STRUCTURAL ANALYSIS OF THE CA++ RELEASE CHANNEL

描述：该提案的总体目标是定义分子 兰尼定敏感细胞钙释放调节的相关事件 细胞内存储并确定缺陷在细胞内存储的机制 这些途径导致恶性体温过低和中央核心病。 该提案所基于的一般假设是， 钙释放通道的活动伴随着整体构象 可以用生物化学和低温电子来表征的变化 显微技术和图像重建。支持这一假说 初步数据显示了闭合和打开的图像 雷帕霉素对钙释放通道和钙释放通道的清除作用 FKBP12。对多肽也提出了广泛的生物化学证据。 测绘、免疫化学鉴定和表征 Ryanodine受体(RyR)在完整的右侧外SR和28S和14S中的表达 蛋白水解物。这些研究将扩展到五个具体的 当前提案的目的。 第一个目标是产生钙释放的不同功能状态 通道(由咖啡因和AMP-PCP开放，由MMCa失活和抑制 Ru红或新霉素)，并用~3-H表征这些状态 兰尼定结合和单通道分析。这些东西的结构 然后，功能状态将通过电子冷冻显微镜和 角度重建。第二种是使用不同的蛋白质分解图谱 表面标记和膜内标记相结合的方法， 免疫学和多肽结合(使用 噬菌体展示文库)，以映射钙释放通道结构域的拓扑结构和 与不同功能状态相关的结构性变化。这个 该蛋白14S蛋白水解性片段的三维结构 含有通道和兰尼定的结合部位也将被确定。 第三个目标是绘制FKBP12的结合位点图并确定其 对钙释放通道结构的影响。3维的 通道的结构将在移除之前和之后确定 用雷帕霉素处理FKBP12，并将纯化的FKBP12重排到 耗尽的航道。FKBP12结合位点将被绘制在钙释放上 通过检查来自125-I-APDP或125-I-SASD的标签传输和 28S复合体的肽图谱和测序。 第四和第五个目标都使用电子冷冻显微镜来比较病例 释放渠道结构。在第四个目标中，三维结构 正常猪和恶性低温猪的钙释放通道 在以下条件下进行比较：(1)兰尼定的亲和力差异为 最大，(2)在没有钙的情况下，其中两个通道都应该关闭， 和(3)在钙和兰尼定存在的情况下，两个通道都应该是 打开。最终的目标是，骨骼的三维结构 并对开放和关闭状态下的心肌钙释放通道进行比较 构象。这可能有助于为差异提供结构性基础。 心肌和骨骼肌之间的EC偶联。中概述的研究 该提议可能导致关于钙通道结构的重要信息， 拓扑和机制，以及确定结构或机制 在疾病状态中发现的变化，如恶性高热和 中央核心病。

DESCRIPTION: The overall goal of the proposal is to define the molecular events involved in the regulation of Ca release from ryanodine sensitive intracellular stores and to determine the mechanisms by which defects in these pathways give rise to Malignant Hypothermia and Central Core Disease. The general hypothesis on which the proposal is based is that changes in the activity of the Ca-release channel are accompanied by global conformation changes that can be characterized biochemically and with cryoelectron microscopy and image reconstruction. In support of this hypothesis preliminary data is included showing the images of closed and open Ca-release channels and Ca-release channels treated with rapamycin to remove FKBP12. Extensive biochemical evidence is also presented for peptide mapping and immunochemical identification and characterization of the ryanodine receptor (RyR) in intact rightside out SR and in 28S and 14S proteolytic complexes. These studies will be extended in the five specific aims of the current proposal. The first aim is to generate different functional states of the Ca release channel (opened by caffeine and AMP-PCP, inactivated by mM Ca and inhibited by ruthenium red or neomycin) and to characterize these states by 3-H ryanodine binding and single channel analysis. The structures of these functional states will then be determined by electron cryomicroscopy and angular reconstruction. The second is to use different proteolytic mapping approaches combined with surface and membrane interior labeling, immunological and peptide binding (high affinity peptides identified using a phage display library) to map the topology of Ca release channel domains and structural changes that relate to different functional states. The 3-dimensional structure of a 14S proteolytic fragment of the protein which contains the channel and the ryanodine binding site will also be determined. The third aim is to map the binding site for FKBP12 and determine its effects on the structure of the Ca release channel. The 3-dimensional structure of the channel will be determined before and after the removal of FKBP12 by rapamycin treatment and upon readdition of purified FKBP12 to the depleted channel. The FKBP12 binding site will be mapped on the Ca release channel by examining label transfer from 125-I-APDP or 125-I-SASD and peptide mapping and sequencing of the 28S complex. The fourth and fifth aims both use electron cryomicroscopy to compare Ca release channel structures. In aim four the 3-dimensional structures of normal pig and malignant hypothermic pig Ca2+ release channels will be compared under conditions where (1) affinity differences for ryanodine are greatest, (2) in the absence of Ca, where both channels should be closed, and (3) in the presence of Ca and ryanodine where both channels should be open. In the final aim, the three dimensional structures of the skeletal and cardiac Ca release channels will be compared in the open and closed conformations. This may help provide a structural basis for the differences between cardiac and skeletal muscle EC coupling. The studies outlined in this proposal may lead to important information on Ca channel structure, topology and mechanism as well as identifying structural or mechanistic alterations found in disease states such as Malignant Hyperthermia and Central Core Disease.