Kinetic mechanism of the Ca2+-dependent switch-on and –off of cardiac troponin in myofibrils

¹ Institut fuer Vegetative Physiologie, University Cologne

^* To whom correspondence should be addressed. E-mail: robert.stehle{at}uni-koeln.de.

Submitted on April 24, 2007
Revised on May 28, 2007
Accepted on 23 July 2007

	Abstract

The kinetics of the Ca²⁺-dependent conformational changes of cardiac troponin (cTn) were studied on isolated cTn and within the sarcomeric environment of myofibrils. Human cTnC was selectively labeled on cystein 84 with IANBD, reconstituted with cTnI and cTnT to the cTn-complex which was incorporated into guinea pig cardiac myofibrils (cMF). These exchanged cMF or the isolated cTn were rapidly mixed in a stopped-flow apparatus with different [Ca2+] or the Ca²⁺-buffer BAPTA to determine the kinetics of the switch-on or switch-off of cTn, respectively. After activation of cMF with high [Ca²⁺] (pCa4.6), fluorescence increased biphasically with rate constants of >2000s^-1 and ~330s^-1, respectively. At low [Ca²⁺ (pCa6.6), the slower rate was reduced to ~25s^-1, but was still ~50-fold faster than the Ca²⁺-induced myofibrillar force development, measured in parallel with a mechanical setup. Decreasing [Ca²⁺] from pCa5.0 to 7.9 led to a fluorescence decay with a rate constant of 39s^-1 which was ~5-fold faster than force relaxation. Modelling the biphasic fluorescence data indicates two sequentially coupled conformational changes of cTnC in cMF: rapid Ca²⁺-binding (k_B120µM-1s-1) and dissociation (k_D550s^-1) followed by a 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 ~4-fold faster than those of isolated cTn. In conclusion, the switch kinetics of cTn are sensitively changed by its structural integration in the sarcomere and do neither directly rate-limit cardiac myofibrillar contraction nor relaxation.

Key Words: calcium-activation, cross-bridge kinetics, muscle relaxation, sarcomere, stopped-flow, thin filament regulation

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