REGULATION OF CALCIUM TRANSPORT IN CARDIAC MUSCLE

我们的长期目标是确定通过心肌肌浆网(SR)膜上的Ca-ATPase调节钙再限制的物理机制。这种活性转运蛋白的功能是调节心脏中心肌松弛的速度和程度。在心脏SR中，调节蛋白磷蛋白(PLB)与Ca-ATPase共表达，并在β-肾上腺素能刺激之前抑制Ca-ATPase的转运活性。我们的假设是，参与Ca-ATPase运输机制的正常催化运动受到原球蛋白或膜脂组成变化的调节，这些调节机制的变化是心脏病的基础。因此，这项研究的一个主要目标是确定将ATP水解与钙转运结合在一起的结构变化，以及PLB和膜成分如何改变催化重要的结构转变。这将涉及使用自旋标记EPR、光学和振动光谱与定点突变相结合的方法来探测Ca-ATPase和PLB上指定位置的蛋白质结构。第二个目标是确定PLB的结构特征，以允许调节Ca-ATPase的运输功能。这些测量的目的是确定PLB和Ca-ATPase之间的相互作用部位，测量PLB结构的变化和与Ca-ATPase的结合，并研究PLB胞液和跨膜结构域之间关于Ca-ATPase功能调节的结构耦合。我们的具体目标包括：(1)确定对钙转运至关重要的Ca-ATPase的动态结构变化；(2)确定PLB调节Ca-ATPase转运功能的机制；(3)确定参与调节Ca-ATPase转运活性的PLB的结构；(4)确定磷脂调节Ca-ATPase离子转运的机制。确定钙-ATPase功能调节的结构机制将有助于设计有效的治疗方法来减轻衰竭心脏的心功能丧失。

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 include: (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.