Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1990 Dec;87(24):9538–9542. doi: 10.1073/pnas.87.24.9538

The low-affinity Ca2(+)-binding sites in cardiac/slow skeletal muscle troponin C perform distinct functions: site I alone cannot trigger contraction.

H L Sweeney 1, R M Brito 1, P R Rosevear 1, J A Putkey 1
PMCID: PMC55207  PMID: 2263608

Abstract

Both troponin C (TnC) and calmodulin share a remarkably similar tertiary motif that may be common to other Ca2(+)-binding proteins with activator activity. TnC plays a critical role in regulating muscle contraction and is particularly well-suited for structural analysis by site-directed mutation. Fast-twitch skeletal muscle TnC has two low-affinity Ca2(+)-binding sites (sites I and II), while in cardiac and slow-twitch skeletal muscle TnC site I is inactive. Recently, using protein engineering, we directly demonstrated that binding of Ca2+ to the low-affinity site(s) initiates muscle contraction. In the present study, we use mutagenesis to determine whether either of the low-affinity sites in cardiac TnC can trigger contraction in slow-twitch skeletal muscle fibers. In one Ca2(+)-binding mutant, Ca2(+)-binding to the dormant low-affinity site I was restored (CBM+I). In a second mutant, site I was activated while site II was inactivated (CBM+I-IIA). Both proteins had the predicted CA2(+)-binding characteristics, and both were able to associate with troponin I and troponin T to form a troponin complex and integrate into permeabilized slow-twitch skeletal muscle fibers. A comparison of NMR spectra shows the aromatic regions in the two proteins to be qualitatively similar without divalent cations but markedly different with Ca2+. Mutant CBM+I supported force generation in skinned slow skeletal muscle fibers but had Sr2+ and Ca2+ sensitivities similar to fast skeletal TnC. Mutant CBM+I-IIA was unable to restore Ca2(+)-dependent contraction to TnC-depleted skinned slow muscle fibers. The data directly demonstrate that low-affinity sites I and II have distinct functions and that only site II in cardiac TnC can trigger muscle contraction in slow-twitch skeletal muscle fibers. This principle of distinct, modular activities for Ca2(+)-binding sites in the same protein may apply to other members of the TnC/calmodulin family.

Full text

PDF
9539

Images in this article

9539Image
on p.9539
9539Image
on p.9539
9541Image
on p.9541

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Babu A., Scordilis S. P., Sonnenblick E. H., Gulati J. The control of myocardial contraction with skeletal fast muscle troponin C. J Biol Chem. 1987 Apr 25;262(12):5815–5822. [PubMed] [Google Scholar]
  2. Babu Y. S., Sack J. S., Greenhough T. J., Bugg C. E., Means A. R., Cook W. J. Three-dimensional structure of calmodulin. Nature. 1985 May 2;315(6014):37–40. doi: 10.1038/315037a0. [DOI] [PubMed] [Google Scholar]
  3. Chabbert M., Kilhoffer M. C., Watterson D. M., Haiech J., Lami H. Time-resolved fluorescence study of VU-9 calmodulin, an engineered calmodulin possessing a single tryptophan residue. Biochemistry. 1989 Jul 11;28(14):6093–6098. doi: 10.1021/bi00440a054. [DOI] [PubMed] [Google Scholar]
  4. Cox J. A., Comte M., Stein E. A. Calmodulin-free skeletal-muscle troponin C prepared in the absence of urea. Biochem J. 1981 Apr 1;195(1):205–211. doi: 10.1042/bj1950205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Craig T. A., Watterson D. M., Prendergast F. G., Haiech J., Roberts D. M. Site-specific mutagenesis of the alpha-helices of calmodulin. Effects of altering a charge cluster in the helix that links the two halves of calmodulin. J Biol Chem. 1987 Mar 5;262(7):3278–3284. [PubMed] [Google Scholar]
  6. Eastwood A. B., Wood D. S., Bock K. L., Sorenson M. M. Chemically skinned mammalian skeletal muscle. I. The structure of skinned rabbit psoas. Tissue Cell. 1979;11(3):553–566. doi: 10.1016/0040-8166(79)90062-4. [DOI] [PubMed] [Google Scholar]
  7. Giulian G. G., Moss R. L., Greaser M. Improved methodology for analysis and quantitation of proteins on one-dimensional silver-stained slab gels. Anal Biochem. 1983 Mar;129(2):277–287. doi: 10.1016/0003-2697(83)90551-1. [DOI] [PubMed] [Google Scholar]
  8. Herzberg O., James M. N. Structure of the calcium regulatory muscle protein troponin-C at 2.8 A resolution. Nature. 1985 Feb 21;313(6004):653–659. doi: 10.1038/313653a0. [DOI] [PubMed] [Google Scholar]
  9. Hoar P. E., Potter J. D., Kerrick W. G. Skinned ventricular fibres: troponin C extraction is species-dependent and its replacement with skeletal troponin C changes Sr2+ activation properties. J Muscle Res Cell Motil. 1988 Apr;9(2):165–173. doi: 10.1007/BF01773738. [DOI] [PubMed] [Google Scholar]
  10. Johnson J. D., Charlton S. C., Potter J. D. A fluorescence stopped flow analysis of Ca2+ exchange with troponin C. J Biol Chem. 1979 May 10;254(9):3497–3502. [PubMed] [Google Scholar]
  11. Kerrick W. G., Zot H. G., Hoar P. E., Potter J. D. Evidence that the Sr2+ activation properties of cardiac troponin C are altered when substituted into skinned skeletal muscle fibers. J Biol Chem. 1985 Dec 15;260(29):15687–15693. [PubMed] [Google Scholar]
  12. Kretsinger R. H., Nockolds C. E. Carp muscle calcium-binding protein. II. Structure determination and general description. J Biol Chem. 1973 May 10;248(9):3313–3326. [PubMed] [Google Scholar]
  13. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  14. Morimoto S., Ohtsuki I. Effect of substitution of troponin C in cardiac myofibrils with skeletal troponin C or calmodulin on the Ca2+- and Sr2+-sensitive ATPase activity. J Biochem. 1988 Jul;104(1):149–154. doi: 10.1093/oxfordjournals.jbchem.a122412. [DOI] [PubMed] [Google Scholar]
  15. Moss R. L., Lauer M. R., Giulian G. G., Greaser M. L. Altered Ca2+ dependence of tension development in skinned skeletal muscle fibers following modification of troponin by partial substitution with cardiac troponin C. J Biol Chem. 1986 May 5;261(13):6096–6099. [PubMed] [Google Scholar]
  16. Persechini A., Kretsinger R. H. The central helix of calmodulin functions as a flexible tether. J Biol Chem. 1988 Sep 5;263(25):12175–12178. [PubMed] [Google Scholar]
  17. Persechini A., Moncrief N. D., Kretsinger R. H. The EF-hand family of calcium-modulated proteins. Trends Neurosci. 1989 Nov;12(11):462–467. doi: 10.1016/0166-2236(89)90097-0. [DOI] [PubMed] [Google Scholar]
  18. Potter J. D., Gergely J. The calcium and magnesium binding sites on troponin and their role in the regulation of myofibrillar adenosine triphosphatase. J Biol Chem. 1975 Jun 25;250(12):4628–4633. [PubMed] [Google Scholar]
  19. Putkey J. A., Draetta G. F., Slaughter G. R., Klee C. B., Cohen P., Stull J. T., Means A. R. Genetically engineered calmodulins differentially activate target enzymes. J Biol Chem. 1986 Jul 25;261(21):9896–9903. [PubMed] [Google Scholar]
  20. Putkey J. A., Ono T., VanBerkum M. F., Means A. R. Functional significance of the central helix in calmodulin. J Biol Chem. 1988 Aug 15;263(23):11242–11249. [PubMed] [Google Scholar]
  21. Putkey J. A., Slaughter G. R., Means A. R. Bacterial expression and characterization of proteins derived from the chicken calmodulin cDNA and a calmodulin processed gene. J Biol Chem. 1985 Apr 25;260(8):4704–4712. [PubMed] [Google Scholar]
  22. Putkey J. A., Sweeney H. L., Campbell S. T. Site-directed mutation of the trigger calcium-binding sites in cardiac troponin C. J Biol Chem. 1989 Jul 25;264(21):12370–12378. [PubMed] [Google Scholar]
  23. Reinach F. C., Karlsson R. Cloning, expression, and site-directed mutagenesis of chicken skeletal muscle troponin C. J Biol Chem. 1988 Feb 15;263(5):2371–2376. [PubMed] [Google Scholar]
  24. Robertson S. P., Johnson J. D., Potter J. D. The time-course of Ca2+ exchange with calmodulin, troponin, parvalbumin, and myosin in response to transient increases in Ca2+. Biophys J. 1981 Jun;34(3):559–569. doi: 10.1016/S0006-3495(81)84868-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Xu G. Q., Hitchcock-DeGregori S. E. Synthesis of a troponin C cDNA and expression of wild-type and mutant proteins in Escherichia coli. J Biol Chem. 1988 Sep 25;263(27):13962–13969. [PubMed] [Google Scholar]
  26. Zot H. G., Potter J. D. A structural role for the Ca2+-Mg2+ sites on troponin C in the regulation of muscle contraction. Preparation and properties of troponin C depleted myofibrils. J Biol Chem. 1982 Jul 10;257(13):7678–7683. [PubMed] [Google Scholar]
  27. van Eerd J. P., Takahashi K. The amino acid sequence of bovine cardiac tamponin-C. Comparison with rabbit skeletal troponin-C. Biochem Biophys Res Commun. 1975 May 5;64(1):122–127. doi: 10.1016/0006-291x(75)90227-2. [DOI] [PubMed] [Google Scholar]
  28. van Eerd J. P., Takahshi K. Determination of the complete amino acid sequence of bovine cardiac troponin C. Biochemistry. 1976 Mar 9;15(5):1171–1180. doi: 10.1021/bi00650a033. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES