Calmodulin regulation of SR calcium release channels

描述（由申请人提供）： 本申请旨在为 Bradley Fruen 博士申请独立科学家发展奖 (K02)。该提案的目标是通过建立肌肉膜生物化学/离子通道生理学方面的专业知识来促进 PI 的独立研究事业，同时促进新型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) 通过称为兰尼碱受体 (RYR) 的大分子通道复合物释放 Ca2 来触发的。长期目标是确定控制骨骼肌 (RYR1) 和心肌 (RYR2) 中表达的 RYR 亚型的分子机制。目前的目标集中于确定钙调蛋白 (CAM) 作为 RYR 调节亚基的机制，调节 Ca2 通道的激活和抑制。目标我将确定 CaM 对 RYR1 和 RYR2 通道同种型特异性调节的潜在机制。目标 II 将定义 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。目标 III 将表征 RYR 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，以解决通道调节蛋白结构动力学问题。新发现的 CaM 点突变体可以选择性地消除通道激活或抑制，这将为定义 CaM 作为调节 RYR 分子开关的机制提供独特的工具。基于诱变和定点标记的结构数据将得到一系列表征从猪心肌和骨骼肌中分离的 RYR 通道功能活性的分析的支持。 RYR1 R615C 猪提供了一个有价值的模型来研究自然发生的 RYR 突变破坏恶性高热和相关通道病中大分子通道复合物的结构功能的机制。定义 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，并且这些调节蛋白的结合改变被认为会导致氧化应激、肌肉疲劳和心脏病期间收缩性能受损。拟议的研究将进一步明确肌肉中钙调节的结构-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.