MODULATION OF CA2+ FLUXES IN HEART AND SMOOTH MUSCLE

我们正在进行的项目的长期目标是研究心脏和流畅 肌肉，重点是膜和分子机制， 调节肌肉收缩和放松。具体地说，我们的目标是：1) 定义参与钙调节的分子机制 在心脏肌浆网(SR)泵送(使肌肉放松)；2) 结晶心脏SR中的钙结合蛋白，这与 在SR中存储钙(也称为钙固缩蛋白)，因此 它的结构可以通过X射线结晶学和电子技术来确定 3)确定了心脏肌浆网钙释放通道的特征。 参与了引发肌肉收缩的钙离子释放。这个 蛋白激酶和磷酸酶对钙离子释放的调节作用 学习。调制的状态可能在一定程度上解释了这种争议 在有关IP激活或缺失或它的文献中；4)获得3- 心脏兰尼定受体/钙释放通道的空间结构 (无可用)和平滑肌钙释放通道 可用(见目标6)；5)将二联体/三联体从心脏中分离出来 描述与骨骼的相似性和差异性 肌肉。这类研究应有助于评估观察到的 兴奋-收缩耦合的宏观差异，即， 骨骼肌去极化诱导钙释放(DICR)与钙 在心脏中释放(CICR)；6)启动一个研究平滑肌的计划 膜，目的是分离和表征膜系统 分子组成涉及钙离子的泵送、储存和释放。 释放机械。这个关于平滑肌肉的节目将与之相媲美 持续进行的心脏(目标1-5)。平滑肌SR似乎有两个 不同类型的钙释放通道，兰尼定受体类型和 一种IP3受体。对这两种受体的定义应进一步提供 心脏和骨骼经络类型的洞察和比较 肌肉。 我们的项目范围是多学科的。它严重依赖于 亚细胞分离以制备限定的膜，及其 功能特性。解离和重组的方法 然后用于分离和表征所涉及的组分 钙离子的运输、储存和释放，以及它们的调制性质。 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，结晶 蛋白质与结构分析、单抗与克隆 技术所涉及的膜机械的基本信息 心脏和心脏对钙离子的摄取、储存和释放及其调节 平滑肌应该为理解心脏提供更好的基础 疾病和高血压，从而为心脏选择性的发展 药物以及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.