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 75% and 50% 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.
Rarick
H M
HM
Department of Physiology and Biophysics, College of Medicine, University of Illinois at Chicago, Chicago, Illinois 60612-7342, USA.
Tu
X H
XH
Solaro
R J
RJ
Martin
A F
AF
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002461:Cell Line, Transformed; D003001:Cloning, Molecular; D004926:Escherichia coli; D006801:Humans; D007700:Kinetics; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000378:metabolism* / Q000648:ultrastructure; D009210:Myofibrils / Q000187:drug effects / Q000502:physiology*; D016133:Polymerase Chain Reaction; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D019210:Troponin I / Q000737:chemistry* / Q000378:metabolism*
D011994:Recombinant Proteins; D019210:Troponin I; D002118:Calcium