Our long term goal is to identify the physical mechanisms that regulate calcium resequestration by the Ca-ATPase in cardiac sarcoplasmic reticulum (SR) membranes. This active transport protein functions to modulate the rate and extent of myocardial relaxation in the heart. The regulatory protein phospholamban (PLB) is co-expressed with the Ca-ATPase in cardiac SR, and prior to beta-adrenergic stimulation functions to inhibit the transport activity of the Ca-ATPase. It is our hypothesis that the normal catalytic motions involved in the transport mechanism of the Ca-ATPase are modulated by either PLB or changes in membrane lipid composition, and that alterations in these regulatory mechanisms underlie heart disease. Therefore, a primary goal of the proposed research is the identification of structural changes that couple ATP hydrolysis to calcium transport, and how PLB and membrane composition modify catalytically important structural transitions. This will involve the use of spin-label EPR, optical and vibrational spectroscopies in conjunction with site-directed mutagenesis to probe protein structure at defined sites on the Ca-ATPase and on PLB. A second goal is the determination of structural features of PLB that permit the regulation of Ca-ATPase transport function. These measurements will aim to define sites of interaction between PLB and the Ca-ATPase, measure changes in PLB structure and binding to the Ca-ATPase, and to investigate the structural coupling between the cytosolic and transmembrane domains of PLB with respect to the modulation of Ca-ATPase function.
Our specific aims i nclude: (1) Identify dynamic structural changes of the Ca-ATPase important to calcium transport, (2) Define mechanisms of PLB regulation of Ca-ATPase transport function, (3) Determine structure of PLB involved in regulation of Ca-ATPase transport activity, and (4) Define mechanisms of phospholipid regulation of Ca-ATPase ion transport. The identification of the structural mechanisms underlying regulation of Ca-ATPase function will permit the design of effective therapies to alleviate the loss of cardiac function in the failing heart.
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