Abstract
The cardiotonic effects of thiadiazinone derivative EMD 57033 are mediated by direct actions on myofilaments (Lues, I., Beier, N., Jonas, R., Klockow, M., and Haeusler, G. J. (1993) Cardiovasc. Pharmacol. 21, 883-892). Cardiac troponin C has been postulated to be a potential target of the drug (White, J., Lee, J. A., Shah, N., and Orchard, C. H. (1993) Circ. Res. 73, 61-70). This study tested whether EMD 57033 interacts directly with recombinant human cardiac TnC (hcTnC). EMD 57033 caused concentration-dependent quenching of tyrosine (Tyr) fluorescence of hcTnC in the presence of Ca2+ (100 microM) and little change of the fluorescence in the presence of Mg2+ (2 mM). Kd for the drug-hcTnC interaction in the presence of Ca2+, determined by Tyr fluorescence titrations, was approximately 40 microM. The binding of EMD 57033 was stereo-selective: the optical isomer of EMD 57033 bound hcTnC much more weakly. The Ca2+ dependence and stereo-selectivity of EMD 57033 binding were substantiated by a dialysis-based direct binding assay. EMD 57033 was found to interfere with Ca(2+)-dependent binding of hydrophobic probe 1,1'-bi-(4-anili-no)naphthalene-5,5'-disulfonate (bis-ANS) to hcTnC. The relationships between [Ca2+] and Tyr fluorescence of hcTnC and between [Ca2+] and bis-ANS fluorescence in the presence of hcTnC were substantially altered by EMD 57033 in the range of [Ca2+] where Ca2+/Mg2+ sites of hcTnC were titrated by Ca2+. EMD 57033 was found to bind as tightly to 2 Ca2+.hcTnC as to 3 Ca(2+).hcTnC. These observations were interpreted as indicating that a EMD 57033-binding site is induced by Ca2+ binding, but not Mg2+ binding, to the Ca2+/Mg2+ sites of hcTnC. The drug-binding site most likely resides in the carboxyl domain of hcTnC.
Highlights
Contractile filaments to Ca2ϩ [2,3,4,5,6,7]
In addition to its well documented structural role in anchoring Troponin C (TnC) to the thin filament, the COOHterminal domain appears to contribute directly to Ca2ϩ regulation by way of interacting with the inhibitory region of TnI and residues of TnI adjacent to the inhibitory region. Both domains of TnC contains a core of hydrophobic residues, which become more exposed to solvent upon Ca2ϩ binding to the domains [25, 26]
Up to 60 M EMD 57439 had no detectable effect on human cardiac TnC (hcTnC) fluorescence, while at the highest [EMD 57033] tested (50 M), only a very small effect (a 5% quenching) was observed
Summary
Contractile filaments to Ca2ϩ [2,3,4,5,6,7]. EMD 57033 has been found to increase Ca2ϩ sensitivity of both myofibrillar ATPase [2, 6] and force development by skinned muscle fibers [2]. In addition to its well documented structural role in anchoring TnC to the thin filament, the COOHterminal domain appears to contribute directly to Ca2ϩ regulation by way of interacting with the inhibitory region of TnI and residues of TnI adjacent to the inhibitory region The interactions of the hydrophobic patches with TnI are believed to be important for transmission of the Ca2ϩ signal and the stability of troponin complex.
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