Recombinant troponin I substitution and calcium responsiveness in skinned cardiac muscle |
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Authors: | John D Strauss Jennifer E Van Eyk Zacharias Barth Lan Kluwe Rudolf J Wiesner K Maéda J Caspar Rüegg |
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Institution: | (1) Department of Physiology II, University of Heidelberg, Im Neuenheimer Feld 326, D-69120 Heidelberg, Germany, DE;(2) Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA, USA, US;(3) Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada, CA;(4) EMBL Research Outstation, Hamburg, Germany , DE;(5) Laboratory for Brain Tumor Biology, Department of Neurosurgery, University Hospital Eppendorf, Hamburg, Germany, DE;(6) International Institute for Advanced Research, Matsushita Electric Industrial Co, Kyoto, Japan, JP |
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Abstract: | Using treatment with vanadate solutions, we extracted native cardiac troponin I and troponin C (cTnI and cTnC) from skinned
fibers of porcine right ventricles. These proteins were replaced by exogenously supplied TnI and TnC isoforms, thereby restoring
Ca2+-dependent regulation. Force then depended on the negative logarithm of Ca2+ concentration (pCa) in a sigmoidal manner, the pCa for 50% force development, pCa50, being about 5.5. For reconstitution we used fast-twitch rabbit skeletal muscle TnI and TnC (sTnI and sTnC), bovine cTnI
and cTnC or recombinant sTnIs that were altered by site-directed mutagenesis. Incubation with TnI inhibited isometric tension
in TnI-extracted fibers in the absence of Ca2+, but restoration of Ca2+ dependence required incubation with both TnI and TnC. Relaxation at low Ca2+ levels and the steepness of the force/pCa relation depended on the concentration of exogenously supplied TnI in the reconstitution
solution (range 20–150 μM), while Ca2+ sensitivity, i.e. the pCa50, was dependent on the isoform, and also on the concentration of TnC in the reconstitution solution. At pH 6.7, skinned fibers
reconstituted with optimal concentrations of sTnC and sTnI (120 μM and 150 μM, respectively) were more sensitive to Ca2+ than those reconstituted with cTnC and cTnI (difference in pCa50 approx. 0.2 units). Rabbit sTnI was cloned and expressed in Escherichia coli using a high yield expression plasmid. We introduced point mutations into the TnI inhibitory region comprising the sequence
of the minimal common TnC/actin binding site (-G104-K-F-K-R-P-P-L-R-R-V-R115-). The four mutants produced by substitution of T for P110, G for P110, G for L111, and G for K105 were chosen, based on previous work with synthetic peptides showing that single amino acid substitution in this region diminished
the capacity of these peptides to inhibit acto-S1 ATPase or contraction of skinned fibers. Therefore, all amino acid residues of the inhibitory region are thought to contribute
to biological activity of TnI. However, each of the recombinant TnIs could substitute for endogenous TnI. In combination with
exogenous TnC, Ca2+ dependence could be restored when gly110sTnI, thr110sTnI or gly111sTnI was used for reconstitution. The mutant gly105sTnI, on the other hand, reduced the ability of skinned fibers to relax at low Ca2+ concentrations and it caused an increase in Ca2+ sensitivity.
Received: 5 October 1995/Received after revision and accepted: 1 December 1995 |
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Keywords: | Troponin I Calcium sensitivity Cardiac muscle contraction Skinned fibers Site-directed mutagenesis |
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