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

描述(申请人提供)：Ryanodine受体(RyR)钙通道功能在横纹肌的钙稳态中起着关键作用。RyRs的功能障碍经常导致肌浆钙循环的失调，这与几种肌病和各种形式的心律失常有关。目前公认的是，钙离子是RyRs最重要的激活剂，通过两个独立的结合部位，一个激活和一个抑制，调节通道功能。然而，尽管有大量的研究，到目前为止，这两个钙结合结构域的位置和分子性质仍然很不清楚。这是一个重大的差距，因为RyRs已经成为一个重要的治疗靶点。本研究通过对新发现的RyRs的钙结合/调节结构域进行全面的结构/功能表征来解决这一差距。该提案挑战了经典的两个钙结合位点的概念，提出了一种假说，即钙介导的RyRs调节涉及一类新的钙结合结构域的贡献，该结构域调节细胞的钙激活位点和整体钙循环特性。我们最近使用一种创新的RyR3/RyR1嵌合受体方法发现了RyRs的一个新的离散功能决定因素(称为CBD区域)，该区域在骨骼肌管的通道功能和钙循环调节中发挥核心作用，这一发现支持了这一假说。这些研究表明，在CBD区域内存在一类新的钙结合位点，它在所有RyRs亚型中高度保守。本研究的目的是从分子水平上确定这一新的钙结合结构域，并对其功能进行表征，明确其在正常和肌病状态下成年肌肉钙调节中的作用。在AIM-1中，我们对新的钙结合结构域进行了全面的结构、生化和功能表征。利用荧光光谱、圆二色谱和核磁共振技术，结合靶向突变的方法，我们将绘制并完全定义RyR1的新的钙结合基序。作为功能相关的研究，我们将利用~3H-Ryanodine结合和单通道研究来探索这个钙结合位点的破坏对1)全长RyR的钙敏感特性的影响，以及2)培养的肌管的钙循环特性。在AIM-2中，我们将利用小鼠FDB纤维探索新的钙结合部位在成年骨骼肌钙循环调节中的作用。我们还将把这些研究扩展到肌病小鼠模型，以探索靶向调节新的钙调节结构域作为潜在的治疗载体的翻译价值，以减轻与RyR功能障碍有关的钙循环障碍的影响。这一系列研究试图为未来开发新的治疗方法提供分子基础，以对抗与RyRs调节失调有关的广泛的骨骼和心肌疾病。因此，该申请直接关系到#司的目标。 肌肉骨骼疾病。

DESCRIPTION (provided by applicant): 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.