MODULATION OF CA FLUXES IN HEART SARCOPLASMIC RETICULUM

我们正在进行的项目的长期目标是研究心脏和平滑 肌肉的重点是膜和分子机制， 调节肌肉的收缩和放松。 具体而言，我们的目标是：1） 定义参与钙调节的分子机制 心脏肌浆网（SR）泵送（使肌肉放松）; 2） 使心脏SR中的钙结合蛋白结晶， 在SR中储存钙（也称为钙螯合蛋白），因此 它的结构可以通过X射线晶体学和电子 3）表征来自SR的心脏Ca 2+释放通道， 参与触发肌肉收缩的Ca 2+释放。 的 通过蛋白激酶和磷酸酶调节Ca 2+释放将是 研究了 调制的状态可能在一定程度上解释了这一争议 在文献中关于IP激活，或缺乏或它; 4）获得3- 心肌Ryanodine受体/Ca ~（2+）释放通道的三维结构 (no可用）和平滑肌Ca 2+释放通道， 可用（见目标6）; 5）从心脏中分离二元组/三元组， 描述与骨骼的相似性和差异 肌肉. 这些研究应有助于评估所观察到的 激发-收缩耦合的宏观差异，即， 骨骼肌去极化诱导的钙释放（DICR）与钙 6）启动研究平滑肌的程序 膜，目的是分离和表征膜系统 以及参与Ca 2+泵送、储存和 解放机械 这个关于平滑肌的项目将与 正在进行心脏病治疗（目标1-5）。 平滑肌SR似乎有两个 不同类型的Ca 2+释放通道，ryanodine受体类型和 IP 3受体。 这两种受体的定义应提供进一步的 对心脏和骨骼手术通道类型的认识和比较 肌肉. 我们的计划是多学科的范围。 它非常依赖 亚细胞分级分离以制备确定的膜，以及它们的 功能特性 分离与重建方法 然后用于分离和表征所涉及的组件 在Ca 2+的运输，储存和释放，以及它们的调制的性质。 方法学包括电子显微镜、酶学、转运动力学， 结合研究，通道电导测量， 蛋白质和结构分析，单克隆抗体和克隆 技术. 所涉及的膜机械的基本信息 心、肺组织Ca ~（2+）的摄取、贮存和释放及其调节 平滑肌应该为理解心脏提供更好的基础 疾病和高血压，从而发展心脏选择性 药物以及用于调节血压的新型药物。

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.