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 Ca²⁺-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 had 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.