Organization of Sarcoplasmic Reticulum in Skeletal Muscle

描述（由申请人提供）：横纹肌的肌浆网（SR）由两个主要隔室组成，即末端池和SR网络，末端池含有打开以启动收缩的Ca 2+通道（兰尼碱受体），SR网络含有大量负责从肌浆中去除Ca 2+的Ca 2 +-ATP酶（SERCA），导致松弛。在哺乳动物骨骼肌中，这些隔室在每个肌节周围立体地排列，在A-I连接处的水平上具有末端池，并且在M带和Z盘周围具有网络SR。这两个部分是如何以这种方式组织起来的，人们还知之甚少。在这里，我们专注于网络SR室，特别是在其组织中的两种蛋白质，一个小（~17 kDa）形式的锚蛋白，和obscurin，一个巨大的（~800 kDa）蛋白的肌联蛋白超家族的可能作用。小锚蛋白，我们称之为sAnk 1（也称为Ank1.5），由ANK 1基因编码，并集中在网络SR中，在那里它通过其疏水性N端序列通过一种尚不清楚的机制靶向。它的C-末端区域暴露于肌浆，在那里它可以结合obscurin。Obscurin集中在M带和Z盘的外围，在那里它理想地位于与SR的网络隔室相互作用。与此一致，obscurin的C-末端以高亲和力结合到sAnk 1的胞质结构域。我们假设，这种结合是必要的，也是足够的网络SR组织周围的肌节。我们将从四个方面来验证这一假设：（1）表征sAnk 1在obscurin上的结合位点;（2）模拟sAnk 1在自由和结合状态下结合位点的三维结构;（3）确定sAnk 1特异性靶向网络SR的基础;（4）确定降低sAnk 1水平或改变其与暗蛋白相互作用的能力对网络SR的结构和功能及其与收缩器官的对齐的影响。我们的实验应该揭示一些负责组织的SR的基本机制。我们的研究结果还应该揭示如何与横纹肌内膜相关的细胞骨架结构的变化可能导致肌病。公共卫生相关性。为了使骨骼肌正常工作，它必须组织和稳定储存和释放钙离子的细胞结构。这些结构的缺陷与肌病和肌营养不良有关，但它们在肌肉中的功能仍然知之甚少。这项计划是研究骨骼肌中储存钙离子的结构。

DESCRIPTION (provided by applicant): The sarcoplasmic reticulum (SR) of striated muscle is comprised of two major compartments, the terminal cisternae, which contain the Ca2+ channels (ryanodine receptors) that open to initiate contraction, and the network SR, which contains much of the Ca2+-ATPase (SERCA) responsible for removing Ca2+ from the myoplasm, leading to relaxation. In mammalian skeletal muscle, these compartments are aligned stereotypically around each sarcomere, with terminal cisternae at the level of the A-I junctions and network SR surrounding M-bands and Z-disks. How these two compartments become organized in this way is poorly understood. Here we focus on the network SR compartment and, in particular, on the possible role in its organization of two proteins, a small (~17 kDa) form of ankyrin, and obscurin, a massive (~800 kDa) protein of the titin superfamily. The small ankyrin, which we refer to as sAnk1 (it is also known as Ank1.5), is encoded by the ANK1 gene and concentrates in the network SR, where it is targeted by its hydrophobic N-terminal sequence thru a mechanism that is not well understood. It is oriented there with its C-terminal region exposed to the myoplasm, where it can bind obscurin. Obscurin is concentrated at the periphery of M-bands and Z-disks, where it is ideally situated to interact with the network compartment of the SR. Consistent with this, the C-terminus of obscurin binds with high affinity to the cytoplasmic domain of sAnk1. We hypothesize that this binding is both necessary and sufficient for the network SR to organize around the sarcomere. We will test this hypothesis in 4 aims: (1) to characterize the binding site on obscurin for sAnk1; (2) to model the 3D structure of obscurin's binding site for sAnk1 in the free and bound states; (3) to determine the basis for the specific targeting of sAnk1 to the network SR; (4) to determine the effect of reducing sAnk1 levels, or altering its ability to interact with obscurin, on the structure and function of the network SR, and its alignment with the contractile apparatus. Our experiments should reveal some of the basic mechanisms responsible for the organization of the SR. Our results should also reveal how changes in cytoskeletal structures associated with internal membranes of striated muscle can lead to myopathies. PUBLIC HEALTH RELEVANCE. For skeletal muscle to function properly, it must organize and stabilize the cellular structures that store and release calcium ions. Defects in these structures have been linked to myopathies and muscular dystrophies, but how they function in muscle is still poorly understood. This proposal is to study the structures in skeletal muscle that store calcium ions.