Cytoskeletal Regulation of SERCA in Muscle

SERCA 在肌肉中的细胞骨架调节

• 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
• 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

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.

横纹肌中的活动由肌浆Ca 2 + [Ca 2 +]i的变化驱动，其主要由Ca 2+流出引起 从肌浆网（SR）通过ryanodine受体启动收缩和Ca 2+的再摄取 通过肌质-内质网Ca 2 + -ATP酶（SERCA）进入SR以启动松弛。SERCA调节[Ca 2 +]i 和整体SR Ca 2+负荷，这反过来又调节收缩强度。SERCA与受磷蛋白结合 (PLN)和肌磷脂（SLN），其降低其对Ca 2+的亲和力。PLN或SLN的磷酸化改变了它们的 与SERCA的相互作用（在短暂的滞后后）在许多分钟的时间内增加其活性。虽然 它们将具有极好的生理意义，在高频下调节SERCA的机制（例如， 收缩到收缩）没有被描述。在这里，我们考虑的假设， 在毫秒时间尺度上调节骨骼肌中的SERCA 1。我们已经证明，暗（Obscn）和小 锚蛋白1（sAnk 1）与PLN和SLN相互作用以调节骨骼肌和心脏中的SERCA。Obscn是一个 肌联蛋白超家族的~800 kDa细胞骨架成员，在M带和Z盘处围绕肌节。 sAnk 1（Ank1.5）是一种约17 kDa的膜整合蛋白，是ANK 1基因的可变剪接产物 集中在M带和Z盘附近的SR中。值得注意的是，sAnk 1结合Obscn、PLN、SLN和 SERCA直接我们发现：（i）sAnk 1、SERCA和SLN的三元复合物部分消除了SLN的抑制作用 （ii）Obscn增加SERCA与sAnk 1和SLN结合的活性;（iii）sAnk 1结合 （iv）肌病Obscn突变体通过强烈结合PLN增加SERCA活性。在这里我们测试 Obscn和sAnk 1是生物力学传感器的新假设， 收缩的机械应力我们发现了从肌节，通过Obscn到sAnk 1的直接联系， 收缩增加了SERCA和SR中的SLN或PLN，使得收缩增加了SERCA的ATPase活性。我们 考虑2种可能的模型：模型1：收缩导致sAnk 1和SLN或PLN从 模型2：收缩诱导复合物中的构象变化以激活 塞卡。我们将在4个具体目标中检验我们的假设和模型：（1）确定sAnk 1，P/SLN和 SERCA形成复合物调节Ca ~（2+）-ATP酶活性：（2）观察Obscn是否增加Ca ~（2+）-ATP酶活性 通过从SERCA中解离sAnk 1和PLN或SLN，或通过诱导SERCA中的构象变化， （3）了解收缩强度和Ca ~（2+）从肌浆中清除的速率是否与心肌收缩强度和Ca ~（2+）从肌浆中清除的速率有关。 受Obscn与sAnk 1和PLN或SLN的相互作用的控制;和（4）评估磷酸化的影响 sAnk 1在调节SERCA活性中的作用。这些实验有可能揭示新的 调节横纹肌中Ca 2+稳态的机制，为SERCA的作用提供新的见解， 在维持肌肉健康方面起着重要作用，并提出了新的方法来操纵SERCA的活动， 肌病