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.

横纹肌的活动是由肌质 Ca2+ [Ca2+]i 的变化驱动的，而肌质 Ca2+ [Ca2+]i 的变化主要由 Ca2+ 外流引起来自肌浆网 (SR) 通过兰尼碱受体启动收缩和 Ca2+ 的再摄取通过肌内质 Ca2+ -ATP 酶 (SERCA) 进入 SR 以启动松弛。总 SR Ca2+ 负荷反过来调节 SERCA 与受磷蛋白结合。 (PLN) 和肌磷脂 (SLN)，降低其对 Ca2+ 的亲和力，PLN 或 SLN 的磷酸化会改变它们的作用。与 SERCA 的相互作用（在短暂的滞后后）会在几分钟内增加其活性。它们将具有出色的生理意义和高频调节 SERCA 的机制（例如，收缩到收缩）尚未描述，这里我们考虑细胞骨架的假设。我们已经证明 obscurin (Obscn) 和小蛋白在骨骼肌中以毫秒为单位调节 SERCA1。锚蛋白 1 (sAnk1) 与 PLN 和 SLN 相互作用，调节骨骼肌和心脏中的 SERCA。约 800 kDa 的肌联蛋白超家族的细胞骨架成员，围绕 M 带和 Z 盘的肌节。 sAnk1 (Ank1.5) 是一种 ~17 kDa 整合膜蛋白，是 ANK1 基因的选择性剪接产物集中在 M 带和 Z 盘周围的 SR 中。值得注意的是，sAnk1 结合 Obscn、PLN、SLN 和。我们直接证明了 SERCA：（i）sAnk1、SERCA 和 SLN 的三向复合物部分消除了 SLN 的抑制作用。 Ca2+-ATPase 活性；(ii) Obscn 增加 SERCA 与 sAnk1 和 SLN 结合的活性； PLN；和（iv）肌病性 Obscn 突变体通过强烈结合 PLN 来增加 SERCA 活性。新假设 Obscn 和 sAnk1 是生物力学传感器，可“调整”SERCA 活动以适应我们假设从肌节通过 Obscn 到 sAnk1 与 sAnk1 复合有直接联系。 SERCA 和 SR 中的 SLN 或 PLN，这样收缩会增加 SERCA 的 ATP 酶活性。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：收缩诱导复合物发生构象变化以激活它 SERCA。我们将在 4 个具体目标中测试我们的假设和模型：(1) 确定 sAnk1、P/SLN 和 SERCA形成复合物调节Ca2+-ATP酶；(2)确定Obscn是否增加Ca2+-ATP酶活性；通过从SERCA解离sAnk1和PLN或SLN，或者通过诱导构象变化 (3) 了解收缩强度和肌浆 Ca2+ 清除率是否存在差异。受 Obscn 与 sAnk1 和 PLN 或 SLN 相互作用的控制；以及 (4) 评估磷酸化的影响；这些实验有可能揭示新的 sAnk1 在调节 SERCA 活性中的作用。调节横纹肌 Ca2+ 稳态的机制，为 SERCA 的作用提供新的见解发挥维持肌肉健康的作用，并提出操纵 SERCA 活动来对抗的新方法肌病。