The proposed project uses a two aim strategy to explore molecular mechanisms of the regulation of ion-motive ATPases. Specifically, we will focus on regulation of calcium transporters in skeletal and cardiac muscle. While it is important in all cells, calcium transport plays a particularly critical role in striated muscle, as the uptake of calcium determines the kinetics of muscle relaxation. In the heart, the calcium transport rate also indirectly determines the strength of the cardiac contraction, since it defines the magnitude of the calcium stores and therefore the size of calcium release.
Aim 1 of the present proposal focuses on regulation of SERCA by newly discovered species of micropeptides that are related to phospholamban. These new regulators include DWORF, endoregulin, myoregulin, and another-regulin. Little is known about the biophysical determinants of their functional regulation of SERCA. We will quantify the stoichiometry and binding affinity of micropeptide regulatory complexes, the dynamics of regulatory interactions, and the structural determinants of regulation. To quantify these key parameters, we will use several complementary approaches including fluorescence spectroscopy/microscopy, live cell physiological measurements, in vitro functional assays, cryoEM, and NMR. Defining the basic building blocks of micropeptide regulatory complexes is a key step in understanding their biophysical function.
Aim 2 will investigate physical and functional coupling of SERCA pumps into dimeric transport complexes. We will investigate the regulation of functional coupling and determine its physiological consequences. In particular, we will test how functional coupling alters SERCA transport rate and the cooperativity of calcium- dependent ATPase activity. The experiments described in the two Aims of this application will provide new insight into fundamental mechanisms of regulation of ion-motive ATPases, and may improve our understanding of the ion transport disorders associated with heart failure. The Principal Investigator has recruited collaborating investigators to provide additional methodological expertise. Calibrated, quantitative calcium uptake measurements will be performed in live cells in the laboratory of Prof. Aleksey Zima, Loyola University Chicago. CryoEM of DWORF-SERCA complexes will be done in the lab of Prof. Howard S. Young, University of Alberta. NMR of DWORF will be done in the lab of Prof. Gianluigi Veglia, University of Minnesota. Each of the collaborators already has a history of productive collaboration with the Principal Investigator. Now they will combine their expertise as a single team to address how micropeptides and SERCA dimerization regulate calcium handling in striated muscle. Additional expertise in molecular biology and animal models of heart failure will be provided by Dr. Toni Pak and Dr. Ivana Kuo, Loyola University Chicago.
Relevance to Public Health: This research project is focused on a protein called SERCA, the main calcium pump in the heart. This pump is at the center of cardiac calcium handling; by increasing the activity of the pump, the strength of the heart is increased to improve performance during exercise. Human heart failure is associated with disordered cardiac calcium handling, so SERCA an important therapeutic target.
