Troponin C (TnC) is the Ca2+ receptor in striated muscle which transfers the Ca2+ signal to troponin I (TnI), and then to troponin T and tropomyosin to ultimately modulate the population of productive myosin crossbridges. The initial event in this transfer, the interaction between TnC and TnI, is of intense research interest. The great majority of studies on the interaction between TnC and TnI have used the fast skeletal isoforms. Interactions between the cardiac isoforms have been largely implied from the skeletal models and on the interactions between a homologous protein, calmodulin, and its target proteins. Unfortunately, cTnC and the interactions between cTnC and cTnT have unique and clinically important features which can not be studied using the skeletal isoforms. For example, depression of contractility in acute myocardial ischemia is likely to involve an effect of pH on the cardiac troponin complex. Cardiac TnI, unlike its skeletal counter part, has specific sites for phosphorylation in response to adrenergic stimulation, which results a decrease in the affinity of the regulatory Ca2+-binding sites in cTnC. Finally, a group of positive inotropic drugs, which have great potential in the treatment of acute myocardial infarction, act by binding to cTnC to increase its sensitivity to Ca2+. Thus, potential ligand binding sites on cTnc, and interactive sites between cardiac Tnc and TnI are logical targets for reagents that could artificially influence the regulation of cardiac muscle contraction under normal or disease states.
The specific aims of this proposal will provide a foundation of biochemical and structural information on the interaction between cTnC and cTnI which can be used in the design and selection of such reagents. The overriding theme is to identify the structural basis for modulating the affinity of the regulatory Ca2+ binding site in cTnC. Using a combination of recombinant DNA, biochemical and biophysical techniques we will first define the relative topology of interaction between cTnC and cTnI. We will then identify those regions in cTnI that are important for the intrinsic increase in Ca2+ binding affinity of the regulatory site II upon association with cTnC, and that may mediate feedback between myosin cross-bridges and cTnC. We will then define the molecular mechanism for how phosphorylation of cTnI modulates Ca2+ binding to the regulatory site of cTnC.
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