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Kinetic Mechanism of the Ca²⁺-Dependent Switch-On and Switch-Off of Cardiac Troponin in Myofibrils

Johannes Solzin ^* , Bogdan Iorga ^* , Eva Sierakowski ^*, Diana P. Gomez Alcazar ^*, Daniel F. Ruess ^*, Torsten Kubacki ^*, Stefan Zittrich ^*, Natascha Blaudeck ^*, Gabriele Pfitzer ^*  and Robert Stehle ^* 

^* Institut fuer Vegetative Physiologie; Center of Molecular Medicine Cologne, University Cologne, Köln, Germany; and Department of Physics and Applied Mathematics, Faculty of Chemistry, University of Bucharest, Bucharest, Romania

Correspondence: Address reprint requests to Dr. Robert Stehle or Dr. Johannes Solzin, Institute of Vegetative Physiology, University of Cologne, Robert-Koch-Str. 39, D-50931 Köln, Germany. Tel.: 49-221-478-6952; Fax: 49-221-478-6965; E-mail: robert.stehle{at}uni-koeln.de or johannes.solzin{at}uni-koeln.de.

The kinetics of Ca²⁺-dependent conformational changes of human cardiac troponin (cTn) were studied on isolated cTn and within the sarcomeric environment of myofibrils. Human cTnC was selectively labeled on cysteine 84 with N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole and reconstituted with cTnI and cTnT to the cTn complex, which was incorporated into guinea pig cardiac myofibrils. These exchanged myofibrils, or the isolated cTn, were rapidly mixed in a stopped-flow apparatus with different [Ca²⁺] or the Ca²⁺-buffer 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid to determine the kinetics of the switch-on or switch-off, respectively, of cTn. Activation of myofibrils with high [Ca²⁺] (pCa 4.6) induced a biphasic fluorescence increase with rate constants of >2000 s^–1 and 330 s^–1, respectively. At low [Ca²⁺] (pCa 6.6), the slower rate was reduced to 25 s^–1, but was still 50-fold higher than the rate constant of Ca²⁺-induced myofibrillar force development measured in a mechanical setup. Decreasing [Ca²⁺] from pCa 5.0–7.9 induced a fluorescence decay with a rate constant of 39 s^–1, which was approximately fivefold faster than force relaxation. Modeling the data indicates two sequentially coupled conformational changes of cTnC in myofibrils: 1), rapid Ca²⁺-binding (k_B 120 µM^–1 s^–1) and dissociation (k_D 550 s^–1); and 2), slower switch-on (k_on = 390s^–1) and switch-off (k_off = 36s^–1) kinetics. At high [Ca²⁺], 90% of cTnC is switched on. Both switch-on and switch-off kinetics of incorporated cTn were around fourfold faster than those of isolated cTn. In conclusion, the switch kinetics of cTn are sensitively changed by its structural integration in the sarcomere and directly rate-limit neither cardiac myofibrillar contraction nor relaxation.

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