Key result
The R205A mutation in cardiac troponin T drastically attenuated Ca2+ activation of the thin filament, reducing S1-ATPase activity to almost half of the wild type.
Why the study?
Does the R205A mutation in cardiac troponin T alter Ca2+ activation and muscle contraction in vitro?
Does the R205A mutation in cardiac troponin T alter Ca2+ activation and muscle contraction in vitro?
Absolute Event Rate: 0.27% vs 0.55%
p-value: p=<0.01
The R205A mutation in cardiac troponin T drastically attenuates Ca2+ activation of the thin filament by altering cooperativity, providing mechanistic insight into dilated cardiomyopathy-linked mutations.
May link troponin T mutations to DCM pathogenesis; leaves open in vivo and clinical translation.
The striated muscle thin filament comprises actin, tropomyosin, and troponin. The Tn complex consists of three subunits, troponin C (TnC), troponin I (TnI), and troponin T (TnT). TnT may serve as a bridge between the Ca2+ sensor (TnC) and the actin filament. In the short helix preceding the IT-arm region, H1(T2), there are known dilated cardiomyopathy-linked mutations (among them R205L). Thus we hypothesized that there is an element in this short helix that plays an important role in regulating the muscle contraction, especially in Ca2+ activation. We mutated Arg-205 and several other amino acid residues within and near the H1(T2) helix. Utilizing an alanine replacement method to compare the effects of the mutations, the biochemical and mechanical impact on the actomyosin interaction was assessed by solution ATPase activity assay, an in vitro motility assay, and Ca2+ binding measurements. Ca2+ activation was markedly impaired by a point mutation of the highly conserved basic residue R205A, residing in the short helix H1(T2) of cTnT, whereas the mutations to nearby residues exhibited little effect on function. Interestingly, rigor activation was unchanged between the wild type and R205A TnT. In addition to the reduction in Ca2+ sensitivity observed in Ca2+ binding to the thin filament, myosin S1-ADP binding to the thin filament was significantly affected by the same mutation, which was also supported by a series of S1 concentration-dependent ATPase assays. These suggest that the R205A mutation alters function through reduction in the nature of cooperative binding of S1. Background: Striated muscle contraction is regulated by Ca2+ binding to troponin. Results: A mutation at position 205 of cardiac troponin T drastically attenuates the Ca2+ activation of the thin filament by altering its properties. Conclusion: The main effect of this mutation is on the cooperativity in the thin filament activation. Significance: Our study sheds light on how mutations in troponin T affect the thin filament regulation.
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Kobayashi et al. (2013) studied this question. R205A mutation in cardiac troponin T vs. Wild-type cardiac troponin T was evaluated on S1-ATPase activity at 1 µM Tn in the presence of Ca2+ (s-1) (p=<0.01). The R205A mutation in cardiac troponin T drastically attenuated Ca2+ activation of the thin filament, reducing S1-ATPase activity to almost half of the wild type.
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