Kinetic mechanism of the Ca2+-dependent switch-on and switch-off of cardiac troponin in myofibrils.
The kinetics of Ca(2+)-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 [Ca(2+)] or the Ca(2+)-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 [Ca(2+)] (pCa 4.6) induced a biphasic fluorescence increase with rate constants of >2000 s(-1) and approximately 330 s(-1), respectively. At low [Ca(2+)] (pCa 6.6), the slower rate was reduced to approximately 25 s(-1), but was still approximately 50-fold higher than the rate constant of Ca(2+)-induced myofibrillar force development measured in a mechanical setup. Decreasing [Ca(2+)] 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 Ca(2+)-binding (k(B) approximately 120 microM(-1) s(-1)) and dissociation (k(D) approximately 550 s(-1)); and 2), slower switch-on (k(on) = 390s(-1)) and switch-off (k(off) = 36s(-1)) kinetics. At high [Ca(2+)], approximately 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.
Solzin
Johannes
J
Institut fuer Vegetative Physiologie, University Cologne, Köln, Germany.
Iorga
Bogdan
B
Sierakowski
Eva
E
Gomez Alcazar
Diana P
DP
Ruess
Daniel F
DF
Kubacki
Torsten
T
Zittrich
Stefan
S
Blaudeck
Natascha
N
Pfitzer
Gabriele
G
Stehle
Robert
R
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D005260:Female; D006168:Guinea Pigs; D006801:Humans; D007700:Kinetics; D009200:Myocardial Contraction / Q000502:physiology*; D032383:Myocytes, Cardiac / Q000502:physiology*; D009210:Myofibrils / Q000502:physiology*; D014336:Troponin / Q000378:metabolism*
D014336:Troponin; D002118:Calcium
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