MODULATION OF CA2+ FLUXES IN HEART AND SMOOTH MUSCLE

心脏和平滑肌中 CA2 通量的调节

• 批准号: 3344145
• 负责人: SIDNEY FLEISCHER
• 金额: $ 22.64万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-12-01 至 1995-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3344145
• 关键词: X ray crystallography; calcium binding protein; calcium channel; calcium flux; calcium transporting ATPase; crystallization; dogs; electron microscopy; heart contraction; inositol phosphates; membrane proteins; membrane structure; molecular cloning; monoclonal antibody; myocardium; protein kinase; protein reconstitution; sarcoplasmic reticulum; smooth muscle; striated muscles; vascular smooth muscle

The long term goal of our ongoing program is to study cardiac and smooth muscle with focus on the membranes and the molecular machinery which regulate muscle contraction and relaxation. Specifically, we aim to: 1) Define the molecular machinery involved in the modulation of calcium pumping (enables muscle to relax) in heart sarcoplasmic reticulum (SR); 2) Crystalize the calcium binding protein from cardiac SR, which is involved in the storage of calcium in SR (also referred to as calsequestrin), so that its structure can be determined by X-ray crystallography and electron diffraction; 3) Characterize the heart Ca2+ release channel from SR, which is involved in Ca2+ release which triggers muscle contraction. The modulation of Ca2+ release by protein kinases and phosphatases will be studied. The state of modulation might, in part, explain the controversy in the literature regarding IP activation, or lack or it; 4) Obtain the 3- dimensional structure of the heart ryanodine receptor/Ca2+ release channel (no available) and smooth muscle Ca2+ release channels when they become available (see aim 6); 5) Isolate dyads/triads from heart in order to characterize similarities and differences with that from the skeletal muscle. Such studies should help to assess the basis of the observed macroscopic difference in excitation-contraction coupling, i.e., depolarization induced calcium release (DICR) in skeletal muscle vs calcium release (CICR) in heart; 6) Initiate a program to study smooth muscle membranes, with the aim to isolate and characterize the membrane systems and the molecular components involved int eh Ca2+ pumping, storage and release machinery. This program on smooth muscle will parallel that ongoing for heart (Aims 1-5). Smooth muscle SR appears to have two different types of Ca2+ release channels, the ryanodine receptor type and an IP3 receptor. Definition of these two receptors should provide further insight and comparison into channel types operative in heart and skeletal muscle. Our program is multidisciplinary in scope. It relies heavily on subcellular fractionation to prepare defined membranes, and their functional characterization. The dissociation and reconstitution approach is then employed for isolation and characterization of components involved in Ca2+ transport, storage and release, and the nature of their modulation. Methodology includes electron microscopy, enzymology, transport kinetics, binding studies, channel conductance measurement, crystallization of proteins and structural analysis, monoclonal antibody and cloning technology. The basic information of the membrane machinery involved in Ca2+ uptake, storage and release and its regulation for both heart and smooth muscle should provide a better basis for the understanding of heart disease and hypertension and thereby for the development of cardioselective drugs as well as new types of drugs for regulation of blood pressure.

我们正在进行的计划的长期目标是研究心脏和平稳 肌肉专注于膜和分子机械的肌肉 调节肌肉收缩和放松。 具体来说，我们的目标是：1） 定义与钙调节有关的分子机械 泵送（使肌肉放松）在心脏肌质网（SR）中； 2） 从涉及的心脏SR中结晶钙结合蛋白 在SR中钙的储存（也称为CalSequestrin）时， 可以通过X射线晶体学和电子来确定其结构 衍射; 3）表征来自SR的心脏Ca2+释放通道 参与Ca2+释放，这会触发肌肉收缩。 这 蛋白激酶和磷酸酶对Ca2+释放的调节将是 研究。 调制状态可能部分解释了争议 在有关IP激活或缺乏或IT的文献中； 4）获得3- 心脏ryanodine受体/Ca2+释放通道的尺寸结构 （没有可用的）和平滑肌Ca2+释放通道 可用（请参阅目标6）； 5）从心脏中孤立二元/三合会 表征与骨骼的相似性和差异 肌肉。 这样的研究应有助于评估观察到的基础 激发收缩耦合的宏观差异，即 去极化诱导骨骼肌与钙的钙释放（DICR） 心脏释放（CICR）； 6）启动一个程序来研究平滑肌 膜，目的是隔离和表征膜系统 分子成分涉及int eh Ca2+泵送，存储和 释放机械。 平滑肌上的程序将平行 持续为心脏（目标1-5）。 平滑肌SR似乎有两个 不同类型的Ca2+释放通道，Ryanodine受体类型和 IP3受体。 这两个受体的定义应进一步提供 洞察力和对心脏和骨骼中操作的通道类型的比较 肌肉。 我们的计划在范围内是多学科的。 它很依赖 亚细胞分馏以制备定义的膜及其 功能表征。 分离和重组方法 然后使用涉及组件的隔离和表征 在CA2+运输，存储和释放及其调制的性质中。 方法论包括电子显微镜，酶学，运输动力学， 结合研究，通道电导测量，结晶 蛋白质和结构分析，单克隆抗体和克隆 技术。 涉及的膜机械的基本信息 CA2+吸收，存储和释放及其对心脏和心脏的调节 平滑肌应该为理解心脏提供更好的基础 疾病和高血压，从而开发心脏选择性 药物以及用于调节血压的新型药物。