Analysis of kinetics of calcium exchange with cardiac troponin using sensitized luminescence

Bennett Thomas Rieck

Though calcium (Ca2+) exchange with cardiac troponin (cTn) is the molecular event that initiates the contraction or relaxation of cardiac muscle, the in vivo kinetics of this event are steeped in controversy. Some researchers have shown indirect evidence that the in situ Ca2+ binding event and the subsequent conformational changes it triggers in cTn are too fast to impact the rate of myocardial contraction, while other studies have argued for the opposite conclusion. Without a fundamental knowledge of the kinetics of Ca2+ exchange with cTn and the cTn regulatory switching events it triggers, it is extremely difficult to rationally design therapeutics to restore systolic or diastolic function. Based on prior in vitro studies, our laboratory has hypothesized that Ca2+ binds rapidly to cTnC, whereas subsequent protein conformational changes in cTnC and other thin filament proteins are orders of magnitude slower. However, in order to test this hypothesis, the experimental methodology employed must be able to distinguish the Ca2+ exchange event from the subsequent changes in protein conformation that it triggers, which is not possible using conventional methods. Currently, the dominant method for testing Ca2+ exchange is to use environmentally sensitive fluorophores. However, this method relies on detecting changes in the polarity of the iii local environment surrounding the fluorophore, which can be affected not only by Ca2+ exchange but changes in the conformation of cTnC. In lieu of these conventional probes, terbium (Tb3+) behaves as a fluorescent Ca2+ analog that can theoretically be used to provide a methodology for precisely measuring Ca2+ equilibria and dissociation rates. Accordingly, the goal of this project was to test the feasibility of a competitive ligand binding assay developed around the concept of using Tb3+ as a fluorescent Ca2+ analog. Stopped-flow experiments were conducted in an effort to measure the kinetics of Ca2+ dissociation from cTnC(V64W/F77W) as a function of cTn reconstitution level and Ca2+ concentration. Unfortunately, the assay as designed could not elicit meaningful kinetic information regarding Ca2+ dissociation from the regulatory site of cTnC. Accordingly, the limitations of the current design are discussed, and some suggestions for future work are offered.

Analysis of kinetics of calcium exchange with cardiac troponin using sensitized luminescence

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