Title

Molecular aspects of the regulation of skeletal muscle contraction by troponin C

Date of Award

1988

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Pharmacology

First Committee Member

James D. Potter, Committee Chair

Abstract

Skeletal muscle contraction is regulated by the binding of Ca$\sp{2+}$ to the troponin C subunit of the thin filament protein troponin. The role of troponin C (TnC) in the regulation of skeletal muscle contraction was studied with regard to three different aspects: (1) the effect of free Mg$\sp{2+}$ on the Ca$\sp{2+}$-dependence of muscle activity and on the binding of Ca$\sp{2+}$ to TnC; (2) the reciprocal coupling between myosin crossbridge attachment to actin and conformational changes of TnC; and (3) the isolation and sequencing of a cDNA clone encoding fast skeletal muscle TnC. First, free Mg$\sp{2+}$ was found to shift the Ca$\sp{2+}$-dependence of myofibrillar ATPase activity, the Ca$\sp{2+}$-dependence of skinned fiber tension development, and the binding of Ca$\sp{2+}$ to the regulatory, low-affinity sites of TnC, all with the same apparent association constant (about 200 M$\sp{-1}$). These results suggest that the effect of Mg$\sp{2+}$ on the Ca$\sp{2+}$-dependence of skeletal muscle activity can be explained by its inhibition of the binding of Ca$\sp{2+}$ to the regulatory sites on TnC. However, this effect is so small that under physiological conditions the regulatory sites of TnC remain essentially Ca$\sp{2+}$-specific. Second, the attachments of both rigor and cycling crossbridges to actin were shown to produce structural changes in TnC in addition to the changes produced by the binding of Ca$\sp{2+}$, based upon changes in fluorescence intensity levels of labelled TnC. A differential coupling between weakly- versus strongly-attached crossbridges and TnC structure was also described. These findings indicate a reciprocal coupling between crossbridge attachment to actin and conformational changes in the amino-terminal region of TnC and provide evidence of a possible differential regulation of weak and strong binding states by changes in TnC. Finally, in order to allow a more detailed, direct investigation of TnC at the molecular level, a cDNA clone containing the entire protein-coding region of rabbit fast skeletal muscle TnC was isolated and its nucleotide sequence was determined in anticipation of future site-directed mutagenesis studies. Comparisons of the sequence of this clone with those of parvalbumin, calmodulin, and slow/cardiac TnC reflected the close evolutionary relationships between these Ca$\sp{2+}$-binding proteins.

Keywords

Biology, General

Link to Full Text

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