Regulation of cardiac Ca2+ release channel

心脏 Ca2 释放通道的调节

• 批准号: 6737571
• 负责人: GERHARD W MEISSNER
• 金额: $ 41.33万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-15 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6737571
• 关键词: adenosine triphosphate; calcium channel; calcium flux; calcium indicator; calmodulin; cardiac myocytes; cell line; dogs; electrophysiology; embryonic stem cell; magnesium; mutant; myocardial ischemia /hypoxia; nitric oxide; oxidation reduction reaction; peptidylprolyl isomerase; protein engineering; protein protein interaction; protein structure function; receptor binding; sarcoplasmic reticulum; single cell analysis; thiols

DESCRIPTION (provided by applicant): The goal of the proposed research is to define the regulation of normal and mutant cardiac muscle Ca2+ release channels (ryanodine receptors, RyR2s) under normal conditions as well as those in ischemic and post-ischemic heart. The cardiac Ca2+ release channel is a 30S protein complex comprised of four 560 kDa RyR2 subunits and four 12.6 kDa FK506 binding protein (FKBP12.6) subunits. Multiple endogenous effector molecules and post-translational modifications regulate RyR2, including Ca2+, Mg2+, ATP, calmodulin, protein phosphorylation, and thiol oxidation/reduction and S-nitrosylation. The principal hypotheses to be tested in the proposed research are that RyR2 is differentially regulated by calmodulin and by redox activation pathways involving free thiol groups. These hypotheses will be addressed using biochemical and electrophysiological methods and by creating mutant embryonic stem (ES) cell lines and mice. The Specific Aims are: (1) Characterize the regulation of RyR2 by calmodulin (CAM) and identify by mutagenesis the apocalmodulin (apoCaM) and Ca2+-calmodulin (CaCaM) regulatory sites in RyR2. (2) Characterize the regulation of RyR2 by redox active and NO-related molecules and identify regulatory redox-sensitive and S-nitrosylation sites by chemical analysis and mutagenesis. (3) Determine the in vivo role of the above RyR2 regulatory mechanisms by creating mutant ES cells differentiated into cardiomyocytes deficient in RyR2 calmodulin binding and S- nitrosylation sites and mice expressing RyR2s deficient in calmodulin binding and S-nitrosylation sites. The functional properties of normal and mutant RyR2s will be determined in intact cells and with isolated membranes and purified channels under normal and simulated ischemic and postischemic conditions. The functional effects of Ca2+, Mg2+, ATP, pH, calmodulin, and redox-active and NO-related molecules will be assessed in Ca2+ imaging, SR vesicle-Ca2+ flux, [3H]ryanodine binding and single channel measurements. The studies will provide new insights into the complex interaction of the cardiac RyR with its regulatory ligands and how these regulatory processes are altered in the ischemic and post-ischemic heart.

描述（由申请人提供）：拟议研究的目标是确定正常条件下以及缺血和缺血后心脏条件下正常和突变心肌 Ca2+ 释放通道（兰尼碱受体，RyR2s）的调节。心脏 Ca2+ 释放通道是一个 30S 蛋白质复合物，由四个 560 kDa RyR2 亚基和四个 12.6 kDa FK506 结合蛋白 (FKBP12.6) 亚基组成。多种内源效应分子和翻译后修饰调节 RyR2，包括 Ca2+、Mg2+、ATP、钙调蛋白、蛋白质磷酸化、硫醇氧化/还原和 S-亚硝基化。在拟议的研究中要测试的主要假设是，RyR2 受到钙调蛋白和涉及游离硫醇基团的氧化还原激活途径的差异调节。这些假设将通过生化和电生理学方法以及创建突变胚胎干（ES）细胞系和小鼠来解决。具体目标是： (1) 表征钙调蛋白 (CAM) 对 RyR2 的调节，并通过诱变鉴定 RyR2 中的脱辅基钙调蛋白 (apoCaM) 和 Ca2+-钙调蛋白 (CaCaM) 调节位点。 (2) 表征氧化还原活性分子和 NO 相关分子对 RyR2 的调节，并通过化学分析和诱变鉴定氧化还原敏感和 S-亚硝基化调节位点。 (3)通过创建分化为缺乏RyR2钙调蛋白结合和S-亚硝基化位点的心肌细胞的突变ES细胞以及表达缺乏钙调蛋白结合和S-亚硝基化位点的RyR2的小鼠，确定上述RyR2调节机制的体内作用。正常和突变 RyR2 的功能特性将在完整细胞中、在正常和模拟缺血和缺血后条件下通过分离的膜和纯化的通道进行测定。 Ca2+、Mg2+、ATP、pH、钙调蛋白以及氧化还原活性和 NO 相关分子的功能影响将在 Ca2+ 成像、SR 囊泡-Ca2+ 通量、[3H]ryanodine 结合和单通道测量中进行评估。这些研究将为心脏 RyR 与其调节配体的复杂相互作用以及这些调节过程在缺血和缺血后心脏中如何改变提供新的见解。