Cytoskeletal Regulation of SERCA in Muscle

SERCA 在肌肉中的细胞骨架调节

基本信息

批准号：
10463614
负责人：
ROBERT J BLOCH
金额：
$ 55.55万
依托单位：
UNIVERSITY OF MARYLAND BALTIMORE
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-08-10 至 2025-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10463614
关键词：
ANK1 gene ATP phosphohydrolase Adrenergic Agents Affinity Alternative Splicing Anisotropy Ankyrins Binding Biological Assay Biomechanics COS Cells Ca(2+)-Transporting ATPase Calcium Cell membrane Chimeric Proteins Complement Complex Cyclic AMP Cyclic AMP-Dependent Protein Kinases Cytoskeletal Proteins Cytoskeleton Dissociation Enzymes Fluorescence Fluorescence Resonance Energy Transfer Frequencies Functional disorder Health Heart Homeostasis In Situ Integral Membrane Protein Kinetics Learning Link Measures Mechanical Stress Membrane Metabolic Clearance Rate Methods Modeling Molecular Conformation Muscle Muscle Contraction Muscle Proteins Myopathy Pathogenicity Pathway interactions Periodicity Phosphorylation Physiological Play Positioning Attribute Protein Splicing Proteins Pump Regulation Relaxation Role Ryanodine Receptor Calcium Release Channel SERCA1 Sarcolemma Sarcomeres Sarcoplasmic Reticulum Signal Transduction Skeletal Muscle Stress Striated Muscles Testing Time Tubular formation combat connectin experimental study insight member millisecond mutant novel obscurin overexpression phospholamban reuptake sarcolipin sensor skeletal therapeutic target uptake

项目摘要

Activity in striated muscle is driven by changes in myoplasmic Ca2+ [Ca2+]i that arise largely from Ca2+ efflux from the sarcoplasmic reticulum (SR) via the ryanodine receptor to initiate contraction, and reuptake of Ca2+ into the SR via the sarco-endoplasmic Ca2+ -ATPase (SERCA) to initiate relaxation. SERCA modulates [Ca2+]i and the overall SR Ca2+ load, which in turn regulates contractile strength. SERCA binds to phospholamban (PLN) and sarcolipin (SLN), which reduce its affinity for Ca2+. Phosphorylation of PLN or SLN alters their interaction with SERCA that (after a short lag) increases its activity over a period of many minutes. Although they would make excellent physiological sense, mechanisms to regulate SERCA at high frequencies (e.g., contraction to contraction) have not been described. Here we consider the hypothesis that the cytoskeleton regulates SERCA1 in skeletal muscle on a msec time scale. We have shown that obscurin (Obscn) and small ankyrin 1 (sAnk1) interact with PLN and SLN to regulate SERCA in skeletal muscle and heart. Obscn is an ~800 kDa cytoskeletal member of the titin superfamily that surrounds sarcomeres at M-bands and Z-disks. sAnk1 (Ank1.5) is a ~17 kDa integral membrane protein and alternatively spliced product of the ANK1 gene that concentrates in the SR around M-bands and Z-disks. Remarkably, sAnk1 binds Obscn, PLN, SLN and SERCA directly. We show: (i) the 3-way complex of sAnk1, SERCA and SLN partially ablates SLN’s inhibition of Ca2+-ATPase activity; (ii) Obscn increases the activity of SERCA bound to sAnk1 and SLN; (iii) sAnk1 binds PLN; and (iv) a myopathic Obscn mutant increases SERCA activity by avidly binding PLN. Here we test the novel hypothesis that Obscn and sAnk1 are biomechanical sensors that “tune” SERCA activity to the mechanical stress of contraction. We posit a direct link from sarcomeres, thru Obscn to sAnk1 complexed with SERCA and either SLN or PLN in the SR, such that contraction increases SERCA’s ATPase activity. We consider 2 possible models: Model 1: Contraction leads to the dissociation of sAnk1 and SLN or PLN from SERCA to activate it; Model 2: Contraction induces a conformational change in the complex to activate SERCA. We will test our hypothesis and models in 4 Specific Aims: (1) To determine if sAnk1, P/SLN and SERCA form complexes to regulate Ca2+-ATPase; (2) To determine if Obscn increases Ca2+-ATPase activity by dissociating sAnk1 and PLN or SLN from SERCA, or by inducing a conformational change in the complex; (3) To learn if the strength of contraction and the rates of Ca2+ clearance from the myoplasm are governed by Obscn’s interactions with sAnk1 and PLN or SLN; and (4) To assess the effect of phosphorylation on sAnk1’s role in regulating SERCA activity. These experiments have the potential to reveal novel mechanisms regulating Ca2+ homeostasis in striated muscle, to offer fresh insights into the role that SERCA plays in maintaining muscle health, and to suggest novel ways to manipulate SERCA’s activity to combat myopathy.

肌肉肌肉中的活性是由大部分来自Ca2+外排的肌质Ca2+ [Ca2+] i的变化驱动的通过ryanodine受体从肌质网（SR）开始收缩，然后再摄取Ca2+ 通过SARCO-内质Ca2+ -ATPase（SERCA）进入SR，以开始放松。 SERCA调制[Ca2+] i 以及总体SR Ca2+负载，反过来调节收缩力。 SERCA与磷兰班结合（PLN）和肌磷脂（SLN），它们减少了对Ca2+的亲和力。 PLN或SLN的磷酸化改变了它们与SERCA的相互作用（短暂滞后后）在几分钟内增加了其活性。虽然它们将具有极好的物理意义，以高频调节SERCA的机制（例如，尚未描述收缩的收缩）。在这里，我们考虑了细胞骨架的假设根据MSEC时间尺度调节骨骼肌中的SERCA1。我们已经证明了晦涩的（淫秽）和小 Ankyrin 1（Sank1）与PLN和SLN相互作用，以调节骨骼肌和心脏中的SERCA。淫秽是一个〜800 kDa titin超家族的细胞骨架成员，周围环境在M频段和Z-Disks上。 SANK1（ANK1.5）是〜17 kDa积分膜蛋白，替代ANK1基因的剪接产物这将集中在M带和Z磁盘周围的SR中。值得注意的是，sank1结合了淫秽，PLN，SLN和直接Serca。我们显示：（i）SANK1，SERCA和SLN的三向综合体部分消灭SLN的抑制作用 Ca2+-ATPase活性；（ii）淫秽增加了与sank1和sln结合的Serca的活性；（iii）sank1结合 PLN; （iv）肌病性淫秽突变体通过狂热的结合PLN增加了SERCA活性。我们在这里测试淫秽和sank1的新型假设是“调整” Serca活性的生物力学传感器收缩的机械应力。我们指出了肉瘤的直接联系 Serca和SR中的SLN或PLN，因此收缩会增加SERCA的ATPase活性。我们考虑2种可能的模型：模型1：收缩导致sank1和sln或PLN从 Serca激活它；模型2：收缩诱导复合物的构象变化以激活 Serca。我们将在4个特定目的中测试我们的假设和模型：（1）确定是否sank1，p/sln和 SERCA形成复合物以调节Ca2+-ATPase；（2）确定淫秽是否增加了Ca2+-ATPase活性通过将sank1和pln或SLN与SERCA分离，或通过引起构象变化复杂的; （3）了解收缩强度和肌质的Ca2+清除率是否是受淫秽与Sank1和PLN或SLN的互动；（4）评估磷酸化的影响关于Sank1在确定SERCA活动中的作用。这些实验有可能揭示新颖的调节条纹肌肉中CA2+稳态的机制，以提供有关Serca角色的新见解在维持肌肉健康方面发挥作用，并提出新的方法来操纵Serca的活动以对抗肌病。