MODULATION OF THE CA2+ SENSITVITY OF CARDIAC TROPONIN C BY PKC PHOSPHORYLATED CARDIAC TROPONIN T AND CA2+ SENSITIZERS
Schlecht, William David
MetadataShow full item record
Cardiac troponin (cTn) exerts temporal control over the cyclic processes of contraction and relaxation in the heart. It does this by binding with calcium which initiates an allosteric chain of protein structural transitions culminating in force generation. Regulation over the sensitivity with which cTn binds calcium is thus of great importance in maintaining proper cardiac output. Unsurprisingly many diseases of the heart are caused by, or associated with, chronic elevation or depression in cTn's calcium sensitivity. Therapies which seek to reverse these chronic alterations in calcium sensitivity may have tremendous value, however insufficient understanding of the mechanisms by which diseases, or the inotropes intended to combat them, actually alter cTn's calcium sensitivity have limited their success. The work contained in this dissertation seeks to contribute to the collective understanding of these mechanisms by looking at two basic types of sensitivity modulators. The first are post translational modifications, specifically protein kinase C (PKC) phosphorylations of the tropomyosin binding subunit of cTn (cTnT) which have been shown to depress calcium sensitivity. The second, calcium sensitizers, are a class of inotrope specifically designed to increase the sensitivity of the calcium binding subunit of cTn (cTnC) for calcium. The spectroscopic technique of fluorescence resonance energy transfer (FRET) was used to monitor regulatory switching, while fluorescence homodimerization induced self quenching was used to study opening of cTnC's hydrophobic cleft. Additionally a newly developed absorbance based technique was used to measure the dynamic equilibrium between open and closed conformations in the hydrophobic cleft of cTnC. Using these techniques the impact on the structure/function of cTn by these two types of modulators determined. Briefly the findings of this work are 1) Most calcium sensitizers work by binding to the hydrophobic cleft and shifting its dynamic equilibrium towards the open state but have varying effects on cTn's relaxation kinetics 2) Altering the dynamic equilibrium of cTnC's hydrophobic cleft will generally result in a change in cTnC's calcium sensitivity 3) Alterations in position/flexibility of the switch region relative to the hydrophobic cleft are sufficient to explain the calcium sensitivity effects of cTnT PKC pseudo-phosphorylation mutants.