REGULATION OF CALCIUM TRANSPORT IN CARDIAC MUSCLE

心肌钙转运的调节

• 批准号: 6652029
• 负责人: THOMAS Comey SQUIER
• 金额: $ 47.86万
• 依托单位: BATTELLE PACIFIC NORTHWEST LABORATORIES
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-04-01 至 2005-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6652029
• 关键词: adenosine triphosphate; calcium flux; calcium transporting ATPase; chemical association; chemical kinetics; electron spin resonance spectroscopy; enzyme induction /repression; fluorescence resonance energy transfer; fluorescent dye /probe; heart function; hydrolysis; infrared spectrometry; interferometry; laboratory rabbit; membrane lipids; membrane structure; molecular dynamics; molecular site; myocardium; phospholamban; phosphorylation; protein binding; protein structure function; sarcoplasmic reticulum; site directed mutagenesis; structural biology

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.

我们的长期目标是确定心脏肌浆网 (SR) 膜中 Ca-ATP 酶调节钙再吸收的物理机制。 这种主动转运蛋白的作用是调节心脏心肌舒张的速率和程度。 调节蛋白受磷蛋白 (PLB) 在​​心脏 SR 中与 Ca-ATP 酶共表达，并且在 β-肾上腺素能刺激之前发挥作用，抑制 Ca-ATP 酶的转运活性。 我们假设 Ca-ATP 酶转运机制中涉及的正常催化运动受到 PLB 或膜脂组成变化的调节，并且这些调节机制的改变是心脏病的基础。 因此，本研究的主要目标是确定将 ATP 水解与钙转运耦合的结构变化，以及 PLB 和膜组成如何改变催化重要的结构转变。 这将涉及使用自旋标记 EPR、光学和振动光谱以及定点诱变来探测 Ca-ATP 酶和 PLB 上指定位点的蛋白质结构。 第二个目标是确定 PLB 的结构特征，以调节 Ca-ATP 酶转运功能。 这些测量旨在确定 PLB 和 Ca-ATPase 之间的相互作用位点，测量 PLB 结构和与 Ca-ATPase 结合的变化，并研究 PLB 的胞质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) 确定对钙转运重要的 Ca-ATP 酶的动态结构变化，(2) 定义 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，以及 (4) 定义磷脂调节 Ca-ATP 酶离子转运的机制。确定 Ca-ATP 酶功能调节的结构机制将允许设计有效的疗法来减轻衰竭心脏中心脏功能的丧失。