Organization of Sarcoplasmic Reticulum in Skeletal Muscle

描述（由申请人提供）：横面肌的肌浆网（SR）由两个主要的隔室组成，末端池，其中包含Ca2+通道（ryanodine受体），开放以启动收缩，网络SR，其中包含大量的Ca2+- atp酶（SERCA）负责从肌质中去除Ca2+，导致松弛。在哺乳动物骨骼肌中，这些隔室在每个肌节周围呈典型排列，在A-I连接处有末端池，在m带和z盘周围有网络SR。这两个部分是如何以这种方式组织起来的，人们知之甚少。在这里，我们将重点关注网络SR区室，特别是两种蛋白质在其组织中的可能作用，一种是小的（~17 kDa）形式的锚蛋白，另一种是大的（~800 kDa）的titin超家族蛋白。小锚蛋白，我们称之为sAnk1（也称为Ank1.5），由ANK1基因编码，并集中在网络SR中，其疏水n端序列通过一种尚不清楚的机制靶向。它在那里定向，其c端区域暴露于肌浆，在那里它可以结合黑素蛋白。Obscurin集中在m波段和z盘的外围，这是与sr的网络区相互作用的理想位置。与此一致的是，Obscurin的c端与sAnk1的细胞质结构域结合具有高亲和力。我们假设这种结合对于SR网络在肌节周围组织是必要和充分的。我们将从四个方面来验证这一假设：(1)表征sAnk1在obscurin上的结合位点；(2)模拟sAnk1在自由和结合状态下的obscurin结合位点的三维结构；(3)确定sAnk1对网络SR的特异性靶向依据；(4)确定降低sAnk1水平或改变其与暗素相互作用的能力对网络SR的结构和功能及其与收缩装置的对齐的影响。我们的实验应该揭示一些负责组织sr的基本机制。我们的结果也应该揭示与横纹肌内膜相关的细胞骨架结构的变化如何导致肌病。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肉萎缩症有关，但它们如何在肌肉中起作用仍然知之甚少。本研究旨在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.