Abstract
There are significant isoform differences between the skeletal and cardiac troponin complexes. Studies of the regulatory properties of these proteins have previously shown only significant differences in the calcium dependence of their regulation. Using a sensitive myosin subfragment 1 (S1) binding assay we show that in the presence of calcium, thin filaments reconstituted with either skeletal or cardiac troponin produce virtually identical S1 binding curves. However in the absence of calcium the S1 binding curves differ considerably. Combined with kinetic measurements, curve fitting to the three-state thin filament regulatory model shows the main difference is that calcium produces a 4-fold change in K(T) (the closed-open equilibrium) for the skeletal system but little change in the cardiac system. The results show a significant difference in the range of regulatory effect between the cardiac and skeletal systems that we interpret as effects upon actin-troponin (Tn)I-TnC binding equilibria. As structural data show that the Ca(2+)-bound TnC structures differ, the additional counter-intuitive result here is that with respect to myosin binding the +Ca(2+) state of the two systems is similar whereas the -Ca(2+) state differs. This shows the regulatory tuning of the troponin complex produced by isoform variation is the net result of a complex series of interactions among all the troponin components.
Highlights
In striated muscle, the troponin-tropomyosin (TnTm)1 complex functions as a calcium-dependent molecular switch that regulates the force-generating interaction between actin and myosin filaments
Using a sensitive myosin subfragment 1 (S1) binding assay we show that in the presence of calcium, thin filaments reconstituted with either skeletal or cardiac troponin produce virtually identical S1 binding curves
Curve fitting to the three-state thin filament regulatory model shows the main difference is that calcium produces a 4-fold change in KT for the skeletal system but little change in the cardiac system
Summary
Isolation of Proteins from Bovine Heart and Rabbit Skeletal Muscle— The skTnTm complex, skTn, Tm, actin, and myosin were isolated from rabbit skeletal muscle. For the stopped-flow experiments thin filaments were preassembled by mixing stock solutions to final concentrations of 10 M actin, 2 M Tm, and 2 M of the appropriate Tn in standard experimental buffer (20 mM MOPS, pH 7.0, 200 mM KCl, 5 mM MgCl2) and leaving to equilibrate for 10 min. To confirm cTnI dephosphorylation, a portion of thin filaments containing 22 g of cTn was rephosphorylated in 20 mM MOPS, pH 7, 300 mM KCl, 2 mM dithiothreitol, with 1 mM (final concentration) [␥-32P]ATP (20 Ci), 3 mM MgCl2 by addition of 20 l of 14 milliunits of protein kinase A catalytic subunit/l (total volume of 100 l). [M] is the concentration of free S1 heads, P ϭ 1 ϩ K1[M](1 ϩ K2) and Q ϭ 1 ϩ K1[M]
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