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Phosphorylation of both serine residues in cardiac troponin I is required to decrease the Ca2+ affinity of cardiac
1Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, Florida 33101, USA.
Abstract:
The phosphorylation of cardiac muscle troponin I (CTnI) at two adjacent N-terminal serine residues by cAMP-dependent protein kinase (PKA) has been implicated in the inotropic response of the heart to beta-agonists. Phosphorylation of these residues has been shown to reduce the Ca2+ affinity of the single Ca(2+)-specific regulatory site of cardiac troponin C (CTnC) and to increase the rate of Ca2+ dissociation from this site (Robertson, S. P., Johnson, J. D., Holroyde, M. J., Kranias, E. G., Potter, J. D., and Solaro, R. J. (1982) J. Biol. Chem. 257, 260-263). Recent studies (Zhang, R., Zhao, J., and Potter, J. D. (1995) Circ. Res. 76, 1028-1035) have correlated this increase in Ca2+ dissociation with a reduced Ca2+ sensitivity of force development and a faster rate of cardiac muscle relaxation in a PKA phosphorylated skinned cardiac muscle preparation. To further determine the role of the two PKA phosphorylation sites in mouse CTnI (serine 22 and 23), serine 22 or 23, or both were mutated to alanine. The wild type and the mutated CTnIs were expressed in Escherichia coli and purified. Using these mutants, it was found that serine 23 was phosphorylated more rapidly than serine 22 and that both serines are required to be phosphorylated in order to observe the characteristic reduction in the Ca2+ sensitivity of force development seen in a skinned cardiac muscle preparation. The latter result confirms that PKA phosphorylation of CTnI, and not other proteins, is responsible for this change in Ca2+ sensitivity. The results also suggest that one of the serines (23) may be constitutively phosphorylated and that serine 22 may be functionally more important.
Insights
Phosphorylation of cardiac troponin I by PKA reduces heart muscle’s calcium sensitivity. Both serine 22 and 23 sites are crucial for this effect, with serine 23 potentially being constitutively phosphorylated.
Area of Science:
- Cardiology
- Molecular Biology
- Biochemistry
Background:
- cAMP-dependent protein kinase (PKA) phosphorylation of cardiac troponin I (CTnI) influences cardiac muscle contractility.
- This phosphorylation affects calcium (Ca2+) binding to cardiac troponin C (CTnC), impacting heart muscle function.
Purpose of the Study:
- To investigate the specific roles of serine 22 and serine 23 in mouse CTnI phosphorylation by PKA.
- To determine the contribution of these phosphorylation sites to altered Ca2+ sensitivity and muscle relaxation.
Main Methods:
- Site-directed mutagenesis of serine 22 and/or 23 to alanine in mouse CTnI.
- Expression and purification of wild-type and mutant CTnI proteins in E. coli.
- Assessment of Ca2+ sensitivity and force development in skinned cardiac muscle preparations.
Main Results:
- Serine 23 phosphorylation occurred faster than serine 22.
- Phosphorylation of both serine 22 and 23 was necessary to achieve the characteristic reduction in Ca2+ sensitivity.
- PKA phosphorylation of CTnI, not other proteins, mediated the observed changes in Ca2+ sensitivity.
Conclusions:
- Both serine 22 and 23 phosphorylation sites on CTnI are essential for the PKA-mediated decrease in cardiac muscle Ca2+ sensitivity.
- Serine 23 may be constitutively phosphorylated, while serine 22 appears functionally more significant.
- CTnI phosphorylation by PKA plays a key role in regulating cardiac muscle relaxation and inotropic responses.