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通过CA-ATPase在心脏肌浆网（SR）膜中调节钙的重量的物理机制。 这种活跃的转运蛋白功能可调节心脏心肌松弛的速度和程度。 调节蛋白磷团（PLB）与心脏SR中的CA-ATPase共表达，并且在β-肾上腺素能刺激功能之前抑制CA-ATPase的转运活性。 我们的假设是，CA-ATPase运输机制所涉及的正常催化运动是由PLB或膜脂质组成的变化调节的，并且这些调节机制的改变是心脏病下的改变。 因此，提出的研究的主要目标是鉴定将ATP水解为钙转运的结构变化，以及PLB和膜组成如何改变催化重要的结构过渡。 这将涉及使用自旋标签EPR，光学和振动光谱与位置定向的诱变结合在CA-ATPase和PLB上定义的位点探测蛋白质结构。 第二个目标是确定PLB的结构特征，该特征允许调节CA-ATPase运输函数。 这些测量结果将旨在定义PLB与CA-ATPase之间的相互作用位点，测量PLB结构的变化并与CA-ATPase结合，并研究PLB的胞质和跨膜结构域之间的结构耦合，相对于CA-ATPase功能的调节。 我们的具体目的包括：（1）确定CA-ATPase对钙转运重要的动态结构变化，（2）定义PLB调节CA-ATPase转运功能的机制，（3）确定与CA-ATPase转运活性调节的PLB结构，以及（4）定义CA-ATPase转运的磷脂调节机制。鉴定CA-ATPase功能调节的结构机制将允许设计有效的疗法，以减轻心脏失败的心脏功能丧失。