Calmodulin regulation of SR calcium release channels

描述（由申请人提供）： 此应用程序是一个独立的科学家发展奖（K 02）博士布拉德利弗鲁恩。该提案的目标是通过建立在肌肉膜生物化学/离子通道生理学方面的专业知识来促进PI的独立研究生涯，同时促进开发新的生物物理方法来理解控制肌肉收缩的通道调节蛋白。肌肉收缩是由肌浆网（SR）通过称为兰尼碱受体（RYR）的大分子通道复合物释放的Ca 2+触发的。长期目标是确定控制骨骼肌（RYR 1）和心肌（RYR 2）中表达的RYR亚型的分子机制。目前的目标集中在定义钙调素（CAM）作为RYRs的调节亚基，调节Ca 2+通道激活和抑制的机制。目的探讨钙调素对RYR 1和RYR 2通道亚型特异性调控的机制。目的II将定义CaM Met残基氧化改变CaM与RYR 1和RYR 2通道的生产性相互作用的机制。目的III将表征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。目的IV设计钙调素的荧光衍生物，用于解析通道调节蛋白的结构动力学。新发现的点突变体的钙调素，选择性地取消通道激活或抑制将提供独特的工具，定义的机制，钙调素作为一个分子开关调节的RYRs。基于诱变和定点标记的结构数据将得到表征从猪心肌和骨骼肌中分离的RYR通道功能活性的一系列测定的支持。RYR 1 R615 C猪提供了一个有价值的模型，以检查机制，其中自然发生的RYR突变破坏结构功能的大分子通道复合物在恶性高热和相关的通道病变。确定RYR调节蛋白的作用是理解控制肌肉中Ca 2+的机制的主要挑战，并且假定这些调节蛋白的改变的结合有助于在氧化应激、肌肉疲劳和心脏病期间受损的收缩性能。拟议的研究将进一步定义肌肉钙调节的结构-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.