The C Terminus of Cardiac Troponin I Is Essential for Full Inhibitory Activity and Ca2+ Sensitivity of Rat Myofibrils

Although the C terminus of troponin I is known to be important in myofilament Ca2+ regulation in skeletal muscle, the regulatory function of this region of cardiac troponin I (cTnI) has not been defined. To address this question, the following recombinant proteins were expressed in Escherichia coli and purified: mouse wild-type cTnI (WT cTnI; 211 residues), cTnI-(1–199) (missing 12 residues), cTnI-(1–188) (missing 23 residues), and cTnI-(1–151) (missing 60 residues). The inhibitory activity of cTnI and the mutants was tested in myofibrils, from which cTnI·cTnC was extracted by exchanging endogenous cardiac troponin with exogenous cTnT causing the Ca2+ sensitivity of the myofibrils to be lost. Addition of increasing amounts of exogenous WT cTnI or cTnI-(1–199) to cTnT-treated myofibrils at pCa 8 caused a concentration-dependent inhibition of the maximum ATPase activity. However, cTnI-(1–188) and cTnI-(1–151) inhibited this activity to about 757 and 507 of that of the WT cTnI, respectively. We also formed a complex of either WT cTnI or each of the mutants with cTnC, reconstituted the complex into the cTnT-treated myofibrils, and measured the Mg2+-ATPase activity as a function of pCa. We found that the cTnI-(1–188)·cTnC complex only partially restored Ca2+ sensitivity, whereas the cTnI-(1–151)·cTnC complex did not restore any Ca2+sensitivity. Each cTnI C-terminal deletion mutant was able to bind to cTnC, as shown by urea-polyacrylamide gel-shift analysis and size exclusion chromatography. Each mutant also co-sedimented with actin. Our results indicate that residues 152–199 (C-terminal to the inhibitory region) of cTnI are essential for full inhibitory activity and Ca2+ sensitivity of myofibrillar ATPase activity in the heart. Although the C terminus of troponin I is known to be important in myofilament Ca2+ regulation in skeletal muscle, the regulatory function of this region of cardiac troponin I (cTnI) has not been defined. To address this question, the following recombinant proteins were expressed in Escherichia coli and purified: mouse wild-type cTnI (WT cTnI; 211 residues), cTnI-(1–199) (missing 12 residues), cTnI-(1–188) (missing 23 residues), and cTnI-(1–151) (missing 60 residues). The inhibitory activity of cTnI and the mutants was tested in myofibrils, from which cTnI·cTnC was extracted by exchanging endogenous cardiac troponin with exogenous cTnT causing the Ca2+ sensitivity of the myofibrils to be lost. Addition of increasing amounts of exogenous WT cTnI or cTnI-(1–199) to cTnT-treated myofibrils at pCa 8 caused a concentration-dependent inhibition of the maximum ATPase activity. However, cTnI-(1–188) and cTnI-(1–151) inhibited this activity to about 757 and 507 of that of the WT cTnI, respectively. We also formed a complex of either WT cTnI or each of the mutants with cTnC, reconstituted the complex into the cTnT-treated myofibrils, and measured the Mg2+-ATPase activity as a function of pCa. We found that the cTnI-(1–188)·cTnC complex only partially restored Ca2+ sensitivity, whereas the cTnI-(1–151)·cTnC complex did not restore any Ca2+sensitivity. Each cTnI C-terminal deletion mutant was able to bind to cTnC, as shown by urea-polyacrylamide gel-shift analysis and size exclusion chromatography. Each mutant also co-sedimented with actin. Our results indicate that residues 152–199 (C-terminal to the inhibitory region) of cTnI are essential for full inhibitory activity and Ca2+ sensitivity of myofibrillar ATPase activity in the heart. The transition of heart muscle from diastole to systole involves a Ca2+-dependent process in which an inhibitory protein, cTnI, 1The abbreviations used are: TnI, troponin I; c, cardiac; TnC, troponin C; TnT, troponin T; Tm, tropomyosin; fs, fast skeletal; WT, wild-type; MOPS, 3-(N-morpholino)propanesulfonic acid; DTT, dithiothreitol; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography. plays a key role in switching on the reaction of myosin cross-bridges with actin. A current model for the switching mechanism has been derived largely from studies with fast skeletal TnI and is as follows. During diastole, cTnI interacts tightly with actin and contributes to the inhibition of the actin-cross-bridge reaction. During systole, with Ca2+binding to cTnC, there is an increased affinity of cTnI for cTnC, which results in weakening of cTnI binding to actin. This leads to changes in Tm position, resulting in activation of contraction through promotion of the transition of cross-bridges from blocked or weak binding states to strong force-generating states (1Solaro R.J. Sperelakis N. Physiology and Pathophysiology of the Heart. 3rd Ed. Kluwer Academic Publishers, Boston1995: 355-366Google Scholar). In the case of heart muscle, Ca2+ signaling appears different from that of fast skeletal muscle, and little is known about regions of cTnI that are essential for the Ca2+ switch. An anti-parallel interaction between cTnI and cTnC has been demonstrated (2Krudy G. Kleerekoper Q. Guo X. Howarth J.W. Solaro R.J. Rosevear P.R. J. Biol. Chem. 1994; 269: 23731-23735Abstract Full Text PDF PubMed Google Scholar). In the anti-parallel arrangement, an interaction of the N-terminal region of cTnI with cTnC acts as an anchor maintaining the two proteins in the correct spatial orientation. The C-terminal region of cTnI is thought to bind to the N terminus of cTnC, which contains the low affinity Ca2+-specific site (site II) (2Krudy G. Kleerekoper Q. Guo X. Howarth J.W. Solaro R.J. Rosevear P.R. J. Biol. Chem. 1994; 269: 23731-23735Abstract Full Text PDF PubMed Google Scholar, 3Kleerekoper Q. Howarth J.W. Guo X. Solaro R.J. Rosevear P.L. Biochemistry. 1995; 34: 13343-13352Crossref PubMed Scopus (44) Google Scholar). Ca2+ binding to site II on cTnC is essential for the regulation of cardiac contraction (4Holroyde M.J. Robertson S.P. Johnson J.D. Solaro R.J. Potter J.D. J. Biol. Chem. 1980; 255: 11688-11693Abstract Full Text PDF PubMed Google Scholar, 5Pan B.-S. Solaro R.J. J. Biol. Chem. 1987; 262: 7839-7849Abstract Full Text PDF PubMed Google Scholar, 6Putkey J.A. Sweeney H.L. Campbell S.T. J. Biol. Chem. 1989; 264: 12370-12378Abstract Full Text PDF PubMed Google Scholar). Interestingly, it has been shown recently by heteronuclear, multidimensional NMR spectroscopy that upon transition from the apo- to the Ca2+-saturated states of the N-domain of cTnC, fewer hydrophobic residues are exposed in cTnC than in fsTnC (7Sia S.K. Li M.X. Spyracopoulos L. Gagne S.M. Liu W. Putkey J.A. Sykes B.D. J. Biol. Chem. 1997; 272: 18216-18221Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 8Spyracopoulos L. Li M.X. Sia S.K. Gagne S.M. Chandra M. Solaro R.J. Sykes B.D. Biochemistry. 1997; (in press)Google Scholar). This hydrophobic region of cTnC is thought to bind to cTnI (9Gagne S.M. Tsuda S. Li M.X. Smilie L.B. Sykes B.D. Nat. Struct. Biol. 1995; 2: 784-788Crossref PubMed Scopus (251) Google Scholar). The finding that the structure of the regulatory N-domain of cTnC is significantly more compact relative to fsTnC (7Sia S.K. Li M.X. Spyracopoulos L. Gagne S.M. Liu W. Putkey J.A. Sykes B.D. J. Biol. Chem. 1997; 272: 18216-18221Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar) may be related to differences in how cTnI and fsTnI interact with TnC and ultimately to differences in the regulation of cardiacversus skeletal muscle contraction (6Putkey J.A. Sweeney H.L. Campbell S.T. J. Biol. Chem. 1989; 264: 12370-12378Abstract Full Text PDF PubMed Google Scholar, 10Putkey J.A. Liu W. Sweeney H.L. J. Biol. Chem. 1991; 266: 14881-14884Abstract Full Text PDF PubMed Google Scholar). Moreover, it is known that TnI contains an inhibitory region (residues 139–150 in mouse cTnI) that binds to both TnC and actin, but not to both simultaneously (11Nozaki S. Kobayashi K. Katayama E. Muramatsu I. Chem. Lett. 1980; 3: 345-348Crossref Google Scholar, 12Talbot J.A. Hodges R.S. J. Biol. Chem. 1981; 256: 2798-2802Abstract Full Text PDF PubMed Google Scholar). This region is believed to be largely responsible for the ability of TnI to inhibit actomyosin ATPase activity and constitutes a key part of the molecular switch turning on the thin filament. Although structural and modulatory functions have been identified within the unique cardiac-specific N-terminal region of cTnI, the functional role of its C-terminal region has yet to be delineated. Our laboratory is using recombinant DNA technology to investigate structure-function relations of cardiac TnI. The C terminus of cTnI is highly conserved among the TnI isoforms and its binding to the N terminus of cTnC containing the low affinity Ca2+-specific site indicates it may be important for Ca2+-dependent regulation of cardiac muscle contraction. Although it has been shown that the C terminus of fsTnI appears to be important for Ca2+ sensitivity in myofilaments in skeletal muscle (13Farah C.S. Miyamoto C.A. Ramos C.H.I. da Silva A.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, 14Jha P.K. Mao C. Sarkar S. Biochemistry. 1996; 35: 11026-11035Crossref PubMed Scopus (26) Google Scholar), there is no evidence for such a role in cardiac muscle. In this study, we generated three deletion mutants of cardiac TnI to examine the function of the C-terminal domain of cTnI in the cardiac myofilament. Our results indicate that regions of cTnI C-terminal to the inhibitory region are essential for full inhibitory activity and Ca2+ sensitivity of cardiac myofilaments. Crude cardiac troponin was prepared from bovine left ventricular ether powder and cTnI and cTnT were subsequently purified by chromatography as described by Potter (15Potter J.D. Methods Enzymol. 1982; 85: 241-263Crossref PubMed Scopus (301) Google Scholar). Purified cTnT or cTnI was resuspended in 50 mm Trizma base, pH 8.0, 1 mm EDTA, 6m urea, 0.1 mm DTT for immediate use or dialyzed against deionized H2O, lyophilized, and stored at −80 °C. F-actin was purified from bovine left ventricular ether powder essentially as described by Pardee and Spudich (16Pardee J.D. Spudich J.A. Methods Enzymol. 1982; 85: PubMed Scopus Google Scholar) and resuspended in mm MOPS, pH 50 mm 1 mm 1 mm mm was purified from the from the cardiac troponin described by chromatography on Purified Tm was resuspended in A mm pH 60 mm mm mm cardiac wild-type TnI was by as described X. J. K. Solaro R.J. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). of cTnI C-terminal deletion and was by using a for The for the and cTnI-(1–151) mutants were as The for the reaction were described X. J. K. Solaro R.J. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). The were into a were for DNA analysis S. A.R. PubMed Scopus Google Scholar). The cTnI and mutant cTnI were from the using and and by gel from the was into the site of the This DNA was used to of WT cTnI or the deletion mutants of A or of was used to 1 of containing 50 of The was at °C. The were at for and the The were stored at −80 °C. The were resuspended in mm pH 8.0, 6m urea, mm EDTA, 1 mm DTT, 1 mm of and on for The was at for at and a with mm pH 8.0, urea, 1 1 mm The WT cTnI and mutant were with a of in the of The WT cTnI and mutant were purified by affinity chromatography on an cTnC with 50 mm pH 8.0, 1 mm 1 mm The WT cTnI and mutant were with 50 mm pH 8.0, urea, mm EDTA, 1 1 mm The of WT cTnI and the mutant were on PubMed Scopus Google Scholar). The molecular of WT cTnI, and cTnI-(1–151) were and respectively. The of a containing the for cTnC and the and of the cTnC was as described by and Johnson B.-S. Johnson J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). of the cTnC was by were from and in The were and myofibrils were prepared from the as described Solaro R.J. Methods in Scholar). Purified myofibrils were resuspended in A and was R.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). proteins were on The cTnI·cTnC containing WT cTnI, or cTnI-(1–151) were formed as described by Johnson J.D. Robertson S.P. Potter J.D. J. Biol. Chem. 1980; 255: Full Text PDF PubMed Google Scholar) with Purified cTnI and cTnC were to a of 8 in mm MOPS, pH urea, 1 mm 1 mm 1 mm DTT and on for 1 The was dialyzed to Potter (15Potter J.D. Methods Enzymol. 1982; 85: 241-263Crossref PubMed Scopus (301) Google Scholar), by the and from to and to The cTnI·cTnC complex was dialyzed against C mm MOPS, pH mm mm mm DTT, 0.1 the cTnI·cTnC complex was and an was on urea-polyacrylamide to complex L. N. PubMed Scopus Google Scholar). WT cTnI or cTnI mutants and cTnC were at a in containing urea, with either mm or mm were on polyacrylamide pH containing essentially as described by L. N. PubMed Scopus Google Scholar). containing cTnC with WT cTnI, or cTnI-(1–151) were prepared at a by as described The cTnI·cTnC in of mm MOPS, pH mm mm 1 mm 1 mm DTT were a gel that was with the were from the with a of and by at A were and by This endogenous cardiac troponin from the myofibrils by with exogenous The myofibrils the exogenous but of cTnI and cTnC the myofibrils to The was by and M. I. J. PubMed Scopus Google Scholar) and was as described by Solaro R.J. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). Purified bovine cTnT was in mm MOPS, pH mm mm mm DTT, 0.1 The at a of was to myofibrils in a of The was to with and the was at for 60 with The cTnT-treated myofibrils were and the was resuspended in of C containing of WT cTnI, of cTnI or cTnI·cTnC This was at for with at The reconstituted myofibrils were and the resuspended in of and in a of 1 of The was by the R.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). ATPase activity was by using a of the of Methods Enzymol. 1982; 85: PubMed Scopus Google Scholar) and J. Biol. Chem. 1991; 266: Full Text PDF PubMed Google Scholar). were in at in an The and are described in the The was and were by the of mm were by the of of was the of the The of was by the of of in the was measured at with a The of and to the of and at pH in the reaction were using in and J. Google Scholar). F-actin Tm and cTnI or cTnI mutants were of in of mm pH mm mm 0.1 mm 1 mm DTT, and for at in a the was and the were resuspended in and were by were at and are as between pCa and ATPase activity were to the using to the and differences of were by a with at To in the C terminus of cTnI that are important for Ca2+ regulation of cardiac muscle we generated three deletion mutants of mouse cardiac cTnI, which were with the WT cTnI in functional The of the mouse WT cTnI of 211 residues and is shown in 1 The WT cTnI and the cTnI C-terminal deletion mutants are shown in 1 The and cTnI-(1–151) mutants have and 60 residues from the C respectively. The cTnI-(1–151) mutant 1 the inhibitory region of The mouse WT cTnI and each cTnI C-terminal deletion mutant were expressed in Escherichia coli and purified as described to The gel in that the recombinant mouse WT cTnI than cardiac TnI purified from bovine of the of of the N-terminal We the binding of each cTnI C-terminal deletion mutant to cTnC by of cTnI·cTnC complex on polyacrylamide and size exclusion chromatography. shown in WT cTnI and each C-terminal deletion mutant of cTnI formed a complex with cTnC on polyacrylamide in the of but not in the of We not any differences between WT cTnI and each cTnI mutant in ability to bind to cTnC in the used for the This that the structural interaction between the N terminus of cTnI and the C terminus of cTnC is the to of the cTnI·cTnC We also tested WT cTnI and each cTnI C-terminal deletion mutant formed a complex with cTnC by size exclusion chromatography. through a gel WT cTnI and each cTnI C-terminal deletion mutant formed a complex with cTnC not We also the ability of WT cTnI and the C-terminal deletion mutants of cTnI to bind to cTnI or cTnI deletion and Tm were and the binding of cTnI or the cTnI deletion mutants to F-actin was by in an The and were by WT cTnI and each cTnI C-terminal deletion mutant co-sedimented with F-actin or not This was only a but it that there was binding to F-actin deletion of to 60 residues from the C terminus of The ability of the cTnI C-terminal deletion mutants to bind to that inhibitory activity. We tested inhibitory by WT cTnI or the cTnI C-terminal deletion mutants to myofibrils endogenous cTnI and The cTnI·cTnC complex was extracted by Solaro R.J. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar) of the by and M. I. J. PubMed Scopus Google Scholar), which exogenous bovine cTnT to the troponin complex from the which analysis of and myofibrils, a in the of endogenous cTnI and cTnC, and the of bovine cTnT of myofilament 1 and In we also of myofibrils with WT cTnI or each cTnI C-terminal deletion mutant with cTnC is to cTnC with but in a Solaro R.J. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar), we have used polyacrylamide gel to the and of cTnC We the of C-terminal deletion of cTnI on its ability to inhibit the Mg2+-ATPase activity of myofibrils the of increasing of the of cTnI on the ATPase activity of the In the of cTnI, as the ATPase was of to that in myofibrils at activity WT cTnI this activity to of maximum with the inhibition a at WT The cTnI-(1–199) mutant an inhibitory to WT However, the inhibitory of the cTnI-(1–188) mutant was to 757 of that of WT cTnI and a at a of 1 by the cTnI-(1–151) mutant was about 507 of that of WT cTnI and a at a of 12 not the inhibitory of this mutant of that of WT cTnI not of 23 residues at the C terminus of cTnI results in of inhibitory activity that is of an We tested the ability of cTnC to the inhibitory of both WT cTnI and the cTnI C-terminal deletion The of cTnC to cTnI tested from to and were in the and of of cTnC at a to cTnI in the of Ca2+ the inhibition caused by both WT cTnI and the cTnI-(1–199) The inhibition caused by cTnI-(1–188) was by cTnC in the of Ca2+ with to within of that for WT However, the inhibition of maximum ATPase activity caused by cTnI-(1–151) was not by the of cTnC (in the of at a of We also formed a complex of WT cTnI or cTnI C-terminal deletion mutants with cTnC into myofibrils, and measured the Mg2+-ATPase activity as a function that the Ca2+ sensitivity was restored to that for myofibrils with WT cTnI·cTnC and the with the cTnI-(1–188)·cTnC complex only partially restored Ca2+ sensitivity not and we a in the for the cTnI-(1–151)·cTnC complex is not shown in with this no was results indicate that in cardiac TnI, residues (C-terminal to the inhibitory region) are essential for Ca2+ sensitivity of the Our results the evidence that the C-terminal region of cardiac TnI, from the inhibitory is essential for the Ca2+-dependent regulation of cardiac myofilament The functional of the C-terminal domain of cTnI with evidence of an anti-parallel between cTnI and cTnC in which the C-terminal region of cTnI binds to the N terminus of This an important to to unique of the mechanism by which Ca2+ on cardiac myofilaments. Our a to the relative of the inhibitory region of cTnI to the regulation of In the has been the inhibitory region of An to the of the inhibitory region of cTnI has the to inhibit ATPase J.A. Hodges R.S. J. Biol. Chem. 1981; 256: 2798-2802Abstract Full Text PDF PubMed Google Scholar, Hodges R.S. J. Biol. Chem. Full Text PDF PubMed Google Scholar, Hodges R.S. Biochemistry. Scopus Google Scholar), not to the as cardiac reconstituted with the cTnI inhibitory are able to J.D. Hodges R.S. Lett. PubMed Scopus Google Scholar). The of the inhibitory in skeletal muscle is also by the of mutant the inhibitory to inhibit ATPase M. Potter J.D. J. 1995; Scholar). However, indicate that in to the inhibitory two in cTnI, C-terminal to the inhibitory are essential for the of maximum analysis of the of C-terminal on the ability of cTnI to inhibit ATPase activity we that the inhibitory region only about 507 of the ATPase activity. between residues and residues each to the inhibition of ATPase as shown by with mutants cTnI-(1–188) and of is that there are two binding in the C-terminal domain of (13Farah C.S. Miyamoto C.A. Ramos C.H.I. da Silva A.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) and Hodges R.S. J. Biol. 1997; PubMed Scopus Google Scholar) also the of an binding site on the C-terminal of the inhibitory region in fsTnI (residues The binding site in cTnI, residues fsTnI residues not to function in skeletal muscle. the on evidence that there is of inhibitory activity with of the inhibitory region (residues in fsTnI (13Farah C.S. Miyamoto C.A. Ramos C.H.I. da Silva A.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, M. Potter J.D. J. 1995; Scholar), we also the that binding of the inhibitory region to changes in that which bind to binding in the C terminus of In a model of of thin J. 1994; PubMed Scopus Google Scholar) that cTnI may be in a of the thin by the and that Ca2+ of cTnI from actin. TnI to be in an and at actin to be in a In a model of Ca2+-saturated complex derived from fsTnI an in the of fsTnC and Ca2+ that is about J. Biochemistry. 1994; PubMed Scopus Google Scholar). An actin is to be about in W. PubMed Scopus Google TnI may of actin. C-terminal to the inhibitory region may be for of TnI to a blocked of the thin in the case of cTnI, which has an at the N In to the of residues C-terminal to the inhibitory region of cTnI in of inhibition of ATPase this C-terminal region of cTnI appears to be essential for Ca2+-dependent regulation of cardiac myofilament contraction. We that the the cTnI-(1–188) mutant ability to Ca2+ sensitivity in myofibrils and the cTnI-(1–151) mutant the ability to Ca2+ sensitivity This of Ca2+-dependent of the myofilament deletion of of the C terminus of cTnI an in the interaction of cTnI and cTnC, through of a binding site for cTnC in the C-terminal region of derived largely from studies on fast skeletal indicates of interaction between fsTnI and fsTnC (13Farah C.S. Miyamoto C.A. Ramos C.H.I. da Silva A.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, B.-S. Potter J.D. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. PubMed Scopus Google Scholar). have that fsTnC may bind C-terminal to the inhibitory region of A fsTnI which about residues C-terminal to the inhibitory region binding to fsTnC than the inhibitory Sykes B.D. Smilie L.B. Biochemistry. 1997; PubMed Scopus Google Scholar). Kobayashi J. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar) found that residues of fsTnI to to in the N terminus of Moreover, an N-terminal part of fsTnC not only to the inhibitory but to residues of fsTnI has also been demonstrated that in the of of fsTnI in the of fsTnC C. J. Biol. 1994; Scholar, E. J. Biochemistry. 1989; PubMed Scopus Google Scholar) and from actin PubMed Scopus Google Scholar). This finding the of the C-terminal part of TnI in the In evidence for a cTnC binding site C-terminal to the inhibitory region from the finding that cTnC was not able to the inhibition of ATPase activity by the cTnI-(1–151) mutant in the of Ca2+ The of inhibitory function deletion of either 23 or 60 residues a interaction of cTnI with which the of for reaction with A interaction also the in Ca2+ sensitivity with the cTnI-(1–188) mutant The evidence for a cTnC binding domain from the inhibitory region is on the in Although inhibition of Mg2+-ATPase activity was in myofibrils reconstituted with the cTnI-(1–188) of cTnC and Ca2+ restored the Mg2+-ATPase activity. However, of cTnC and Ca2+ to myofibrils reconstituted with the cTnI-(1–151) mutant not the inhibition of the Mg2+-ATPase activity results indicate the of a Ca2+-dependent cTnC binding domain within residues this region contains both and cTnC binding The of and cTnC binding C-terminal to the inhibitory region to the of thin and may also be an important of the Ca2+ regulation of thin Although has on the role of the C-terminal domain of cTnI in Ca2+ regulation of cardiac it is also important to that cTnI contains an unique N-terminal that by which the for activation of the myofilaments. The mechanism involves of cTnI at and an in the of of the N terminus of cTnC S.P. Johnson J.D. M.J. Potter J.D. Solaro R.J. J. Biol. Chem. 1982; Full Text PDF PubMed Google Scholar). This appears to changes in the cTnI that an interaction between the C-terminal domain of cTnI with cTnC Chandra M. J. M. Solaro R.J. Biochemistry. 1997; PubMed Scopus Google Scholar) have demonstrated this by using to a in between and C-terminal regions of cTnI by A Moreover, Chandra M. B.-S. Solaro R.J. Biochemistry. 1997; (in press)Google Scholar) have demonstrated that of the N terminus of cTnI by A is able to Ca2+ binding to an N-terminal of cTnC at the N terminus of cTnI are by the C terminus of cTnI, which appears able to the interaction of the N-terminal domain of cTnC with In results to the of the mechanism by which cTnI in the Ca2+ switch of the and are important in the of both cardiac and is evidence that in the C-terminal region of the cTnI are in in the M. M. S. N. J.A. M. Nat. 1997; PubMed Scopus Google Scholar). Our results that such in cTnI have on the of cardiac myofilaments by Moreover, the region of cTnI in the cTnI-(1–151) which interacts with the N terminus of cTnC, be important with to of in heart We for in the of the cTnI C-terminal deletion and for with the actin