Calmodulin regulation of SR calcium release channels

钙调蛋白对 SR 钙释放通道的调节

• 批准号: 6917114
• 负责人: BRADLEY R. FRUEN
• 金额: $ 8.57万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-15 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6917114
• 关键词: FK506; binding proteins; binding sites; biophysics; calcium channel; calmodulin; fatigue; fluorescence resonance energy transfer; muscle contraction; oxidative stress; phosphorylation; protein binding; protein isoforms; protein kinase A; protein protein interaction; protein purification; protein structure function; recombinant proteins; sarcoplasmic reticulum; spectrometry; swine; time resolved data; transcription factor

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.

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