RECOMBINANT TROPONIN-I SUBSTITUTION AND CALCIUM RESPONSIVENESS IN SKINNED CARDIAC-MUSCLE

Using treatment with vanadate solutions, we extracted native cardiac t roponin I and troponin C (cTnI and cTnC) from skinned fibers of porcin e right ventricles. These proteins were replaced by exogenously suppli ed TnI and TnC isoforms, thereby restoring Ca2+-dependent regulation. Force then depended on the negative logarithm of Ca2+ concentration (p Ca) in a sigmoidal manner, the pCa for 50% force development, pCa(50), being about 5.5. For reconstitution we used fast-twitch rabbit skelet al muscle TnI and TnC (sTnI and sTnC), bovine cTnI and cTnC or recombi nant sTnIs that were altered by site-directed mutagenesis. Incubation with TnI inhibited isometric tension in TnI-extracted fibers in the ab sence 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 exogenousl y supplied TnI in the reconstitution solution (range 20-150 mu M), whi le Ca2+ sensitivity, i.e. the pCa(50), was dependent on the isoform, a nd also on the concentration of TnC in the reconstitution solution. At pH 6.7, skinned fibers reconstituted with optimal concentrations of s TnC and sTnI (120 mu M and 150 mu M, respectively) were more sensitive to Ca2+ than those reconstituted with cTnC and cTnI (difference in pC a(50) approx. 0.2 units). Rabbit sTnI was cloned and expressed in Esch erichia coli using a high yield expression plasmid. We introduced poin t mutations into the TnI inhibitory region comprising the sequence of the minimal common TnC/actin binding site (-G(104)-K-F-K-R-P-P-L-R-R-V -R(115)-). The four mutants produced by substitution of T for P-110, G for P-110, G for L(111), and C for K-105 were chosen, based on previo us work with synthetic peptides showing that single amino acid substit ution in this region diminished the capacity of these peptides to inhi bit acto-S-1 ATPase or contraction of skinned fibers. Therefore, all a mino acid residues of the inhibitory region are thought to contribute to biological activity of TnI. However, each of the recombinant TnIs c ould substitute for endogenous TnI. In combination with exogenous TnC, Ca2+ dependence could be restored when (gly110)sTnI, (thr110)sTnI or (gly111)sTnI was used for reconstitution. The mutant (gly105)sTnI, On the other hand, reduced the ability of skinned fibers to relax at low Ca2+ concentrations and it caused an increase in Ca2+ sensitivity.