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

http://access.library.miami.edu/login?url=http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:8827878

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