REGULATORY MECHANISMS OF CARDIAC MUSCLE

心肌的调节机制

• 批准号: 2668724
• 负责人: HERBERT C CHEUNG
• 金额: $ 21.53万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-03-01 至 2001-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2668724
• 关键词: calcium; chemical binding; chemical kinetics; chickens; conformation; fluorescence spectrometry; genetic regulation; laboratory rabbit; muscle contraction; myocardium; phosphoproteins; protein structure function; troponin

Muscle contraction results from cyclic interactions between actin and myosin. The magnitude of developed force is a function of the number actin-myosin contacts that are formed. This interaction in vertebrate striated muscle is regulated by the binding of activator calcium to the NH/2-domain of troponin C (TnC), and this binding results in a series of changes in both the structures of the proteins associated with the thin filament and the energetics of interactions among those proteins. The details of the immediate molecular events resulting from activator calcium binding are still poorly understood. Modeling studies suggest that activator calcium induces substantial reorientations of several helices located in the NH/2-terminal domain of TnC, resulting in an "open" conformation. This conformation in turn provides specific sites for a strong interaction of TnC with troponin I (TnI). This strong interaction is believed to be responsible for triggering the actin-myosin interaction, thus activating actomyosin ATPase and contractile events. TnI from cardiac muscle has an activity which is related to phosphorylation of the protein in the NH/2-terminal segment and which is not found in skeletal muscle TnI. While the sites of phosphorylation are known, little is known about how this segment may modulate Ca2+ signaling in thin-filament regulation. This application addresses certain aspects of the calcium regulatory mechanism in cardiac muscle. The proposed work has four major goals. (1) The first is identification of pairs of specific residues or helices on TnC that move relative to each other resulting from activator calcium binding. Equilibrium experiments will be done using fluorescence resonance energy transfer (FRET) to establish such calcium-induced conformational changes, followed by transient kinetic experiments to time- resolve the conformational transitions. TnC mutants from cardiac muscle and cardiac-skeletal chimeras will be used for these experiments. (2) The kinetic mechanism of reversible binding of activator calcium to cardiac TnC and the cardiac regulatory system will be studied using transient kinetic methods. It is expected that these kinetic results additionally can be used to track movements of helices in the NH/2-domain. (3) Cardiac TnI has an extension at the NH/2-terminus which is absent in skeletal TnI. We will investigate the relationship of phosphorylation of this extension with its overall conformation. Full-length recombinant mutants and truncated mutants of TnI will be used in FRET, hydrodynamic, and binding studies. (4) The last goal will investigate the relationships of amino acid sequences of the calcium-binding loops and flanking helices in the NH/2-domain of TnC with binding affinity and biological activities. Cardiac muscle TnC mutants with specific alterations in amino acid sequences will be designed and used for this goal. The proposed studies are expected to advance our knowledge of the molecular determinants that are important in thin filament regulation of cardiac muscle. We also expect that the anticipated results may provide a basis to understand the extent to which the calcium triggering mechanism plays a role in abnormal cardiac functions.

肌肉收缩是肌动蛋白和肌动蛋白循环作用的结果 肌球蛋白。发展的力量的大小是数字的函数 肌动蛋白-肌球蛋白接触形成。这种相互作用在脊椎动物中 横纹肌受激活剂钙结合的调节 肌钙蛋白C(TNC)的NH/2-结构域，这种结合导致了一系列 与薄荷相关的蛋白质结构的变化 细丝和这些蛋白质之间相互作用的能量学。这个 激活剂钙引起的即时分子事件的细节 人们对结合仍然知之甚少。建模研究表明， 激活剂钙能显著改变几个螺旋的方向 位于TNC的NH/2-末端结构域中，导致了一种“开放” 构象。这种构象反过来又为 TnC与肌钙蛋白I(TnI)有很强的相互作用。这种强烈的相互作用 被认为是触发肌动蛋白-肌球蛋白相互作用的原因， 从而激活肌动蛋白ATPase和收缩事件。TNI发件人 心肌有一种与磷酸化有关的活性。 NH/2-末端片段中的蛋白质，在骨骼中没有发现 肌肉TnI。虽然磷酸化的部位是已知的，但人们对此知之甚少。 关于这个片段如何调节细丝中的钙信号 监管。 本申请涉及钙调节的某些方面 心肌中的作用机制。拟议的工作有四个主要目标。(1) 第一个是鉴定特定残基或螺旋对。 由激活剂钙引起的相对于彼此移动的TNC 有约束力的。将使用荧光法进行平衡实验 建立这种钙诱导的共振能量转移(FRET) 构象变化，然后是时间的瞬变动力学实验- 解决构象转变。来自心肌的TNC突变体 心脏-骨骼嵌合体将用于这些实验。(2) 激活剂钙与心脏可逆结合的动力学机制 TNC和心脏调节系统将使用瞬变 动力学方法。预计这些动力学结果还会 可用于跟踪NH/2-结构域中螺旋的运动。(3)心脏 TnI在NH/2末端有一个延伸，这在骨骼TnI中是不存在的。 我们将研究这一延伸的磷酸化关系。 它的整体构象。全长重组突变体和 TnI的截短突变体将用于FRET、流体力学和结合 学习。(4)最后一个目标是研究氨基的相互关系。 钙结合环和侧翼螺旋的酸序列 TNC的NH/2结构域具有结合亲和力和生物活性。 氨基酸特异性改变的心肌TNC突变体 将为这一目标设计和使用序列。建议进行的研究 有望促进我们对分子决定因素的了解 在心肌细丝调节中起重要作用。我们也 期望预期的结果可以为理解 钙离子触发机制在异常中的作用程度 心脏功能。