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

• 批准号: 9045571
• 负责人: Claudio F Perez
• 金额: $ 8.62万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-01 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9045571
• 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; Health; Heart Diseases; Homeostasis; Ions; Knockout Mice; 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; receptor; skeletal; therapeutic target

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.

描述（由申请人提供）：Ryanodine受体（RyR）Ca 2+通道功能在横纹肌的Ca 2+稳态中起关键作用。RyR功能障碍通常导致肌浆Ca 2+循环失调，这与几种肌病和各种形式的致心律失常性心脏疾病有关。 目前公认的是，离子Ca 2+是RyR的单一最重要的激活剂，并且通过两个独立的结合位点（一个激活位点和一个抑制位点）调节通道功能。然而，尽管有许多研究，到目前为止，任何Ca 2+结合结构域的位置和分子特性在很大程度上仍然未知。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。这项研究解决了这一差距，提出了一个全面的结构/功能表征新发现的钙离子结合/调控域的RyRs。该提案挑战了两个钙离子结合位点的经典概念，提出了一个假设，即钙离子介导的调节RyRs涉及一类新的钙离子结合结构域的贡献，调节钙离子激活位点和整体的钙离子循环特性的细胞。这一假设得到了我们最近的研究结果的支持，使用创新的RyR 3/RyR 1嵌合受体的方法，确定了一个新的离散功能决定因素的RyR（命名为CBD区域），发挥了核心作用的通道功能和钙离子循环调节骨骼肌管。这些研究表明，在CBD区域内存在一类新的Ca 2+结合位点，该位点在所有RyR亚型中高度保守。 本提案的目的是分子定义和功能特性，这个新的钙离子结合域，并定义其在正常和肌病条件下的成人肌肉钙离子调节的作用。在目的-1，我们提出了一个全面的结构，生化和功能表征的新的钙离子结合域。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RyR 1的新Ca 2+结合基序。作为功能相关性，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+感应特性和2）培养的肌管的Ca 2+循环特性的影响。在目的-2中，我们将探讨新的钙离子结合位点的钙离子循环调节成年骨骼肌使用小鼠FDB纤维的作用。我们还将这些研究扩展到肌病小鼠模型，以探索新的Ca 2+调节结构域作为潜在治疗载体的靶向调节的翻译价值，以减轻与RyR功能障碍相关的Ca 2+循环失调的影响。这一系列研究旨在为未来开发新的治疗方法提供分子基础，以对抗与RyR失调相关的各种骨骼和心脏肌病。因此，这一申请直接关系到海法司的目标， 肌肉骨骼疾病。

项目成果

Functional and structural characterization of a novel malignant hyperthermia-susceptible variant of DHPR-β1a subunit (CACNB1).

DHPR-β1a 亚基 (CACNB1) 的新型恶性高热敏感变体的功能和结构特征。

• DOI: 10.1152/ajpcell.00187.2017
• 发表时间: 2018
• 期刊: American journal of physiology. Cell physiology
• 作者: Perez,ClaudioF;Eltit,JoseM;Lopez,JoseR;Bodnár,Dóra;Dulhunty,AngelaF;Aditya,Shouvik;Casarotto,MarcoG
• 通讯作者: Casarotto,MarcoG