The role of the four Ca2+ binding sites of troponin C in the regulation of skeletal muscle contraction.
In order to study the role of the Ca2+-specific sites (I and II) and the high affinity Ca2+-Mg2+ sites (III and IV) of TnC in the regulation of muscle contraction, we have constructed four mutants and the wild type (WTnC) of chicken skeletal TnC, with inactivated Ca2+ binding sites I and II (TnC1,2-), site III (TnC3-), site IV (TnC4-), and sites III and IV (TnC3,4C-). All Ca2+ binding site mutations were generated by replacing the Asp at the X-coordinating position of the Ca2+ binding loop with Ala. The binding of these mutated proteins to TnC-depleted skinned skeletal muscle fibers was investigated as well as the rate of their dissociation from these fibers. The proteins were also tested for their ability to restore steady state force to TnC-depleted fibers. We found that although the NH2-terminal mutant of TnC (TnC1,2-) bound to the TnC-depleted fibers (with a lower affinity than wild type TnC (WTnC)), it was unable to reactivate Ca2+-dependent force. This supports earlier findings that the low affinity Ca2+ binding sites (I and II) in TnC are responsible for the Ca2+-dependent activation of skeletal muscle contraction. All three COOH-terminal mutants of TnC bound to the TnC-depleted fibers, had different rates of dissociation, and could restore steady state force to the level of unextracted fibers. Although both high affinity Ca2+ binding sites (III and IV) are important for binding to the fibers, site III appears to be the primary determinant for maintaining the structural stability of TnC in the thin filament. Moreover, our results suggest an interaction between the NH2- and COOH-terminal domains of TnC, since alteration of sites I and II lowers the binding affinity of TnC to the fibers, and mutations in sites III and IV affect the Ca2+ sensitivity of force development.
Szczesna
D
D
Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, Miami, Florida 33101, USA.
Guzman
G
G
Miller
T
T
Zhao
J
J
Farokhi
K
K
Ellemberger
H
H
Potter
J D
JD
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000502:physiology*; D002135:Calcium-Binding Proteins / Q000502:physiology*; D009119:Muscle Contraction; D009124:Muscle Proteins / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D011817:Rabbits; D013329:Structure-Activity Relationship; D014336:Troponin / Q000502:physiology*; D019209:Troponin C
D002135:Calcium-Binding Proteins; D009124:Muscle Proteins; D014336:Troponin; D019209:Troponin C; D002118:Calcium