Organization of Sarcoplasmic Reticulum in Skeletal Muscle

骨骼肌肌浆网的组织

• 批准号: 7646300
• 负责人: ROBERT J BLOCH
• 金额: $ 29.7万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-01 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7646300
• 关键词: 5' Untranslated Regions; Actinin; Actins; Affinity; Amino Acids; Ankyrins; Binding; Binding Sites; C-terminal; Ca(2+)-Transporting ATPase; Calcium ion; Cellular Structures; Charge; Complex; Cytoplasmic Tail; Defect; Development; Docking; Figs - dietary; Genes; Integral Membrane Protein; Lead; Learning; Ligands; Link; Lipid Bilayers; Mediating; Membrane; Methods; Modeling; Molecular; Molecular Models; Morphology; Muscle; Muscle Cells; Muscular Dystrophies; Mutagenesis; Myofibrillogenesis; Myopathy; Myosin ATPase; N-terminal; Nature; Pharmacy Schools; Physiological; Physiology; Proteins; Relaxation; Role; Ryanodine Receptor Calcium Release Channel; Ryanodine Receptors; Sarcomeres; Sarcoplasmic Reticulum; School Nursing; Site-Directed Mutagenesis; Skeletal Muscle; Small Interfering RNA; Specific qualifier value; Striated Muscles; Structural Protein; Structure; Surface Plasmon Resonance; Testing; Transmembrane Domain; base; connectin; molecular modeling; mutant; network models; obscurin; public health relevance; research study; skeletal; three dimensional structure; uptake; ward

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.

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，其中包含打开以启动收缩的 Ca2+ 通道（兰尼碱受体），以及网络 SR，其中包含许多Ca2+-ATP酶 (SERCA) 负责从肌质中去除 Ca2+，从而导致松弛。在哺乳动物骨骼肌中，这些区室在每个肌节周围按照刻板方式排列，末端池位于 A-I 连接处，网络 SR 围绕 M 带和 Z 盘。人们对这两个隔间如何以这种方式组织起来知之甚少。在这里，我们重点关注网络 SR 区室，特别是两种蛋白质在其组织中可能发挥的作用：一种小型（约 17 kDa）锚蛋白和暗蛋白（肌联蛋白超家族的一种大型（约 800 kDa）蛋白质） 。小锚蛋白，我们称为 sAnk1（也称为 Ank1.5），由 ANK1 基因编码，集中在网络 SR 中，通过其疏水性 N 端序列通过以下机制进行靶向：不太理解。它的 C 末端区域暴露于肌质，在那里它可以结合暗蛋白。 Obscurin 集中在 M 波段和 Z 盘的外围，这里是与 SR 网络隔室交互的理想位置。与此一致的是，obscurin 的 C 末端以高亲和力与 sAnk1 的细胞质结构域结合。我们假设这种结合对于网络 SR 围绕肌节组织起来既必要又充分。我们将在 4 个目标中检验这一假设：(1) 表征 sAnk1 在 obscurin 上的结合位点； (2) 模拟 sAnk1 在游离态和结合态下 obscurin 结合位点的 3D 结构； (3)确定sAnk1具体针对网络SR的依据； (4) 确定降低 sAnk1 水平或改变其与 obscurin 相互作用的能力对 SR 网络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 SR 组织的基本机制。我们的结果还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。为了使骨骼肌正常发挥作用，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症有关，但它们在肌肉中的功能仍知之甚少。该提案旨在研究骨骼肌中储存钙离子的结构。