Calmodulin regulation of SR calcium release channels

描述(由申请人提供)： 本申请是为Bradley Fruen博士颁发的独立科学家发展奖(K02)。这项建议的目标是通过建立肌膜生物化学/离子通道生理学的专业知识来促进PI的独立研究事业，同时促进新的生物物理方法的发展，以了解控制肌肉收缩的通道调节蛋白。肌肉收缩是由肌浆网(SR)通过被称为兰尼定受体(RyR)的大分子通道复合体(RyR)释放钙而触发的。一个长期的目标是确定控制在骨骼肌(RYR1)和心肌(RYR2)中表达的RyR亚型的分子机制。目前的目标集中在确定钙调蛋白(CAM)作为RYRs的调节亚单位的机制，调节钙离子对通道的激活和抑制。目的研究钙调素(CaM)对RYR1和RYR2通道异构体特异性调控的机制。AIM II将确定CaM Met残基氧化改变CaM与RYR1和RYR2通道的生产性相互作用的机制。目的III将描述RYRs上CaM和FKBP位点之间的变构相互作用，以及肾上腺素能刺激对它们的调节。AIM IV将设计CaM的荧光衍生物来解析通道调节蛋白的结构动力学。最近发现的选择性地取消通道激活或抑制的CaM点突变将为确定CaM作为调节RYRs的分子开关的机制提供独特的工具。基于突变和定点标记的结构数据将得到一系列分析的支持，这些分析表征了从猪心肌和骨骼肌分离的RyR通道的功能活性。RYR1R615C猪提供了一个有价值的模型来研究在恶性高热和相关的通道病变中自然发生的RyR突变破坏大分子通道复合体的结构和功能的机制。确定RyR调节蛋白的作用是理解肌肉中钙离子控制机制的主要挑战，这些调节蛋白的结合改变被认为是在氧化应激、肌肉疲劳和心脏病期间导致收缩性能受损的原因。拟议的研究将进一步确定作为肌肉钙调节基础的结构-功能关系，并有助于开发治疗神经肌肉和心血管疾病的新策略。

DESCRIPTION (provided by applicant): This application is for an Independent Scientist Development Award (K02) for Dr. Bradley Fruen. The goal of this proposal is to promote the PI's independent research career by building on expertise in muscle membrane biochemistry/ion channel physiology, while fostering the development of novel biophysical approaches to understanding channel regulatory proteins that control muscle contraction. Muscle contraction is triggered by Ca2+ release from the sarcoplasmic reticulum (SR) via a macromolecular channel complex known as the ryanodine receptor (RYR). A long-term objective is to define the molecular mechanisms that control the RYR isoforms expressed in skeletal muscle (RYR1) and cardiac muscle (RYR2). Current aims focus on defining mechanisms by which calmodulin (CAM) acts as a regulatory subunit of the RYRs, modulating both channel activation and inhibition by Ca2+. Aim I will determine the mechanism underlying the isoform-specific regulation of RYR1 and RYR2 channels by CaM. Aim II will define the mechanism by which CaM Met residue oxidation alters productive interactions of CaM with RYR1 and RYR2 channels. Aim III will characterize allosteric interactions between CaM and FKBP sites on the RYRs, and their modulation by adrenergic stimulation. Aim IV will engineer fluorescent derivatives of CaM for resolving channel regulatory protein structural dynamics. Newly identified point mutants of CaM that selectively abolish either channel activation or inhibition will provide unique tools for defining the mechanism by which CaM functions as a molecular switch modulating the RYRs. Structural data based on mutagenesis and site-directed labeling will be supported by a battery of assays characterizing the functional activity of RYR channels isolated from pig cardiac and skeletal muscle. The RYR1 R615C pig provides a valuable model to examine mechanisms by which naturally occurring RYR mutations disrupt structure-function of the macromolecular channel complex in malignant hyperthermia and related channelopathies. Defining the role of RYR regulatory proteins is major challenge in understanding of the mechanisms that control Ca 2+ in muscle, and altered binding of these regulatory proteins is postulated to contribute to impaired contractile performance during oxidative stress, muscle fatigue, and heart disease. Proposed studies will further define structure-function relationships that underlie Ca regulation m muscle, and aid in the development of new strategies for treating neuromuscular and cardiovascular disease.