JBC, Vol. 250, Issue 12, 4628-4633, Jun, 1975

The calcium and magnesium binding sites on troponin and their role in the regulation of myofibrillar adenosine triphosphatase

J. D. Potter and J. Gergely

Purified troponin (Tn), the complex of the Ca-2+ binding subunit (TnC), the inhibitory subunit (TnI), and the tropomyosin binding subunit (TnT) binds 4 mol of Ca-2+ per mol. Two sites bind Ca-2+ with a binding constant of 5 times 10-8 M- minus 1, and two with a binding constant of 5 times 10-6 M- minus 1. In the presence of 2 mM MgCl2 the binding to four sites can be characterized with a single affinity constant of 5 times 10-6 M- minus 1. Purified TnC also binds 4 mol of Ca-2+ per mol; two sites have a binding constant of 2 times 10-7 M- minus 1 and two have one of 2 times 10-5 M- minus 1. In the presence of 2 mM MgCl2 the binding constant of the sites of higher affinity is reduced to 2 times 10-6 M- minus 1, while Ca-2+ binding to the sites of lower affinity is unaffected. Assuming competition between Mg-2+ and Ca-2+ for the high affinity sites on TnC and Tn, the changes in Ca-2+ binding can be accounted for with KMg values of 5 times 10-3 M- minus 1 and 5 times 10-4 M- minus 1, respectively. Tn and TnC bind 4 mol of Mg-2+ per mol in the absence of Cs-2+. The fact that at [Ca-2+] similar to 10- minus 5 M four Ca-2+ and only two Mg-2+ are bound per mol of TnC in the presence of 2 mM Mg-2+ further supports the view that there is direct competition between Mg-2+ and Ca-2+ for the high affinity Ca-2+ binding sites on TnC and Tn. These results then suggest that Tn and TnC contain six divalent cation binding sites: two high affinity Ca-2+ binding sites that also bind Mg-2+ competitively (Ca-2+-Mg-2+ sites); two sites with lower affinity for Ca-2+ that do not bind Mg-2+ (Ca-2+-specific sites); and two sites that bind Mg-2+ but not Ca-2+ (Mg-2+-specific sites). The complex of TnC and TnI (1:1 molar ratio) has the same binding properties as Tn, suggesting a conformational change in TnC upon interaction with TnI. Studies on myofibrillar ATPase activity as a function of free Ca-2+ concentration at two different free Mg-2+ concentrations suggest that full activation by Ca-2+ occurs only upon binding of Ca-2+ to the two Ca-2+-specific binding sites in Tn but does not require binding of Ca-2+ to the Ca-2+-Mg-2+ sites.
CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				L. H. Tay, O. Griesbeck, and D. T. Yue Live-Cell Transforms between Ca2+ Transients and FRET Responses for a Troponin-C-Based Ca2+ Sensor Biophys. J., December 1, 2007; 93(11): 4031 - 4040. [Abstract] [Full Text] [PDF]

				Y.-B. Sun, B. Brandmeier, and M. Irving Structural changes in troponin in response to Ca2+ and myosin binding to thin filaments during activation of skeletal muscle PNAS, November 21, 2006; 103(47): 17771 - 17776. [Abstract] [Full Text] [PDF]

				M. Mank, D. F. Reiff, N. Heim, M. W. Friedrich, A. Borst, and O. Griesbeck A FRET-Based Calcium Biosensor with Fast Signal Kinetics and High Fluorescence Change Biophys. J., March 1, 2006; 90(5): 1790 - 1796. [Abstract] [Full Text] [PDF]

				S. A. Niederer, P. J. Hunter, and N. P. Smith A Quantitative Analysis of Cardiac Myocyte Relaxation: A Simulation Study Biophys. J., March 1, 2006; 90(5): 1697 - 1722. [Abstract] [Full Text] [PDF]

				W. Ohashi, S. Inouye, T. Yamazaki, and H. Hirota NMR Analysis of the Mg2+-Binding Properties of Aequorin, a Ca2+-Binding Photoprotein J. Biochem., November 1, 2005; 138(5): 613 - 620. [Abstract] [Full Text] [PDF]

				T. M. A. Blumenschein, D. B. Stone, R. J. Fletterick, R. A. Mendelson, and B. D. Sykes Calcium-dependent Changes in the Flexibility of the Regulatory Domain of Troponin C in the Troponin Complex J. Biol. Chem., June 10, 2005; 280(23): 21924 - 21932. [Abstract] [Full Text] [PDF]

				K. K. REDDY, F. M. OITOMEN, G. P. PATEL, and J. BAG Perinuclear localization of slow troponin C m RNA in muscle cells is controlled by a cis-element located at its 3' untranslated region RNA, March 1, 2005; 11(3): 294 - 307. [Abstract] [Full Text] [PDF]

				A. V. Gomes, G. Venkatraman, J. P. Davis, S. B. Tikunova, P. Engel, R. J. Solaro, and J. D. Potter Cardiac Troponin T Isoforms Affect the Ca2+ Sensitivity of Force Development in the Presence of Slow Skeletal Troponin I: INSIGHTS INTO THE ROLE OF TROPONIN T ISOFORMS IN THE FETAL HEART J. Biol. Chem., November 26, 2004; 279(48): 49579 - 49587. [Abstract] [Full Text] [PDF]

				S. B. Tikunova and J. P. Davis Designing Calcium-sensitizing Mutations in the Regulatory Domain of Cardiac Troponin C J. Biol. Chem., August 20, 2004; 279(34): 35341 - 35352. [Abstract] [Full Text] [PDF]

				J. P. Davis, J. A Rall, C. Alionte, and S. B. Tikunova Mutations of Hydrophobic Residues in the N-terminal Domain of Troponin C Affect Calcium Binding and Exchange with the Troponin C-Troponin I96-148 Complex and Muscle Force Production J. Biol. Chem., April 23, 2004; 279(17): 17348 - 17360. [Abstract] [Full Text] [PDF]

				G. L. Gay, D. A. Lindhout, and B. D. Sykes Using lanthanide ions to align troponin complexes in solution: Order of lanthanide occupancy in cardiac troponin C Protein Sci., March 1, 2004; 13(3): 640 - 651. [Abstract] [Full Text] [PDF]

				D. A. Smith and M. A. Geeves Cooperative Regulation of Myosin-Actin Interactions by a Continuous Flexible Chain II: Actin-Tropomyosin-Troponin and Regulation by Calcium Biophys. J., May 1, 2003; 84(5): 3168 - 3180. [Abstract] [Full Text] [PDF]

				F. Fortes de Valencia, A. A. Paulucci, R. B. Quaggio, A. C. Rasera da Silva, C. S. Farah, and F. de Castro Reinach Parallel Measurement of Ca2+ Binding and Fluorescence Emission upon Ca2+ Titration of Recombinant Skeletal Muscle Troponin C. MEASUREMENT OF SEQUENTIAL CALCIUM BINDING TO THE REGULATORY SITES J. Biol. Chem., March 21, 2003; 278(13): 11007 - 11014. [Abstract] [Full Text] [PDF]

				C. Sheldahl, J. Xing, W.-J. Dong, S. C. Harvey, and H. C. Cheung The Calcium-Saturated cTnI/cTnC Complex: Structure of the Inhibitory Region of cTnI Biophys. J., February 1, 2003; 84(2): 1057 - 1064. [Abstract] [Full Text] [PDF]

				J. P. Davis, J. A. Rall, P. J. Reiser, L. B. Smillie, and S. B. Tikunova Engineering Competitive Magnesium Binding into the First EF-hand of Skeletal Troponin C J. Biol. Chem., December 13, 2002; 277(51): 49716 - 49726. [Abstract] [Full Text] [PDF]

				P. Kischel, B. Bastide, L. Stevens, and Y. Mounier Expression and functional behavior of troponin C in soleus muscle fibers of rat after hindlimb unloading J Appl Physiol, March 1, 2001; 90(3): 1095 - 1101. [Abstract] [Full Text] [PDF]

				K. Allen, Y. Y. Xu, and W. G. L. Kerrick Ca2+ measurements in skinned cardiac fibers: effects of Mg2+ on Ca2+ activation of force and fiber ATPase J Appl Physiol, January 1, 2000; 88(1): 180 - 185. [Abstract] [Full Text] [PDF]

				P. C. Geiger, M. J. Cody, and G. C. Sieck Force-calcium relationship depends on myosin heavy chain and troponin isoforms in rat diaphragm muscle fibers J Appl Physiol, November 1, 1999; 87(5): 1894 - 1900. [Abstract] [Full Text] [PDF]

				E. Yang and H. Schulman Structural Examination of Autoregulation of Multifunctional Calcium/Calmodulin-dependent Protein Kinase II J. Biol. Chem., September 10, 1999; 274(37): 26199 - 26208. [Abstract] [Full Text] [PDF]

				C. H. I. Ramos Mapping Subdomains in the C-terminal Region of Troponin I Involved in Its Binding to Troponin C and to Thin Filament J. Biol. Chem., June 25, 1999; 274(26): 18189 - 18195. [Abstract] [Full Text] [PDF]

				K. McArdle, T. StC. Allen, and E. A. Bucher Ca2+-dependent Muscle Dysfunction Caused by Mutation of the Caenorhabditis elegans Troponin T-1 Gene J. Cell Biol., November 30, 1998; 143(5): 1201 - 1213. [Abstract] [Full Text] [PDF]

				J. R. Patel, K. S. McDonald, M. R. Wolff, and R. L. Moss Ca2+ Binding to Troponin C in Skinned Skeletal Muscle Fibers Assessed with Caged Ca2+ and a Ca2+ Fluorophore. INVARIANCE OF Ca2+ BINDING AS A FUNCTION OF SARCOMERE LENGTH J. Biol. Chem., February 28, 1997; 272(9): 6018 - 6027. [Abstract] [Full Text] [PDF]

				D. Szczesna, G. Guzman, T. Miller, J. Zhao, K. Farokhi, H. Ellemberger, and J. D. Potter The Role of the Four Ca[IMAGE] Binding Sites of Troponin C in the Regulation of Skeletal Muscle Contraction J. Biol. Chem., April 5, 1996; 271(14): 8381 - 8386. [Abstract] [Full Text] [PDF]

				W.-j. Dong, S. S. Rosenfeld, C.-K. Wang, A. M. Gordon, and H. C. Cheung Kinetic Studies of Calcium Binding to the Regulatory Site of Troponin C from Cardiac Muscle J. Biol. Chem., January 12, 1996; 271(2): 688 - 694. [Abstract] [Full Text] [PDF]

				B.-S. Pan and R. G. Johnson Jr. Interaction of Cardiotonic Thiadiazinone Derivatives with Cardiac Troponin C J. Biol. Chem., January 12, 1996; 271(2): 817 - 823. [Abstract] [Full Text] [PDF]

				J.-M. Francois, Z. Sheng, D. Szczesna, and J. D. Potter The Functional Role of the Domains of Troponin-C Investigated with Thrombin Fragments of Troponin-C Reconstituted into Skinned Muscle Fibers J. Biol. Chem., August 18, 1995; 270(33): 19287 - 19293. [Abstract] [Full Text] [PDF]

				J. K. Krueger, R. C. Padre, and J. T. Stull Intrasteric Regulation of Myosin Light Chain Kinase J. Biol. Chem., July 14, 1995; 270(28): 16848 - 16853. [Abstract] [Full Text] [PDF]

				X. Zhao, T. Kobayashi, H. Malak, I. Gryczynski, J. Lakowicz, R. Wade, and J. H. Collins Calcium-induced Troponin Flexibility Revealed by Distance Distribution Measurements between Engineered Sites J. Biol. Chem., June 30, 1995; 270(26): 15507 - 15514. [Abstract] [Full Text] [PDF]

				J. D. Potter, Z. Sheng, B.-S. Pan, and J. Zhao A Direct Regulatory Role for Troponin T and a Dual Role for Troponin C in the Ca[IMAGE] Regulation of Muscle Contraction J. Biol. Chem., February 10, 1995; 270(6): 2557 - 2562. [Abstract] [Full Text] [PDF]

				S. King and R. Patel-King Identification of a Ca(2+)-binding light chain within Chlamydomonas outer arm dynein J. Cell Sci., January 12, 1995; 108(12): 3757 - 3764. [Abstract] [PDF]

				M Sundaralingam, R Bergstrom, G Strasburg, S. Rao, P Roychowdhury, M Greaser, and B. Wang Molecular structure of troponin C from chicken skeletal muscle at 3-angstrom resolution Science, February 22, 1985; 227(4689): 945 - 948. [Abstract] [PDF]

				P. D. Ellis Cadmium-113 Magnetic Resonance Spectroscopy Science, September 16, 1983; 221(4616): 1141 - 1146. [Abstract] [PDF]

				J. R. Pearlstone, M. Chandra, M. M. Sorenson, and L. B. Smillie Biological Function and Site II Ca2+-induced Opening of the Regulatory Domain of Skeletal Troponin C Are Impaired by Invariant Site I or II Glu Mutations J. Biol. Chem., November 3, 2000; 275(45): 35106 - 35115. [Abstract] [Full Text] [PDF]

				R. C. Padre and J. T. Stull Functional Assembly of Fragments from Bisected Smooth Muscle Myosin Light Chain Kinase J. Biol. Chem., August 25, 2000; 275(35): 26665 - 26673. [Abstract] [Full Text] [PDF]

				M. J. Calvert, D. G. Ward, H. R. Trayer, and I. P. Trayer The Importance of the Carboxyl-terminal Domain of Cardiac Troponin C in Ca2+-sensitive Muscle Regulation J. Biol. Chem., October 13, 2000; 275(42): 32508 - 32515. [Abstract] [Full Text] [PDF]

				S. Morimoto, Q.-W. Lu, K. Harada, F. Takahashi-Yanaga, R. Minakami, M. Ohta, T. Sasaguri, and I. Ohtsuki Ca2+-desensitizing effect of a deletion mutation Delta K210 in cardiac troponin T that causes familial dilated cardiomyopathy PNAS, January 22, 2002; 99(2): 913 - 918. [Abstract] [Full Text] [PDF]

				D. P. Dobesh, J. P. Konhilas, and P. P. de Tombe Cooperative activation in cardiac muscle: impact of sarcomere length Am J Physiol Heart Circ Physiol, March 1, 2002; 282(3): H1055 - H1062. [Abstract] [Full Text] [PDF]