Distinct regions of troponin I regulate Ca2+-dependent activation and Ca2+ sensitivity of the acto-S1-TM ATPase activity of the thin filament

Jennifer E. Van Eyk, Lorie T. Thomas, Brian Tripet, Rudolf J. Wiesner, Joyce R. Pearlstone, Chuck S. Farah, Fernando C. Reinach, Robert S. Hodges

Research output: Contribution to journalArticle

Abstract

The regions of troponin I (TnI) responsible for Ca2+-dependent activation and Ca2+ sensitivity of the actin-myosin subfragment 1- tropomyosin ATPase (acto-S1-TM) activity have been determined. A colorimetric ATPase 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 single amino acid substitutions within the inhibitory region (residues 104-115) were assayed to determine their roles on the regulatory function of TnI. TnI-(104-115) is sufficient for achieving maximum inhibition of the acto-S1-TM ATPase activity and its importance was clearly shown by the reduced potency of TnI mutants with single amino acid substitutions within this region. However, the function of the inhibitory region is modulated by other regions of TnI as observed by the poor inhibitory activity of TnI-(1-116) and the increased potency of the inhibitory region by Tni-(96-148). The regulatory complex composed of Tni-(96-148) plus troponin T-troponin C complex (TnTC) displays the same Ca2+ sensitivity (pCa50) as intact troponin (Tn) or TnI plus TnTC while those regulatory complexes composed of TnTC plus either TnI-(104- 115) or TnI-(1-116) had an increase in their pCa50 values. This indicates that the Ca2+ sensitivity or responsiveness of the thin filament is 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-S1 rate was mimicked by the regulatory complex composed of TnI-(l-116) plus TnTC and was not seen with complexes composed with either 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 the ATPase activity. Although the TnI inhibitory region (104-115) is the Ca2+-sensitive switch which changes binding sites from actin-TM to TnC 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.

Original languageEnglish (US)
Pages (from-to)10529-10537
Number of pages9
JournalJournal of Biological Chemistry
Volume272
Issue number16
DOIs
StatePublished - Apr 18 1997
Externally publishedYes

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Myosin Subfragments
Tropomyosin
Troponin I
Adenosine Triphosphatases
Actins
Chemical activation
Troponin
Troponin C
Troponin T
pioglitazone
actin subfragments
Amino Acid Substitution
Substitution reactions
Calcium
Amino Acids

ASJC Scopus subject areas

  • Biochemistry

Cite this

Van Eyk, J. E., Thomas, L. T., Tripet, B., Wiesner, R. J., Pearlstone, J. R., Farah, C. S., ... Hodges, R. S. (1997). Distinct regions of troponin I regulate Ca2+-dependent activation and Ca2+ sensitivity of the acto-S1-TM ATPase activity of the thin filament. Journal of Biological Chemistry, 272(16), 10529-10537. https://doi.org/10.1074/jbc.272.16.10529

Distinct regions of troponin I regulate Ca2+-dependent activation and Ca2+ sensitivity of the acto-S1-TM ATPase activity of the thin filament. / Van Eyk, Jennifer E.; Thomas, Lorie T.; Tripet, Brian; Wiesner, Rudolf J.; Pearlstone, Joyce R.; Farah, Chuck S.; Reinach, Fernando C.; Hodges, Robert S.

In: Journal of Biological Chemistry, Vol. 272, No. 16, 18.04.1997, p. 10529-10537.

Research output: Contribution to journalArticle

Van Eyk, JE, Thomas, LT, Tripet, B, Wiesner, RJ, Pearlstone, JR, Farah, CS, Reinach, FC & Hodges, RS 1997, 'Distinct regions of troponin I regulate Ca2+-dependent activation and Ca2+ sensitivity of the acto-S1-TM ATPase activity of the thin filament', Journal of Biological Chemistry, vol. 272, no. 16, pp. 10529-10537. https://doi.org/10.1074/jbc.272.16.10529
Van Eyk, Jennifer E. ; Thomas, Lorie T. ; Tripet, Brian ; Wiesner, Rudolf J. ; Pearlstone, Joyce R. ; Farah, Chuck S. ; Reinach, Fernando C. ; Hodges, Robert S. / Distinct regions of troponin I regulate Ca2+-dependent activation and Ca2+ sensitivity of the acto-S1-TM ATPase activity of the thin filament. In: Journal of Biological Chemistry. 1997 ; Vol. 272, No. 16. pp. 10529-10537.
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abstract = "The regions of troponin I (TnI) responsible for Ca2+-dependent activation and Ca2+ sensitivity of the actin-myosin subfragment 1- tropomyosin ATPase (acto-S1-TM) activity have been determined. A colorimetric ATPase 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 single amino acid substitutions within the inhibitory region (residues 104-115) were assayed to determine their roles on the regulatory function of TnI. TnI-(104-115) is sufficient for achieving maximum inhibition of the acto-S1-TM ATPase activity and its importance was clearly shown by the reduced potency of TnI mutants with single amino acid substitutions within this region. However, the function of the inhibitory region is modulated by other regions of TnI as observed by the poor inhibitory activity of TnI-(1-116) and the increased potency of the inhibitory region by Tni-(96-148). The regulatory complex composed of Tni-(96-148) plus troponin T-troponin C complex (TnTC) displays the same Ca2+ sensitivity (pCa50) as intact troponin (Tn) or TnI plus TnTC while those regulatory complexes composed of TnTC plus either TnI-(104- 115) or TnI-(1-116) had an increase in their pCa50 values. This indicates that the Ca2+ sensitivity or responsiveness of the thin filament is 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-S1 rate was mimicked by the regulatory complex composed of TnI-(l-116) plus TnTC and was not seen with complexes composed with either 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 the ATPase activity. Although the TnI inhibitory region (104-115) is the Ca2+-sensitive switch which changes binding sites from actin-TM to TnC 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.",
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AU - Wiesner, Rudolf J.

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N2 - The regions of troponin I (TnI) responsible for Ca2+-dependent activation and Ca2+ sensitivity of the actin-myosin subfragment 1- tropomyosin ATPase (acto-S1-TM) activity have been determined. A colorimetric ATPase 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 single amino acid substitutions within the inhibitory region (residues 104-115) were assayed to determine their roles on the regulatory function of TnI. TnI-(104-115) is sufficient for achieving maximum inhibition of the acto-S1-TM ATPase activity and its importance was clearly shown by the reduced potency of TnI mutants with single amino acid substitutions within this region. However, the function of the inhibitory region is modulated by other regions of TnI as observed by the poor inhibitory activity of TnI-(1-116) and the increased potency of the inhibitory region by Tni-(96-148). The regulatory complex composed of Tni-(96-148) plus troponin T-troponin C complex (TnTC) displays the same Ca2+ sensitivity (pCa50) as intact troponin (Tn) or TnI plus TnTC while those regulatory complexes composed of TnTC plus either TnI-(104- 115) or TnI-(1-116) had an increase in their pCa50 values. This indicates that the Ca2+ sensitivity or responsiveness of the thin filament is 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-S1 rate was mimicked by the regulatory complex composed of TnI-(l-116) plus TnTC and was not seen with complexes composed with either 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 the ATPase activity. Although the TnI inhibitory region (104-115) is the Ca2+-sensitive switch which changes binding sites from actin-TM to TnC 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.

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