Mutagenesis of cardiac troponin I. Role of the unique NH2-terminal peptide in myofilament activation

• Published in: The Journal of biological chemistry Vol. 269; no. 21; pp. 15210 - 15216
• Main Authors: XIAODU GUO, WATTANAPERMPOOL, J, PALMITER, K. A, MURPHY, A. M, SOLARO, R. J
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 27.05.1994
• Subjects: Actin Cytoskeleton - metabolism; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; Base Sequence; Biological and medical sciences; Cloning, Molecular; Contractile proteins; DNA, Complementary; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Holoproteins; Hydrogen-Ion Concentration; Mice; Molecular Sequence Data; Mutagenesis; Myocardium - metabolism; Peptide Fragments - metabolism; Phosphorylation; Proteins; Sequence Deletion; Spectrometry, Fluorescence; Troponin - chemistry; Troponin - genetics; Troponin - metabolism; Troponin I; Heart; Filament; Rodentia; Contractile protein; Primary structure; Activation; N terminal peptide; Structure function relationship; Vertebrata; Troponin; Mammalia; Mouse; Molecular cloning
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(17)36593-6

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.