimori K. Smillie L.B. Reinach F.C. J. Biol. Chem. 1994; 269: 5230-5240Abstract Full Text PDF PubMed Google Scholar). Furthermore, Tripetet al. (17Tripet B. Van Eyk J.E. Hodges R.S. J. Mol. Biol. 1997; 271: 728-750Crossref PubMed Scopus (184) Google Scholar) demonstrated that residues 116–131 are not important for inhibition, but are critical for the interaction with TnC and designated this region to be the second TnC-binding site (17Tripet B. Van Eyk J.E. Hodges R.S. J. Mol. Biol. 1997; 271: 728-750Crossref PubMed Scopus (184) Google Scholar). Several studies have shown that residues 96–116 of TnI are primarily responsible for the binding to the COOH-terminal domain of TnC and residues 117–148 for the binding of TnI to the NH2-terminal domain of TnC (19Pearlstone J.R. Sykes B.D. Smillie L.B. Biochemistry. 1997; 36: 7601-7606Crossref PubMed Scopus (56) Google Scholar, 20Van Eyk J.E. Thomas L.T. Tripet B. Wiesner R.J. Pearlstone J.R. Farah C.S. Reinach F.C. Hodges R.S. J. Biol. Chem. 1997; 272: 10529-10537Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Reconstituted TnI fragments containing the inhibitory region of TnI (residues 96–116) plus either the NH2-terminal (TnI-(1–116)) or COOH-terminal (TnI-(96–148)) region of TnI were shown to be responsible for either maintaining the maximal level of actomyosin ATPase activity or the Ca2+ dependence of ATPase, respectively (20Van Eyk J.E. Thomas L.T. Tripet B. Wiesner R.J. Pearlstone J.R. Farah C.S. Reinach F.C. Hodges R.S. J. Biol. Chem. 1997; 272: 10529-10537Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). In summary, the regulatory complex containing TnT, TnC, and TnI-(96–148) retained all of the full regulatory properties of troponin, suggesting that TnI-(96–148) contains the major sequence of TnI responsible for inhibitory activity (17Tripet B. Van Eyk J.E. Hodges R.S. J. Mol. Biol. 1997; 271: 728-750Crossref PubMed Scopus (184) Google Scholar, 20Van Eyk J.E. Thomas L.T. Tripet B. Wiesner R.J. Pearlstone J.R. Farah C.S. Reinach F.C. Hodges R.S. J. Biol. Chem. 1997; 272: 10529-10537Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Based upon these experiments, an extended inhibitory region of TnI has been proposed, containing residues 96–148 (17Tripet B. Van Eyk J.E. Hodges R.S. J. Mol. Biol. 1997; 271: 728-750Crossref PubMed Scopus (184) Google Scholar, 19Pearlstone J.R. Sykes B.D. Smillie L.B. Biochemistry. 1997; 36: 7601-7606Crossref PubMed Scopus (56) Google Scholar, 20Van Eyk J.E. Thomas L.T. Tripet B. Wiesner R.J. Pearlstone J.R. Farah C.S. Reinach F.C. Hodges R.S. J. Biol. Chem. 1997; 272: 10529-10537Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 21McKay R.T. Tripet B.P. Hodges R.S. Sykes B.D. J. Biol. Chem. 1997; 272: 28494-28500Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 22McKay R.T. Pearlstone J.R. Corson D.C. Gagne S.M. Smillie L.B. Sykes B.D. Biochemistry. 1998; 37: 12419-12430Crossref PubMed Scopus (49) Google Scholar).This study was undertaken to determine the effect of the NH2- or COOH-terminal deletions of inhibitory region 96–116 of TnI on the contractile properties of skinned skeletal muscle fibers. We have expressed and purified wild-type TnI (WTnI) and three deletion mutants of TnI: TnI-(Δ95–106), TnI-(Δ105–115), and TnI-(Δ96–116). We have applied the improved method of Shiraishi and Yamamoto (23Shiraishi F. Yamamoto K. J. Biochem. ( Tokyo ). 1994; 115: 171-173Crossref PubMed Scopus (5) Google Scholar) and Hatakenaka and Ohtsuki (24Hatakenaka M. Ohtsuki I. Eur. J. Biochem. 1992; 205: 985-993Crossref PubMed Scopus (56) Google Scholar) to displace the endogenous Tn complex with high concentrations of added TnT, followed by functional reconstitution of the fibers with preformed complexes of TnC and the TnI mutants. This system allowed us to study the effect of these deletions on the Ca2+ regulation of force development. These proteins were also tested for their ability to inhibit actin-Tm-activated myosin ATPase activity as well as their ability to regulate ATPase activity when complexed with TnT and TnC. Our results suggest that the COOH-terminal end of the inhibitory region of TnI (residues 105–115) is much more critical for the biological activity of TnI than its NH2-terminal region (residues 95–106). Presumably, the COOH-terminal domain of the inhibitory region of TnI is a component of the Ca2+-sensitive molecular switch that regulates muscle contraction.MATERIALS AND METHODSProtein PurificationRabbit skeletal muscle troponin, tropomyosin, and actin and myosin were isolated and purified according to Potter (25Potter J.D. Methods Enzymol. 1982; 85: 241-263Crossref PubMed Scopus (290) Google Scholar), Strzelecka-Golaszewska et al. (26Strzelecka-Golaszewska H. Jakubiak H. Drabikowski W. Eur. J. Biochem. 1975; 55: 221-230Crossref PubMed Scopus (49) Google Scholar), and Stepkowski et al. (27Stepkowski D. Szczesna D. Wrotek M. Kakol I. Biochim. Biophys. Acta. 1985; 831: 321-329Crossref PubMed Scopus (37) Google Scholar), respectively.Wild-type TnI and the TnI inhibitory region deletion mutants were expressed in Escherichia coli BL21(DE3) (Novagen) using the protocol provided by the manufacturer. The expression was checked by SDS-15% PAGE (28Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205998) Google Scholar) and Western blotting. The culture for bacterial expression was collected and centrifuged at 7000 rpm (J-10, Beckman). Bacterial pellets were dissolved in a solution containing 6m urea, 10 mm sodium citrate, pH 7.0, 1 mm dithiothreitol, 2 mm EDTA, and 0.01% NaN3 and sonicated (Sonicator Heat Systems, Inc.) twice at setting 8 for 2 min at 4 °C. After the sonication, the pH of the solution was adjusted to 5.0, and the mixtures were then centrifuged again at 18,000 rpm (J-20, Beckman) at 4 °C for 30 min. Supernatants, containing the TnI proteins, were loaded onto a CM52 ion-exchange column equilibrated with the same buffer used to dissolve bacterial pellets, except that the pH was 5.0. The proteins were eluted with a linear salt gradient of 0–0.6 m KCl in the equilibration buffer. The fractions containing the TnI proteins (identified by SDS-PAGE and Western blotting) were pooled together and dialyzed against 0.5 m NaCl, 50 mm Tris, 2 mm CaCl2, and 1 mm dithiothreitol, pH 7.5, and loaded onto a TnC affinity column equilibrated with the same buffer. Pure TnI proteins were eluted with a double gradient of urea (0–6 m) and EDTA (0–3 mm). The average yield was 3–5 mg of pure TnI/liter of culture.Actin-Tm-activated Myosin ATPase AssaysWild-type TnI and the TnI mutants were first tested for their ability to inhibit actin-Tm-activated myosin ATPase activity, and then the whole Tn complexes (TnI + TnC + TnT formed at a molar ratio 1:1:1) were examined for their ability to regulate (±Ca2+) the ATPase activity. The ATPase assays were performed with actin (3.5 μm), Tm (1 μm), and myosin (0.6 μm) in the presence (0.5 mm CaCl2) or absence (1 mmEGTA) of Ca2+. The ATPase reaction (in 10 mmMOPS, 50 mm KCl, and 4 mm MgCl2, pH 7.0) was initiated by the addition of 2.5 mm ATP and terminated after 6 min with 5% trichloroacetic acid. Inorganic phosphate was measured according to Fiske and SubbaRow (29Fiske C.H. SubbaRow Y. J. Biol. Chem. 1925; 66: 375-400Abstract Full Text PDF Google Scholar).Airfuge Binding AssaysThe binding of the TnI proteins or troponins containing TnI, TnT, and TnC (1:1:1) to actin-Tm was measured at a molar ratio of 7 actin (14 μm)/1 Tm (2 μm)/1 Tn (2 μm) or 2 TnI (4 μm) in a buffer containing 10 mm MOPS, 50 mm KCl, and 4 mm MgCl2, pH 7.0. The complexes of Tn and actin-Tm were additionally mixed with either 0.5 mm CaCl2 or 1 mm EGTA. Protein complexes were incubated for 10 min at room temperature and Airfuged (maximum speed) for 30 min. In parallel, the control samples of the TnI proteins alone or complexed with TnT and TnC without actin-Tm were sedimented. The samples of TnI proteins, troponins, and their complexes with actin-Tm (prior to sedimentation) as well as the supernatants and pellets were run on SDS-15% polyacrylamide gels (28Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205998) Google Scholar) and analyzed.Displacement of the Endogenous Troponin Complex in Skinned Skeletal Muscle Fibers with TnT: Steady-state Force and the Ca2+ Sensitivity of Force DevelopmentDisplacement of the endogenous Tn complex in skinned skeletal muscle fibers was performed according to method of Shiraishi and Yamamoto (23Shiraishi F. Yamamoto K. J. Biochem. ( Tokyo ). 1994; 115: 171-173Crossref PubMed Scopus (5) Google Scholar) and Hatakenaka and Ohtsuki (24Hatakenaka M. Ohtsuki I. Eur. J. Biochem. 1992; 205: 985-993Crossref PubMed Scopus (56) Google Scholar). We have slightly modified this method (described below) to achieve complete Tn displacement, as judged by SDS-PAGE, and the measurements of Ca2+-unregulated force. Briefly, rabbit psoas skinned muscle fiber bundles (three to five fibers) were mounted on a force transducer and treated with apCa 8 relaxing solution containing 1% Triton X-100 for 20 min. The composition of the pCa 8 solution was 10−8m Ca2+, 1 mmMg2+, 7 mm EGTA, 5 mmMgATP2+, 20 mm imidazole, pH 7.0, 20 mm creatinine phosphate, and 15 units/ml creatinine phosphokinase, I = 150 mm. The Ca2+ dependence of force development was tested twice to make certain that it was stable. Following the initial testing, the fibers were then incubated in a solution containing 250 mmKCl, 20 mm MOPS, pH 6.2, 5 mmMgCl2, 5 mm EGTA, 0.5 mmdithiothreitol, and ≈1.6–2 mg/ml rabbit skeletal TnT for 1 h at room temperature. The fibers were then washed with the same solution without the protein (10 min at room temperature) and tested for the Ca2+-unregulated force that results from the displacement of the endogenous Tn complex from the fibers. The Ca2+regulation of steady-state force development was then restored with a preformed TnI·TnC complex. The reconstitution with the TnI·TnC complex (20 μm) was performed in the pCa 8 solution, generally for 1 h at room temperature or long enough for the force to reach a stable level. Wild-type TnI and the various TnI mutants were complexed with TnC and used to reconstitute Ca2+-regulated force. Control fibers were run in parallel and treated with the same solutions minus the proteins. The purpose of this was to get an estimate of fiber rundown and to be able to determine the extent of reconstitution of the TnT-treated fibers. The Ca2+ dependence of force development was determined before the TnT treatment and after TnI·TnC reconstitution, and the data were analyzed with the Hill equation (30Hill A.V. J. Physiol. ( Lond. ). 1910; 40: 190-224Crossref PubMed Scopus (187) Google Scholar): % relative force = 100 × [Ca2+]n/([Ca2+]n +pCa50n), wherepCa50 is the pCa of a solution in which 50% of the change is produced and n is the Hill coefficient.RESULTSTo identify domains of physiological significance in inhibitory region 96–116 of TnI, we generated WTnI and three inhibitory region deletion mutants: an NH2-terminal deletion mutant, TnI-(Δ95–106); a COOH-terminal deletion mutant, TnI-(Δ105–115); and a deletion of the complete inhibitory region, TnI-(Δ96–116). Fig. 1 shows amino acid sequences of the inhibitory region of TnI that have been deleted.Inhibition of Actin-Tm-activated Myosin ATPase Activity by TnI and Its Inhibitory Region Deletion MutantsThe inhibitory properties of rabbit skeletal TnI, WTnI, and its deletion mutants are presented in Fig. 2. Actin-Tm-activated myosin ATPase activity was measured as a function of increasing concentrations of the TnI proteins. As illustrated, recombinant TnI (WTnI) had essentially the same activity as rabbit skeletal TnI, and both inhibited ≈90–95% of the ATPase activity. The NH2-terminal inhibitory deletion mutant, TnI-(Δ95–106), only partially inhibited actin-Tm-activated myosin ATPase activity, and ≈40–50% inhibition occurred at a 3–4-fold molar excess of TnI-(Δ95–106) over actin. The COOH-terminal deletion mutant, TnI-(Δ105–115), and the entire inhibitory region deletion mutant, TnI-(Δ96–116), completely lost the ability to inhibit ATPase activity. The latter also gave a slight activation. In summary, the TnI mutants alone could not inhibit actin-Tm-activated myosin ATPase activity when the COOH-terminal region or the entire inhibitory region was deleted.Figure 2Effect of the TnI inhibitory region deletion mutants on the inhibition of actin-Tm-activated myosin ATPase activity. The concentration of actin was 3.5 μm, that of Tm was 1 μm, and that of myosin was 0.6 μm. The error bars represent S.D. values from four to six experiments. RSTnI, rabbit skeletal TnI.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Regulation of Actin-Tm-activated Myosin ATPase Activity by Tn Containing TnT, TnC, and the TnI Inhibitory MutantsFig.3 (A and B) illustrates the effect of the TnI inhibitory region deletion mutations on actin-Tm-activated myosin ATPase activity in reconstituted thin filaments. TnI and its deletion mutants were complexed with TnT and TnC, and the actomyosin ATPase activity was measured in the presence (Fig. 3 A) or absence (Fig. 3 B) of Ca2+. As shown in Fig. 3, the troponin complex containing wild-type TnI or rabbit skeletal TnI regulated the ATPase activity in a similar way (±Ca2+), suggesting that recombinant TnI is functional. Tn containing the NH2-terminal inhibitory region deletion mutant, TnI-(Δ95–106), activated the ATPase activity in the presence of Ca2+, but its inhibitory function (in the absence of Ca2+) was lost. The COOH-terminal inhibitory region deletion mutant, TnI-(Δ105–115), activated the ATPase activity in the presence or absence of Ca2+, with ∼1.3-fold higher activation in Ca2+. A similar, Ca2+-independent activation of ATPase activity was observed for the complete inhibitory region deletion mutant, TnI-(Δ96–116); however, the extent of activation was not as high as for TnI-(Δ105–115). In summary, in the presence of Ca2+(Fig. 3 A), all the activation curves were not significantly different, whereas the inhibition curves (in the absence of Ca2+) were dramatically different among the various TnI mutants (Fig. 3 B). TnI-(Δ105–115) as well as TnI-(Δ96–116) not only did not inhibit the ATPase activity in the absence of Ca2+, but gave a 1.4-fold activation of the ATPase. TnI-(Δ95–106) did not inhibit the ATPase activity in the absence of Ca2+ and also lacked the activation seen with the other mutants (Fig. 3 B).Figure 3Activation (+Ca2+) (A) and inhibition (−Ca2+) (B) of actin-Tm-activated myosin ATPase activity by the troponin complex containing TnT, TnC, and TnI inhibitory region deletion mutants.The concentration of actin was 3.5 μm, that of Tm was 1 μm, and that of myosin was 0.6 μm. Data points are the average of five to seven experiments, and theerror bars are the S.D.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Sedimentation Studies (Airfuge)To examine the binding of TnI proteins alone or complexed with TnC and TnT to actin-Tm, the complexes were Airfuged, and the pellets and supernatants were analyzed by SDS-PAGE. In parallel, the controls of the TnI proteins alone or complexed with TnT and TnC without actin-Tm were sedimented. No pellets of the TnI proteins alone or troponins containing the TnI mutants were observed (data not shown). Fig.4 A shows the binding of WTnI and TnI mutants to actin-Tm, whereas Fig. 4 B demonstrates the binding of the Tn complexes in the presence or absence of Ca2+. The analysis of the pellets and supernatants indicated that all of the troponin complexes containing either WTnI or the TnI deletion mutants bound well to actin-Tm in the presence or absence of Ca2+ (Fig. 4 B). Their binding to actin-Tm was weaker for the TnI mutants alone than when they were complexed with TnT and TnC (Fig. 4, A and B).Figure 4SDS-PAGE pattern of the binding of WTnI and its inhibitory region deletion mutants to actin-Tm alone (A) and complexed with TnT and TnC (B). The complexes were formed at a molar ratio of 7:1:1 actin (14 μm)/Tm (2 μm)/Tn (2 μm) in a buffer containing 10 mm MOPS, 50 mm KCl, and 4 mm MgCl2, pH 7.0. After incubation for 10 min at room temperature, they were Airfuged (maximum speed) for 30 min. SDS-PAGE was performed on 15% polyacrylamide gel according to Laemmli (28Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205998) Google Scholar). The first lanes represent either TnI (A) or Tn (B) complexes containing TnT, TnC, and TnI inhibitory region deletion mutants (St). The second lanes represent the Tn complexes mixed with actin-Tm (Mix). The third lanes represent supernatants (S). The fourth lanes represent pellets (P) after centrifugation. Thearrows indicate the bands of the TnI proteins. For quantitation, the amount of the protein mixture applied to the gel (second lane) was equal to the amount of the protein complex centrifuged in the Airfuge. The resolubilized pellets and supernatants were loaded in full.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Force Activation, Relaxation, and the Ca2+ Sensitivity of Force in Skinned Skeletal Muscle Fibers Reconstituted with the TnI Inhibitory Region Deletion MutantsThe physiological significance of the NH2- and COOH-terminal domains of the inhibitory region of TnI was examined using rabbit psoas skinned fibers, in which steady-state force, relaxation, and the Ca2+ sensitivity of force development were measured. Following Tn displacement, the fibers were reconstituted with preformed complexes of TnC and WTnI and its deletion mutants. Fig. 5 illustrates a typical experiment on the TnT-treated fiber (panel A) compared with the control buffer-treated fiber (panel B), which had been tested in parallel to estimate the time-dependent rundown of the fibers. Fig.6 illustrates the Tn displacement procedure. As shown, incubation of the fibers with TnT resulted in a complete loss of Ca2+ dependence of force, and the fibers became unregulated (Fig. 5 A). This is illustrated in Fig. 6by a transition from step 1 to step 2. When Tn-displaced fibers were incubated with a preformed TnI·TnC complex, dissolved in the relaxing solution (pCa 8), the fibers underwent a gradual relaxation as the Tn activity was reconstituted (Fig. 5 A). This step (step 3) restored the Tn complex (Fig. 6), and the fibers became entirely regulated by Ca2+. The level of force relaxation depended on the TnI mutant used for the TnI·TnC complex. In Fig. 5 A, WTnI or rabbit skeletal TnI was used, and full relaxation in the pCa 8 solution was achieved. Fig. 7summarizes the effect of the TnI mutations on the level of relaxation and force development following reconstitution of the fibers with preformed TnI·TnC complexes. The dashed line in Fig. 7represents the level of Ca2+-unregulated force after RSTnT treatment (this step and the level of Ca2+-unregulated force are also shown in Fig. 5 A). Arrows indicate the percentage of force inhibition relative to Ca2+-unregulated force. As shown, the relaxation in the fibers could be restored upon incubation with TnI·TnC; however, the level of relaxation varied depending on the TnI mutant utilized in the reconstitution. The NH2-terminal inhibitory region deletion mutant, TnI-(Δ95–106), inhibited ≈54.9 ± 6% of the force following reconstitution. On the other hand, the COOH-terminal inhibitory region deletion mutant, TnI-(Δ105–115), as well as the complete inhibitory region deletion mutant, TnI-(Δ96–116), inhibited only 28.3 ± 10 and 28.0 ± 7% of the force, respectively. Interestingly, all three TnI deletion mutants gave an elevated maximal level of force recovery in the high Ca2+ solution (pCa 4) (Fig. 7). The extent of activation over that recovered with WTnI·TnC was ≈114 ± 16, 116 ± 15, and 123 ± 5% for TnI-(Δ96–116), TnI-(Δ105–115), and TnI-(Δ95–106), respectively (Fig. 7). The Ca2+sensitivity of force development measured after fiber reconstitution is demonstrated in Fig. 8. All three deletion mutants of TnI increased slightly the Ca2+dependence of force (ΔpCa50 ≅ 0.1–0.2) compared with WTnI·TnC-reconstituted fibers. A Western blot of the experimental fibers performed with the TnI antibodies is presented in Fig. 9. As shown, all of the experimental fibers were reconstituted with the respective TnI deletion mutants. Amino acid deletions within the inhibitory region of TnI did not prevent the binding of TnI-(Δ95–106)·TnC, TnI-(Δ105–115)·TnC, or TnI-(Δ96–116)·TnC complexes to the Tn-displaced fibers.Figure 5Protocol for the RSTnT-treated (troponin-displaced) skinned skeletal muscle fibers. A, fibers treated with RSTnT for 1 h at room temperature and reconstituted with preforme
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