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Regulation of Calcium Transport in Cardiac Muscle

Thomas Squier, Principal Investigator

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Funding Agency: National Institutes of Health, National Heart, Lung, and Blood Institute

The long-term goal of the Regulation of Calcium Transport in Cardiac Muscle project team at Pacific Northwest National Laboratory (PNNL) is to identify the physical mechanisms that regulate calcium resequestration by the Ca-ATPase in cardiac sarcoplasmic reticulum (SR) membranes. This active transport protein modulates 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 by changes in membrane lipid composition and that alterations in these regulatory mechanisms underlie heart disease.

The primary goal of our research is to identify structural changes that couple ATP hydrolysis to calcium transport and to discern how PLB and membrane composition modify catalytically important structural transitions. To support this goal, we are using spin-label electron paramagnetic resonance (EPR) as well as optical and vibrational spectroscopies in conjunction with site-directed mutagenesis to probe protein structure at defined sites on Ca-ATPase and on PLB. A second goal is to determine the structural features of PLB that permit the regulation of the Ca-ATPase transport function. To this end, we are defining sites of interaction between PLB and Ca-ATPase, measuring changes in PLB structure upon binding to the Ca-ATPase, and investigating the structural coupling between the cytosolic and transmembrane domains of PLB with respect to the modulation of Ca-ATPase function. 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.

Release of Ca-ATPase Inhibition Following Stabilization of Helix in Switch Region.
Release of Ca-ATPase Inhibition Following Stabilization of Helix in Switch Region. Motional restriction of catalytically important motions of the nucleotide binding domain (N) of the Ca-ATPase occurs through the binding of either PLB (A) or an autoinhibitory domain (shown in red), which underlies regulation of calcium transport in the sarcoplasmic reticulum or plasma membrane of the heart. In both cases, the stabilization of helical structure within a conformational switch region results in a shortening of the physical dimensions and the necessary release of the inhibitory interactions with the N-domain. View full image.