SMALL ANKYRINS IN EXCITABLE CELLS

肌浆网(SR)嵌于肌浆中，包围着每个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可能会在收缩周期中受到破坏，导致其储存的钙不受控制地释放，从而对肌肉结构和功能产生深远的影响。当然，在健康的肌肉中，这种情况不会发生，部分原因是肌原纤维的SR和Z盘间存在规则的联系。在这里，我们建议对一种可能形成这些联系的蛋白质进行表征。我们已经在横纹肌肉细胞中发现了小的、可选择性剪接的结构蛋白--锚蛋白。小粘蛋白(缩写为sAnk1)在肌原纤维的M盘和Z盘两侧的SR中特别丰富。哺乳动物红细胞中的锚蛋白通过连接到基于血影蛋白的细胞骨架来稳定细胞膜。我们假设，sAnk1通过将SR连接到横纹肌细胞的细胞骨架--收缩装置来稳定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。(I)sAnk1有一个独特的、高度疏水的氨基末端序列，很可能将其锚定在膜上，并将其特异性地靶向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。(Ii)sAnk1可能由于与这些结构中存在的蛋白质特异性结合而集中在M和Z圆盘附近的SR中。我们将使用酵母双杂交筛选来鉴定这些配体，并在共焦和超微结构水平上进行免疫标记，以将它们定位于肌节。(3)如果sAnk1将SR与收缩装置联系在一起，它及其配体将在发育肌肉中SR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是如何表达和组织的。(4)如果sAnk1对肌浆组织是重要的，抑制它的活性应该破坏SR与收缩装置的关系，改变钙稳态。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纵对SR的组织和功能的影响。因此，我们的研究应该有助于阐明未成熟和成年横纹肌中SR的形成和组织机制。这些机制可能在兴奋-收缩偶联以及维持未成熟、成年和衰老骨骼肌SR的结构和功能方面发挥重要作用。

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.