Mutagenesis of cardiac troponin I. Role of the unique NH2-terminal peptide in myofilament activation.
Phosphorylation of Ser residues in the NH2-terminal extension unique to cardiac troponin I (cTnI) is known to occur through protein kinase A and to alter myofilament Ca2+ activation (Robertson, S. P., Johnson, J. D., Holroyde, M. J., Kranias, E. G., Potter, J. D., and Solaro, R. J. (1982) J. Biol. Chem. 257, 260-263). Yet, how the NH2-terminal extension may itself affect thin filament Ca2+ signaling is unknown. To approach this question we have used molecular cloning, mutagenesis, and bacterial synthesis of a full-length cTnI and a truncated mutant (cTnI/NH2) missing the 32 amino acids. Using reconstituted preparations we could show no differences between cTnI and cTnI/NH2 either in inhibition of actomyosin ATPase activity, in Ca(2+)-reversible inhibitory activity, or in the relation between pCa and Ca2+ binding to the regulatory site of cTnC at either pH 7.0 or 6.5. There were also no significant differences at either pH in the pCa-MgATPase activity relation of myofibrils into which the various species of TnI has been exchanged. Our results indicate: 1) that phosphorylation most likely induces a new state of TnI activity rather than altering an intrinsic effect of the NH2-terminal peptide on Ca2+ activation; and 2) that domains outside the NH2-terminal extension are important with regard to differences in effects of acidic pH on Ca2+ activation on cardiac and skeletal myofilaments.
Guo
X
X
Department of Physiology and Biophysics, College of Medicine, University of Illinois, Chicago 60612-7342.
Wattanapermpool
J
J
Palmiter
K A
KA
Murphy
A M
AM
Solaro
R J
RJ
D008841:Actin Cytoskeleton / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009206:Myocardium / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D017384:Sequence Deletion; D013050:Spectrometry, Fluorescence; D014336:Troponin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D019210:Troponin I
D018076:DNA, Complementary; D010446:Peptide Fragments; D014336:Troponin; D019210:Troponin I