Abstract |
This spectroscopic study examined the steady-state and kinetic parameters governing the cross-bridge effect on the increased Ca(2+) affinity of hypertrophic cardiomyopathy-cardiac troponin C (HCM-cTnC) mutants. Previously, we found that incorporation of the A8V and D145E HCM-cTnC mutants, but not E134D into thin filaments (TFs), increased the apparent Ca(2+) affinity relative to TFs containing the WT protein. Here, we show that the addition of myosin subfragment 1 (S1) to TFs reconstituted with these mutants in the absence of MgATP(2-), the condition conducive to rigor cross-bridge formation, further increased the apparent Ca(2+) affinity. Stopped-flow fluorescence techniques were used to determine the kinetics of Ca(2+) dissociation (k(off)) from the cTnC mutants in the presence of TFs and S1. At a high level of complexity (i.e. TF + S1), an increase in the Ca(2+) affinity and decrease in k(off) was achieved for the A8V and D145E mutants when compared with WT. Therefore, it appears that the cTnC Ca(2+) off-rate is most likely to be affected rather than the Ca(2+) on rate. At all levels of TF complexity, the results obtained with the E134D mutant reproduced those seen with the WT protein. We conclude that strong cross-bridges potentiate the Ca(2+)-sensitizing effect of HCM-cTnC mutants on the myofilament. Finally, the slower k(off) from the A8V and D145E mutants can be directly correlated with the diastolic dysfunction seen in these patients.
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Authors | Jose Renato Pinto, Daniel P Reynaldo, Michelle S Parvatiyar, David Dweck, Jingsheng Liang, Michelle A Jones, Martha M Sorenson, James D Potter |
Journal | The Journal of biological chemistry
(J Biol Chem)
Vol. 286
Issue 2
Pg. 1005-13
(Jan 14 2011)
ISSN: 1083-351X [Electronic] United States |
PMID | 21056975
(Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)
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Chemical References |
- Cross-Linking Reagents
- Myosin Subfragments
- Troponin C
- Calcium
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Topics |
- Actin Cytoskeleton
(physiology)
- Animals
- Calcium
(metabolism)
- Cardiomyopathy, Hypertrophic
(genetics, metabolism, physiopathology)
- Cross-Linking Reagents
(chemistry, metabolism)
- Humans
- Kinetics
- Mutagenesis
- Myocardial Contraction
(physiology)
- Myocytes, Cardiac
(cytology, physiology)
- Myosin Subfragments
(metabolism)
- Protein Conformation
- Rabbits
- Swine
- Troponin C
(chemistry, genetics, metabolism)
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