SMALL ANKYRINS IN EXCITABLE CELLS

可兴奋细胞中的小锚蛋白

• 批准号: 6390622
• 负责人: ROBERT J BLOCH
• 金额: $ 28.85万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-06-01 至 2003-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6390622
• 关键词: ankyrins; calcium ion; cell component structure /function; chickens; chimeric proteins; confocal scanning microscopy; cytoskeleton; developmental genetics; electron microscopy; gene expression; genetically modified animals; laboratory mouse; laboratory rabbit; laboratory rat; ligands; myofibrils; myogenesis; protein binding; protein localization; protein sequence; sarcomeres; sarcoplasmic reticulum; site directed mutagenesis; striated muscles; transfection; yeast two hybrid system

The sarcoplasmic reticulum (SR) is embedded in the myoplasm, where it surrounds the contractile structures of each myofibril. Without a mechanism to stabilize the membrane and to link it at regular intervals to the contractile apparatus, the SR might be damaged during the contractile cycle, causing the uncontrolled release of its stores of Ca2+ and profound affects on muscle structure and function. In healthy muscle, of course, this does not occur, due in part to the presence of regular links between the SR and the Z disks of myofibrils. Here we propose to characterize a protein that may form these connections. We have discovered small, alternatively spliced forms of the structural protein, ankyrin, in striated muscle cells. The small ankyrins (abbreviated sAnk1) are enriched in the SR specifically at sites flanking the M and Z disks of nearby myofibrils. Ankyrin in mammalian erythrocytes stabilizes the cell membrane by linking it to the spectrin-based cytoskeleton. We hypothesize that sAnk1 stabilizes the SR by attaching it to the cytoskeleton of striated muscle cells -- the contractile apparatus. We propose 4 specific aims to test this idea and its developmental and functional consequences. (i) sAnk1 has an unique, highly hydrophobic amino terminal sequence that is likely to anchor it to membranes and target it specifically to the SR. We will perform biochemical and cellular transfection experiments to determine how sAnk1 associates preferentially with the SR. (ii) sAnk1 may concentrate in the SR near M and Z disks as a result of specific binding to proteins present in those structures. We will use the yeast two-hybrid screen to identify these ligands, and immunolabeling at the confocal and ultrastructural levels to localize them in the sarcomere. (iii) If sAnk1 links the SR to the contractile apparatus, it and its ligands should play an important role in the organization and differentiation of the SR in developing muscle. We will use biochemical, immunological and structural approaches to determine how sAnk1 and its ligands are expressed and organized during development. (iv) If sAnk1 is important for sarcoplasmic organization, inhibiting its activity should disrupt the relationship of the SR with the contractile apparatus and alter Ca2+ homeostasis. We will use transgenic techniques to manipulate the levels of sAnk1 and its ligands in muscle cells, and physiological and ultrastructural techniques to assess the effects of these manipulations on the organization and function of the SR. Our studies should therefore help to elucidate the mechanisms responsible for the formation and organization of the SR in immature and adult striated muscle. These mechanisms may have an important role in excitation-contraction coupling, and in maintaining the structure and function of the SR in immature, adult, and aging skeletal muscle.

肌浆网（SR）嵌入肌浆中，包围着每根肌原纤维的收缩结构。如果没有一种机制来稳定膜并将其定期连接到收缩装置，SR可能在收缩周期中受损，导致其Ca2+储存的不受控制的释放，并对肌肉结构和功能产生深远的影响。当然，在健康肌肉中，这种情况不会发生，部分原因是肌原纤维的SR盘和Z盘之间存在规律的联系。在这里，我们提出表征可能形成这些连接的蛋白质。我们在横纹肌细胞中发现了结构蛋白锚蛋白的小的、可选择的剪接形式。小锚定蛋白（缩写为sAnk1）在SR中富集，特别是在附近肌原纤维的M和Z盘两侧。哺乳动物红细胞中的锚蛋白通过将细胞膜连接到基于谱蛋白的细胞骨架来稳定细胞膜。我们假设sAnk1通过将SR附着在横纹肌细胞的细胞骨架（收缩装置）上来稳定SR。我们提出了4个具体目标来测试这一想法及其发展和功能后果。(1) sAnk1具有独特的、高度疏水的氨基末端序列，可能将其锚定在膜上，并特异性靶向SR。我们将进行生化和细胞转染实验，以确定sAnk1如何优先与SR结合。(2)由于与这些结构中存在的蛋白质特异性结合，sAnk1可能集中在靠近M和Z盘的SR中。我们将使用酵母双杂交筛选来识别这些配体，并在共聚焦和超微结构水平上进行免疫标记以定位它们在肌节中的位置。（iii）如果sAnk1将SR连接到收缩器，那么它及其配体应该在SR在肌肉发育过程中的组织和分化中发挥重要作用。我们将使用生化、免疫学和结构方法来确定sAnk1及其配体在发育过程中如何表达和组织。（iv）如果sAnk1对肌浆组织很重要，抑制其活性应破坏SR与收缩器的关系并改变Ca2+稳态。我们将使用转基因技术来操纵肌肉细胞中sAnk1及其配体的水平，并使用生理和超微结构技术来评估这些操作对SR的组织和功能的影响。因此，我们的研究将有助于阐明未成熟和成年横纹肌中SR的形成和组织的机制。这些机制可能在未成熟、成年和衰老骨骼肌的兴奋-收缩耦合以及SR的结构和功能维持中发挥重要作用。