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.

横纹肌的活动是由肌浆内钙离子[Ca~(2+)]i的变化驱动的，这种变化主要是由于Ca~(2+)外流 通过兰尼定受体从肌浆网(SR)启动收缩，并重新摄取钙离子 通过肌浆网钙-ATPase(SERCA)进入SR，启动松弛。SERCA对[钙]i的调节作用 和总的肌浆网钙负荷，这反过来又调节收缩强度。SERCA与磷蛋白结合 (PLN)和肌磷脂(SLN)，这降低了它对钙的亲和力。PLN或SLN的磷酸化改变它们的 与SERCA的相互作用(在短暂的滞后之后)在许多分钟的时间内增加其活动。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)还没有被描述。这里我们考虑一种假设，即细胞骨架 在毫秒的时间尺度上调节骨骼肌中的SERCA1。我们已经证明了暗黑(Obscn)和小的 Ankyrin 1(SAnk1)与PLN和SLN相互作用，调节骨骼肌和心肌中的SERCA。Obscn是一种 ~800 kDa的细胞骨架，是肌动蛋白超家族中的一员，包围着M带和Z盘处的肌节。 SAnk1(Ank1.5)是ANK1基因的一个~17 kDa的完整膜蛋白和选择性剪接产物 它集中在M波段和Z盘周围的SR中。值得注意的是，sAnk1与Obscn、PLN、SLN和 直接使用SERCA。结果表明：(1)sAnk1、SERCA和SLN的三向复合体可部分阻断SLN的抑制作用 (Ii)Obscn增加sAnk1和SLN结合的SERCA的活性；(Iii)sAnk1结合 和(Iv)一个肌病Obscn突变体通过与PLN的强烈结合而增加SERCA活性。我们在这里测试 新的假设认为Obscn和sAnk1是生物力学传感器，可以将SERCA活性与 收缩的机械应力。我们假设从肌节到sAnk1的直接联系，通过Obscn复杂 SERCA和SR中的SLN或PLN，因此收缩使SERCA的ATPase活性增加。我们 考虑两种可能的模型：模型1：收缩导致sAnk1和SLN或PLN从 SERCA激活它；模型2：收缩导致复合体的构象变化激活 SERCA。我们将在4个具体目标上检验我们的假设和模型：(1)确定sAnk1、P/SLN和 SERCA形成复合体调节Ca~(2+)-ATPase；(2)确定Obscn是否增加Ca~(2+)-ATPase活性 通过将sAnk1和PLN或SLN从SERCA中分离出来，或通过诱导SERCA的构象变化 复杂；(3)了解肌浆的收缩强度和钙清除速率是否 由Obscn与sAnk1和PLN或SLN的相互作用控制；以及(4)评估磷酸化的效果 SAnk1、S在调节SERCA活性中的作用这些实验有可能揭示新奇的东西 调节横纹肌钙稳态的机制，为SERCA的作用提供新的见解 在保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。