Evidence that both Ca(2+)-specific sites of skeletal muscle TnC are required for full activity.
To investigate the role of the Ca2(+)-specific (I and II) sites of fast skeletal muscle troponin C (TnC) in the regulation of contraction, we have produced two TnC mutants which have lost the ability to bind Ca2+ at either site I (VG1) or at site II (VG2). Both mutants were able to partially restore force to TnC-depleted skinned muscle fibers (approximately 25% for VG1 and approximately 50% for VG2). In contrast, bovine cardiac TnC (BCTnC), which like VG1 binds Ca2+ only at site II, could fully reactivate the contraction of TnC-depleted fibers. Higher concentrations of both mutants were required to restore force to the TnC-depleted fibers than with wild type TnC (WTnC) or BCTnC. VG1 and VG2 substituted fibers could not bind additional WTnC, indicating that all of the TnC-binding sites were saturated with the mutant TnC's. The Ca2+ concentration required for force activation was much higher for VG1 and VG2 substituted fibers than for WTnC or BCTnC substituted fibers. Also, the steepness of force activation was much less in VG1 and VG2 versus WTnC and BCTnC substituted fibers. These results suggest cooperative interactions between sites I and II in WTnC. In contrast, BCTnC has essentially the same apparent Ca2+ affinity and steepness of force activation as does WTnC. Thus, cardiac TnC must have structural differences from WTnC which compensate for the lack of site I, while in WTnC, both Ca2(+)-specific sites are probably crucial for full functional activity.
Sheng
Z
Z
Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, Florida 33101.
Strauss
W L
WL
Francois
J M
JM
Potter
J D
JD
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000502:physiology*; D002417:Cattle; D002645:Chickens; D004252:DNA Mutational Analysis; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009119:Muscle Contraction; D009132:Muscles / Q000502:physiology; D014336:Troponin / Q000502:physiology*; D019209:Troponin C
D014336:Troponin; D019209:Troponin C; D002118:Calcium