Effects of phosphorylation and mutation R145G on human cardiac troponin I function

  • Deng Y
  • Schmidtmann A
  • Redlich A
 et al. 
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We have studied functional consequences of the mutations R145G, S22A, and S23A of human cardiac troponin I (cTnI) and of phosphorylation of two adjacent N-terminal serine residues in the wild-type cTnI and the mutated proteins. The mutation R145G has been linked to the development of familial hypertrophic cardiomyopathy. Cardiac troponin was reconstituted from recombinant human subunits including either wild-type or mutant cTnI and was used for reconstitution of thin filaments with skeletal muscle actin and tropomyosin. The Ca(2+)-dependent thin filament-activated myosin subfragment 1 ATPase (actoS1-ATPase) activity and the in vitro motility of these filaments driven by myosin were measured as a function of the cTnI phosphorylation state. Bisphosphorylation of wild-type cTnI decreases the Ca(2+) sensitivity of the actoS1-ATPase activity and the in vitro thin filament motility by about 0.15-0.21 pCa unit. The nonconservative replacement R145G in cTnI enhances the Ca(2+) sensitivity of the actoS1-ATPase activity by about 0.6 pCa unit independent of the phosphorylation state of cTnI. Furthermore, it mimics a strong suppressing effect on both the maximum actoS1-ATPase activity and the maximum in vitro filament sliding velocity which has been observed upon bisphosphorylation of wild-type cTnI. Bisphosphorylation of the mutant cTnI-R145G itself had no such suppressing effects anymore. Differential analysis of the effect of phosphorylation of each of the two serines, Ser23 in cTnI-S22A and Ser22 in cTnI-S23A, indicates that phosphorylation of Ser23 may already be sufficient for causing the reduction of maximum actoS1-ATPase activity and thin filament sliding velocity seen upon phosphorylation of both of these serines.

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