title: The low-affinity Ca2(+)-binding sites in cardiac/slow skeletal muscle troponin C perform distinct functions: site I alone cannot trigger contraction.
abstract: Both troponin C (TnC) and calmodulin share a remarkably similar tertiary motif that may be common to other Ca2(+)-binding proteins with activator activity. TnC plays a critical role in regulating muscle contraction and is particularly well-suited for structural analysis by site-directed mutation. Fast-twitch skeletal muscle TnC has two low-affinity Ca2(+)-binding sites (sites I and II), while in cardiac and slow-twitch skeletal muscle TnC site I is inactive. Recently, using protein engineering, we directly demonstrated that binding of Ca2+ to the low-affinity site(s) initiates muscle contraction. In the present study, we use mutagenesis to determine whether either of the low-affinity sites in cardiac TnC can trigger contraction in slow-twitch skeletal muscle fibers. In one Ca2(+)-binding mutant, Ca2(+)-binding to the dormant low-affinity site I was restored (CBM+I). In a second mutant, site I was activated while site II was inactivated (CBM+I-IIA). Both proteins had the predicted CA2(+)-binding characteristics, and both were able to associate with troponin I and troponin T to form a troponin complex and integrate into permeabilized slow-twitch skeletal muscle fibers. A comparison of NMR spectra shows the aromatic regions in the two proteins to be qualitatively similar without divalent cations but markedly different with Ca2+. Mutant CBM+I supported force generation in skinned slow skeletal muscle fibers but had Sr2+ and Ca2+ sensitivities similar to fast skeletal TnC. Mutant CBM+I-IIA was unable to restore Ca2(+)-dependent contraction to TnC-depleted skinned slow muscle fibers. The data directly demonstrate that low-affinity sites I and II have distinct functions and that only site II in cardiac TnC can trigger muscle contraction in slow-twitch skeletal muscle fibers. This principle of distinct, modular activities for Ca2(+)-binding sites in the same protein may apply to other members of the TnC/calmodulin family.
authors: Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia 19104-6085.
authors: H L
authors: HL
authors: Sweeney
authors: R M
authors: RM
authors: Brito
authors: P R
authors: PR
authors: Rosevear
authors: J A
authors: JA
authors: Putkey
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002122:Calcium Chloride / Q000378:metabolism; D006321:Heart / Q000502:physiology*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009119:Muscle Contraction; D009132:Muscles / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D009200:Myocardial Contraction; D014336:Troponin / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D019209:Troponin C
chemical_list: D014336:Troponin; D019209:Troponin C; D002122:Calcium Chloride; D002118:Calcium