The goal of this research is to determine the molecular mechanisms of catalysis and regulation ~ active calcium transport in sarcoplasmic reticulum (SR) in skeletal and cardiac muscle. The focus is on the Ca- ATPase, the integral membrane enzyme that pumps calcium into the SR and thus relaxes the muscle, and phospholamban (PL3), the integral membrane peptide involved in regulating the Ca-ATPase in the heart. Our previous work on skeletal muscle, which contains a very similar Ca-ATPase but no PLB, indicates that Ca-ATPase activity is quite dependent on the molecular dynamics and interactions of the SR membrane, including lipid chain motions, lipid-protein interactions, protein dynamics, and protein-protein interactions. In our future work, we will test specific mechanistic hypotheses for these correlations, and we will extend this work to the study of cardiac SR. In particular, we will test specific hypotheses for the physical basis of the functional differences between cardiac and skeletal calcium transport. We will continue to focus on spectroscopic probe methods to analyze molecular dynamics and interactions, and we will expand our use of biochemical kinetics and molecular genetics to determine more precisely the relationship between physics and function. We will pursue the following specific aims: (a) Develop improved EPR and optical (fiuorecence, phosphorescence, transient absorption) spectroscopic methods for studying membrane molecular dynamics, using sarcopiasmic reticulum (SR) as a model system to demonstrate these techniques. (b)Use spectroscopy and biochemical kinetics to determine what changes in molecular motions and interactions are coupled to the Ca-ATPase reaction cycle. (c) Investigate the regulation and modulation of the Ca-ATPase, as affected by intrinsic physiological effectors such as phospholamban, as well as extrinsic perturbants such anesthetics and toxins. (d) Use spectroscopy to probe phospholamban directly, in order to determine the aspects of its structure, dynamics, and interactions that are crucial to the regulation of the Ca-ATPase. The proposed research brings together a powerful combination of techniques, from biophysics to molecular genetics, to solve the molecular mechanism of calcium transport regulation, which is fundamental to understanding muscle function and malfunction. More generally, the techniques we develop and the lessons we learn about this well-defined system will have broad implications for studying the role of molecular dynamics and interactions in membrane energy transduction mechanisms.
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