Abstract
Although regulatory Ca2+-binding domains of calmodulin (CaM) and troponin C (TnC) are similar, it is interesting that agents that act as CaM antagonists appear to be TnC "agonists" in that they sensitize cardiac myofilaments to activation by Ca2+ (El-Saleh, S., and Solaro, R. J. (1987) Biophys. J. 51, 325 (abstr.). This indicates that the effects of agents that react with Ca2+-binding proteins may depend on protein-protein interactions involved in a particular Ca2+-dependent process. In experiments described here, we have explored this idea by testing effects of calmidazolium (CDZ), a potent calmodulin antagonist on striated muscle myofilaments regulated by cardiac TnC, skeletal TnC, and CaM. CDZ was shown to increase submaximal calcium activation of myofilament force and ATPase activity in both cardiac and skeletal muscle, but the effect was greater in the case of the cardiac preparations. In the presence of 10 microM CDZ, the free Ca2+ giving half-maximal activation was reduced to about 60% of the control value in the case of cardiac myofilaments. Analogous differential effects of CDZ were also seen in studies in which we measured direct effects of CDZ on Ca2+-dependent fluorescence changes of cardiac TnC and skeletal TnC labeled with probes reporting Ca2+ binding to the regulatory sites. Measurements were also done with myofibrillar preparations of psoas muscle in which the native skeletal TnC was removed and exchanged with cardiac TnC and CaM, both of which could substitute for skeletal TnC as a regulatory protein. CDZ was more effective in sensitizing Ca2+-dependent MgATPase activity of skeletal myofibrils containing CaM than in preparations containing the native TnC. However, CDZ was most effective in its Ca2+-sensitizing effect in the case of the preparations containing cardiac TnC. Our results indicate that effects of agents that bind to Ca2+-binding proteins depend not only on the particular variant, but also on the specific environment in which the Ca2+-binding proteins operate.
Highlights
This indicates that the effeocftasgents that react withmains, have opposite effects on the processes dependent on
In the presence of 10 M M CDZ, the free Ca2+ giving half-maximal activation was reduced to. These results suggest at least two important possibilities: i) that CaM antagonists with presumed specificity of action, may affect processes dependent on other Ca2+-binding proteins in unexpected ways and ii) that the effects of agents, which react with Ca2+-binding proteinsmay depend on the particular type of Ca2+-binding protein, and on about 60%of the control value in the case of cardiac interactions of theCa2+-bindingprotein withneighboring myofilaments
Our results show that a etal myofibrils containing CaM than in preparations single agent which reacts with Ca2+-bindingproteins may containing the native TnC
Summary
Reconstitution with skeletal TnC, cardiac TnC, and CaM was achieved by the addition of each protein to depleted myofibrils a t a ratio of 5 pmol of protein/g of myofibrils in 30 mM imidazole, pH 7.0, 60 mM KCl, 7.0 mM MgCI,, 2.0 mM EGTA,and 0.2 mM DTT.Excessunboundprotein was diameter) were mounted between an AME 801 force transducer (Aksjeselskapet, Horten, Norway) and a glass rod extending from a micromanipulator. Recon- with Y being the sumof binding totwo classes of binding sitesor the stituted myofibrils were assayed for their ATPase activities as sum of the fraction of the maximal change of fluorescence arising described earlier.Skeletal myofibrils reconstitutedwithCaMand from Ca2+ titration of TnCDANZ or TnCIA. TnC was labeled with the fluorescent probeDANZ according to the The Hill equation in its form: method described by Johnson et al (13).DANZ was incorporated at 0.6 mol/mol of TnC
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