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(申请人提供)：横纹肌的肌浆网(SR)由两个主要部分组成：终端池和网络SR，前者包含开放以启动收缩的钙通道(Ryanodine受体)，后者包含大量负责从肌浆中清除钙离子从而导致松弛的钙离子-ATPase(SERCA)。在哺乳动物骨骼肌中，这些间隔通常围绕每个肌节排列，末端池位于A-I连接水平，网络SR围绕M带和Z盘。这两个隔间是如何以这种方式组织起来的，人们知之甚少。在这里，我们集中在网络SR间隔，特别是在它的两个蛋白质的组织中可能发挥的作用，一个小的(~17 kDa)的锚蛋白，和一个巨大的(~800 kDa)的Titin超家族的蛋白质Oblcurin。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，在那里它通过一种尚不清楚的机制被其疏水的N末端序列靶向。它定位在那里，其C-末端区域暴露在肌浆中，在那里它可以结合遮盖素。Obscurin集中在M-带和Z-盘的外围，在那里它是与SR的网络隔间相互作用的理想位置。与此一致的是，Oblcurin的C末端与sAnk1的细胞质结构域具有高亲和力。我们假设这种结合是网络SR围绕肌节组织的必要条件和充分条件。我们将从四个方面验证这一假说：(1)表征sAnk1在Oblcurin上的结合部位；(2)模拟sAnk1在自由和结合状态下结合部位的3D结构；(3)确定sAnk1特异性靶向网络SR的基础；(4)确定降低sAnk1水平或改变其与Oblcurin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准的影响。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。与公共卫生相关。为了使骨骼肌功能正常，它必须组织和稳定储存和释放钙离子的细胞结构。这些结构的缺陷被认为与肌病和肌营养不良症有关，但它们在肌肉中的功能仍然知之甚少。这项建议是为了研究骨骼肌中储存钙离子的结构。