STRUCTURE-FUNCTION OF PHOSPHOLAMBAN IN HEART

磷脂酰心苷在心脏中的结构与功能

• 批准号: 2225518
• 负责人: LARRY R. JONES
• 金额: $ 13.45万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-01-01 至 1996-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2225518
• 关键词: affinity labeling; calcium transporting ATPase; crystallization; enzyme activity; heart function; membrane proteins; phosphorylation; protein isoforms; protein purification; protein structure function; sarcoplasmic reticulum; stoichiometry; tissue /cell culture; transfection

Phospholamban (PLB) is the principal membrane protein of the heart phosphorylated in response to P-adrenergic stimulation. The protein is localized to cardiac sarcoplasmic reticulum (SR), where it exists as a pentamer of small, tightly associated, 52-amino acid subunits. When dephosphorylated, PLB inhibits the Ca pump and Ca transport by cardiac SR. Phosphorylation of PLB at Ser16 and Thr17 disinhibits the Ca pump, stimulates active Ca sequestration, and increases the rate of myocardial relaxation during beta adrenergic activation. Precisely how this detailed regulation occurs is currently unknown. In this application we propose structure/function studies on PLB to define a molecular mechanism of action. Aim 1 will address protein structure in native SR vesicles. The membrane protein topology, pentameric organization, and molar stoichiometry with the Ca pump will be determined. These studies will lay the framework for expression studies correlating protein structure with function. In Aim 2, PLB will be expressed in atrial tumor cells, which contain Ca pumps, but no PLB. Both wild-type and mutated subunits will be expressed. Using this cellular reconstitution system, we will determine which amino acids stabilize the pentamer, and if the pentameric structure is required for Ca pump regulation. Whether the Ca channel activity of PLB is involved in this process will be investigated, and the mechanism of regulation of active Ca transport by the phosphorylation sites will be determined. In Aim 3, mg quantities of PLB will be expressed and purified from insect cells. The goal here is to produce sufficient material for detailed biochemical studies, including protein crystallization to determine the three dimensional structure. Co- expression of PLB with the Ca pump in insect cells will also be attempted. In Aim 4, expression of wild-type and mutant PLB subunits will be targeted to mouse atrium and ventricle in transgenic animals. By overexpressing wild-type PLB, the role of the PLB/Ca pump stoichiometry in controlling the rate of myocardial relaxation will be assessed. By expressing mutant PLB subunits in mouse hearts, we hope to identify transdominant mutations that disrupt the function of the wild- type pentamer. Mutations that destabilize the pentamer and/or alter the phosphorylation sites will be investigated. Thus, the studies will address the cellular and molecular physiology of PLB, from the purified protein level to the whole animal. By correlating experimental results from Aims 1-4 a complete picture of PLB structure and function will emerge. The results will be important for a precise understanding of catecholamine regulation of the heart.

受磷蛋白（PLB）是心脏的主要膜蛋白 在响应于β-肾上腺素能刺激时磷酸化。 述蛋白质是 定位于心肌肌浆网（SR），在那里它作为一种 由52个氨基酸亚基组成的小的、紧密结合的五聚体。 当 去磷酸化后，PLB抑制心肌钙泵和钙转运。 Sr. PLB在Ser 16和Thr 17处的磷酸化抑制钙泵， 刺激活性钙螯合，并增加心肌收缩率， β肾上腺素能激活期间的松弛。 准确地说， 详细的规定目前还不得而知。 在本申请中，我们 建议对PLB进行结构/功能研究，以确定分子机制 的行动。 目的1将解决天然SR囊泡中的蛋白质结构。 膜蛋白拓扑结构，五聚体组织，和摩尔 将确定与Ca泵的化学计量。 这些研究将 为蛋白质结构相关的表达研究奠定框架 功能。 在目标2中，PLB将在心房肿瘤细胞中表达， 其中包含Ca泵，但不包含PLB。野生型和突变亚基 将被表达。 利用这种细胞重组系统，我们将 确定哪些氨基酸使五聚体稳定，并且如果五聚体 钙泵调节需要结构。 Ca通道 活动的公共小巴参与这一进程将被调查， 磷酸化调节活性钙转运的机制 网站将被确定。 在目标3中，PLB的毫克量将是 从昆虫细胞中表达和纯化。 我们的目标是 足够的材料进行详细的生物化学研究，包括蛋白质 结晶以确定三维结构。 共- 在昆虫细胞中用Ca泵表达PLB也将是 企图。 在目的4中，表达野生型和突变型PLB亚基 将靶向转基因动物的小鼠心房和心室。 通过过度表达野生型PLB，PLB/Ca泵的作用被证实。 化学计量学在控制心肌舒张速率中的作用将是 评估。 通过在小鼠心脏中表达突变的PLB亚基，我们希望 识别破坏野生型基因功能的转显性突变， 型五聚体。 使五聚体不稳定和/或改变五聚体的结构的突变可导致突变。 将研究磷酸化位点。因此，研究将 解决PLB的细胞和分子生理学，从纯化的 蛋白质水平对整个动物。 通过关联实验结果 根据目标1-4，公共小巴结构和功能的全貌将 出现。 这些结果对于准确理解 心脏的儿茶酚胺调节。