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The Low-Affinity Ca2+-Binding Sites in Cardiac/Slow Skeletal Muscle Troponin C Perform Distinct Functions: Site I Alone Cannot Trigger Contraction
H. Lee Sweeney, Rui M. M. Brito, Paul R. Rosevear and John A. Putkey
Proceedings of the National Academy of Sciences of the United States of America
Vol. 87, No. 24 (Dec., 1990), pp. 9538-9542
Published by: National Academy of Sciences
Stable URL: http://www.jstor.org/stable/2356447
Page Count: 5
You can always find the topics here!Topics: Skeletal muscle, Proteins, Muscle contraction, Protein isoforms, Slow twitch muscle fibers, Amino acids, Biochemistry, Muscles, Muscle fibers, Myocardium
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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 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.
Proceedings of the National Academy of Sciences of the United States of America © 1990 National Academy of Sciences