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Ca2+ regulation in muscle by a new class of Ca2+-binding domain of RyRs

RyRs 的一类新型 Ca2 结合域对肌肉中的 Ca2 进行调节

基本信息

批准号：
8704477
负责人：
Claudio F Perez
金额：
$ 8.61万
依托单位：
BRIGHAM AND WOMEN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-04-01 至 2017-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8704477
关键词：
Abate Address Adult Affect Amino Acids Attenuated Binding Binding Sites Biochemical Biological Assay Calcium Channel Cardiac Cardiovascular Diseases Cells Circular Dichroism Complement Dependence Development Drug Targeting Environment Family Fiber Fluorescence Fluorescence Spectroscopy Functional disorder Future Goals Heart Diseases Homeostasis Ions Lead Length Link Location Maps Mediating Metals Modeling Molecular Molecular Structure Monitor Mus Muscle Muscle Contraction Muscle Fibers Muscle function Musculoskeletal Diseases Mutate Myocardium Myopathy NMR Spectroscopy Names Nuclear Magnetic Resonance Play Property Protein Isoforms Public Health Regulation Research Role RyR1 RyR3 Ryanodine Ryanodine Receptor Calcium Release Channel Ryanodine Receptors Site Skeletal Muscle Striated Muscles Structure Testing Therapeutic Tryptophan base innovation mouse model novel novel therapeutic intervention novel therapeutics public health relevance receptor skeletal therapeutic target

项目摘要

Ryanodine receptor (RyR) Ca2+ channel function plays a critical role in Ca2+ homeostasis of striated muscles. Dysfunction of RyRs often result in dysregulation of myoplasmic Ca2+ cycling that has been associated to several myopathies and various forms of arrhythmogenic cardiac disorders. It is currently accepted that ion Ca2+ is the single most important activator of RyRs and modulate channel function through two independent binding sites, one activatory and one inhibitory. However, despite numerous studies, to this date, the location and molecular properties of either Ca2+-binding domain remains largely unknown. This represents a major gap since RyRs have become an important therapeutic target. This study addresses this gap by proposing a comprehensive structural/functional characterization of a newly found Ca2+-binding/regulatory domain of RyRs. The proposal challenges the classic concept of two Ca2+-binding sites by proposing the hypothesis that Ca2+-mediated regulation of RyRs involves the contribution of a new class of Ca2+-binding domain that modulate the Ca2+-activation site and overall Ca2+-cycling properties of the cell. This hypothesis is supported by our recent findings using an innovative RyR3/RyR1 chimeric receptor approach that identified a new discrete functional determinant of RyRs (named as the CBD region) that plays a central role in channel function and Ca2+-cycling regulation of skeletal myotubes. These studies indicate that within the CBD region resides a new class of Ca2+-binding site that is highly conserved among all isoforms of RyRs. The objective of this proposal is to molecularly define and functional characterize this new Ca2+-binding domain and define its role in Ca2+ regulation of adult muscle under normal and myopathic conditions. In Aim-1 we propose a comprehensive structural, biochemical and functional characterization of the new Ca2+- binding domain. Using Fluorescence Spectroscopy, Circular Dichroism and Nuclear Magnetic Resonance in combination with a targeted mutational approach we will map and fully define the new Ca2+-binding motif of RyR1. As functional correlate we will explore the effects of disruption of this Ca2+-binding site on 1 ) Ca2+-sensing properties of full length RyRs using 3H-ryanodine binding and single channels studies and 2) Ca2+-cycling properties of cultured myotubes. In Aim-2 we will explore the role of the new Ca2+-binding site in Ca2+-cycling regulation of adult skeletal muscles using mouse FDB fibers. We will also extend these studies to a myopathic mouse model to explore the translational value of targeted modulation of the new Ca2+-regulatory domain as potential therapeutic vehicle to abate the effects of Ca2+-cycling dysregulation linked to RyR dysfunction. This line of research seeks to generate the molecular basis for future development of new therapeutic approaches against a wide range of skeletal and cardiac myopathies linked to dysregulation of RyRs. Therefore, this application directly relates to the goals of the Division of Musculoskeletal Diseases.

Ryanodine receptor（RyR）Ca ~（2+）通道功能在纹状体内Ca ~（2+）稳态中起着重要作用肌肉. RyR的功能障碍通常导致肌浆Ca 2+循环的失调，与几种肌病和各种形式的致心律失常性心脏病相关。目前认为Ca 2+是RyRs的唯一最重要的激活剂，并调节通道功能通过两个独立的结合位点，一个激活和一个抑制。但尽管迄今为止，许多研究表明，Ca 2+结合结构域的位置和分子特性仍然存在，大部分未知。这代表了一个主要的差距，因为RyR已成为一个重要的治疗靶点。这一项研究通过提出一种新发现的蛋白质的全面结构/功能表征来解决这一差距。 RyR的Ca 2+结合/调节结构域。该提案挑战了两个Ca 2+结合位点的经典概念通过提出Ca 2+介导的RyRs调节涉及一类新的 Ca 2+结合结构域，其调节细胞的Ca 2+激活位点和整体Ca 2+循环特性。这我们最近使用一种创新的RyR 3/RyR 1嵌合受体的发现支持了这一假设该方法确定了RyR的一个新的离散功能决定簇（命名为CBD区域），在骨骼肌肌管的通道功能和钙循环调节中起核心作用。这些研究这表明CBD区域内存在一类新的Ca 2+结合位点，该位点在CBD中高度保守。 RyR的所有同种型。该提案的目的是从分子上定义和功能上表征这种新的Ca 2+结合结构域，并确定其在正常和肌病条件下的成人肌肉中的Ca 2+调节作用。在Aim-1中我们提出了一个全面的结构，生物化学和功能特性的新的钙2 +- 结合域使用荧光光谱、圆二色性和核磁共振结合靶向突变方法，我们将绘制并完全定义新的Ca 2+结合基序 RyR1作为功能相关物，我们将探索该Ca 2+结合位点的破坏对1）使用3 H-兰尼碱结合和单通道研究的全长RyR的Ca 2+敏感特性， 2)培养肌管的钙循环特性。在Aim-2中，我们将探索新的Ca 2+结合的作用。使用小鼠FDB纤维对成人骨骼肌Ca 2+循环调节的位点。我们还将扩大这些对肌病小鼠模型的研究，以探索靶向调节新的 Ca 2+调节结构域作为潜在的治疗载体，以减轻与Ca 2+循环失调相关的影响 RyR功能障碍这一系列的研究旨在为未来的发展产生分子基础，针对与调节异常相关的广泛骨骼和心肌病的新治疗方法的RyR。因此，这项申请直接关系到肌肉骨骼疾病部门的目标。