DISTINCT REGIONS OF TROPONIN-I REGULATE CA2-DEPENDENT ACTIVATION AND CA2+ SENSITIVITY OF THE ACTO-S1-TM ATPASE ACTIVITY OF THE THIN FILAMENT()

The regions of troponin I (TnI) responsible for Ca2+-dependent activat ion and Ca2+ sensitivity of the actin-myosin subfragment 1-tropomyosin ATPase (acto-S1-TM) activity have been determined. A colorimetric ATP ase assay at pH 7.8 has been applied to reconstituted skeletal muscle thin filaments at actin:S1:TM ratios of 6:1:2. Several TnI fragments ( TnI-(104-115), TnI-(1-116), and TnI-(96-148)) and TnI mutants with sin gle amino acid substitutions within the inhibitory region (residues 10 4-115) were assayed to determine their roles on the regulatory functio n of TnI. TnI-(104-115) is sufficient for achieving maximum inhibition of the acto-S1-TM ATPase activity and its importance was clearly show n by the reduced potency of TnI mutants with single amino acid substit utions within this region. However, the function of the inhibitory reg ion is modulated by other regions of TnI as observed by the poor inhib itory activity of TnI-(1-116) and the increased potency of the inhibit ory region by TnI-(96-148). The regulatory complex composed of TnI-(96 -148) plus troponin T-troponin C complex (TnT.C) displays the same Ca2 + sensitivity (pCa(50)) as intact troponin (Tn) or TnI plus TnT.C whil e those regulatory complexes composed of TnT.C plus either TnI-(104-11 5) or TnI-(1-116) had an increase in their pCa(50) values. This indica tes that the Ca2+ sensitivity or responsiveness of the thin filament i s controlled by TnI residues 96-148. The ability of Tn to activate the acto-S1-TM ATPase activity in the presence of calcium to the level of the acto-sl rate was mimicked by the regulatory complex composed of T nI-(1-116) plus TnT.C and was not seen with complexes composed with ei ther TnI-(104-115) or TnI-(96-148). This indicates that the N terminus of TnI in conjunction with TnT controls the degree of activation of t he ATPase activity. Although the TnI inhibitory region (104-115) is th e Ca2+-sensitive switch which changes binding sites from actin-TM to T nC in the presence of calcium, its function is modulated by both the C -terminal and N-terminal regions of TnI. Thus, distinct regions of TnI control different aspects of Tn's biological function.