Regulation of Cardiac Calcium Transport

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Project Summary/Abstract: The proposed project uses a two Aim strategy to investigate the regulation of cardiac calcium transport. The goals of the planned experiments are to uncover novel mechanisms of calcium handling and determine how these mechanisms are disrupted in disease. Aim 1 is focused on how the affinity of phospholamban (PLB) for its regulatory target, SERCA2a, changes during exercise. Aim 1a experiments will reveal the structural determinants of the functional differences between inhibitory and non-inhibitory regulators of SERCA. We will explore how PLB stabilizes specific enzymatic intermediate states in the SERCA catalytic cycle, leading to transport inhibition. Aim 1b addresses the dynamics of regulatory interactions in health and disease. The experiments will test how competing interactions limit the rate of recovery of inhibition of SERCA after a prolonged period of calcium elevation. Aim 1 will reveal how SERCA and PLB molecular interactions change as the heart is stimulated from a resting heart rate to an elevated heart rate during exercise, followed by post-exercise recovery. Aim 2 will investigate alternative modes of binding for the PLB-SERCA regulatory complex. Our previous computational study identified multiple non-canonical binding sites on SERCA where PLB can interact in several alternative orientations. Thus, the PLB-SERCA interaction represents a “fuzzy complex”. The structural hypotheses generated from that previous study will be tested directly in Aim 2a of the proposed project using photoactivatable cross-linking and mass spectroscopy experiments. We will determine the functional role of the alternative interactions using live cell Ca uptake measurements and in vitro assays of SERCA ATPase function. In Aim 2b we will explore the pathophysiology of the SERCA fuzzy complex. Heart disease produces peptide fragments of membrane proteins that may interact with and disrupt SERCA activity or regulation. We will identify candidate “poison peptides” by mass spectrometry and test their ability to bind SERCA, inhibit Ca transport, or displace native regulatory partners. The experiments will provide new insight into a novel pathophysiological mechanism in heart failure and ischemic heart disease. Seth Robia (PI) and collaborating investigators bring complementary expertise and methods. Howard Young, University of Alberta, will provide expertise in cryo-EM and SERCA structure/function. Aleksey Zima is an expert in cardiac calcium handling and will assist with quantitative physiological experiments. Drs. Young and Zima participated in the previous, highly productive funding cycle for this project. Jonathan Kirk will bring mass spectrometry and proteomics expertise. Peter Kekenes-Huskey will contribute physiological models of the SERCA transport cycle and the regulatory interactions that govern transport function. Preliminary collaborative experiments suggest a high likelihood of future success for this research team. We are in a unique position to make scientifically significant advances in our understanding of the regulation of cardiac calcium transport.