Kinetic Mechanism of the Ca 2+-Dependent Switch-On and Switch-Off of Cardiac Troponin in Myofibrils

Abstract

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 ∼330 s −1, respectively. At low [Ca 2+] (pCa 6.6), the slower rate was reduced to ∼25 s −1, but was still ∼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 ≈ 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 [Ca 2+], ∼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.