REGULATION OF CALCIUM TRANSPORT IN CARDIAC MUSCLE

我们的长期目标是确定心肌肌浆网（SR）膜上Ca-ATP酶调节钙再沉积的物理机制。 这种主动转运蛋白的功能是调节心脏中心肌舒张的速率和程度。 受磷蛋白（PLB）与Ca-ATP酶在心脏SR中共表达，并且在β-肾上腺素能刺激之前起作用以抑制Ca-ATP酶的转运活性。 这是我们的假设，正常的催化运动参与运输机制的Ca-ATP酶的调制PLB或膜脂质组成的变化，这些监管机制的改变是心脏病的基础。 因此，所提出的研究的主要目标是鉴定将ATP水解与钙转运偶联的结构变化，以及PLB和膜组合物如何修饰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诱变结合，以探测Ca-ATP酶和PLB上定义位点的蛋白质结构。 第二个目标是确定PLB的结构特征，允许Ca-ATP酶转运功能的调节。 这些测量旨在定义PLB和Ca-ATP酶之间的相互作用位点，测量PLB结构和与Ca-ATP酶结合的变化，并研究PLB的胞质和跨膜结构域之间的结构偶联对Ca-ATP酶功能的调节。 我们的具体目标包括：（1）确定对钙转运重要的Ca-ATP酶的动态结构变化，（2）确定PLB调节Ca-ATP酶转运功能的机制，（3）确定参与调节Ca-ATP酶转运活性的PLB结构，（4）确定磷脂调节Ca-ATP酶离子转运的机制。Ca-ATP酶功能调节的结构机制的鉴定将允许设计有效的治疗方法来减轻衰竭心脏中的心功能丧失。

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.