SUBSPECIALIZATION OF CARDIAC SARCOPLASMIC RETICULUM

目标是在分子水平上了解钙是如何积累的 并由心肌肌浆网(SR)释放。交汇性 SR囊泡和游离SR囊泡将从心脏中分离出来，其 分析了不同的钙吸收和钙释放特性。特例 重点是钙诱导的钙释放的特征 通道定位于连接的SR小泡，这似乎是一种 细胞内钙通道主要负责细胞内钙的升高 肌肉收缩时的钙浓度。三种关键的心脏SR蛋白， 高分子量(HMW)钙调素蛋白(推定成分 SR脚)，和磷蛋白将被提纯到均一性和 详细地描述了。钙调素和HMW蛋白被定位 心脏以交界性SR为主，而磷蛋白均一 分布在整个SR中。这三种蛋白质都有很强的牵连 在SR的钙稳态中，但它们的确切功能仍未明确。 重点对这三个化合物的一级结构进行了测定 蛋白质、超微结构特征及其测定 磷酸化对蛋白质构象和功能的影响。我们会 确定纯化的磷蛋白是否作为通道和/或 直接与肌质网钙泵相互作用来调节钙的运输，并且 HMW蛋白和Calequestrin(以及共分泌110-160 kDa) 蛋白质)相互作用、其他蛋白质和/或脂质相互作用 双层以改变SR的离子渗透性。我们还将确定是否 HMW蛋白是SR足部不可或缺的蛋白质组分。实验 将专注于定义潜在的蛋白质-蛋白质和蛋白质-脂肪 净化-重组系统中的相互作用。以这种方式，函数 可能在分子水平上更好地理解这三种蛋白质中的一种。 将在研究中使用的重要技术包括融合 心脏SR囊泡的平面脂双分子膜测量单个钙通道 活性；气相蛋白测序和重组DNA方法 测定一级结构；分光光度法、共价探针、单克隆法 抗体和流体动力学技术检测高阶结构和 膜-蛋白质拓扑学和膜-蛋白质重组研究 蛋白质的功能。上述研究的完成将增加我们的 对SR在心力衰竭基本机制中的作用的认识 心肌的兴奋-收缩偶联。

The goal is to understand, at the molecular level, how Ca is accumulated and released by sarcoplasmic reticulum (SR) in cardiac muscle. Junctional SR vesicles and free SR vesicles will be isolated from heart, and their different Ca uptake and Ca release properties analyzed. Particular emphasis will be placed on characterization of the Ca-induced Ca release channel localized to junctional SR vesicles, which appears to be an intracellular Ca channel primarily responsible for elevating intracellular Ca concentration during muscle contraction. Three key cardiac SR proteins, calsequestrin, high molecular weight (HMW) proteins (putative components of the SR feet), and phospholamban will be purified to homogeneity and characterized in detail. Calsequestrin and HMW proteins are localized predominantly to junctional SR in heart, whereas phospholamban is uniformly distributed throughout the SR. All three proteins are strongly implicated in SR Ca homeostasis, but their precise functions remain poorly defined. Emphasis is placed on primary structure determination of these three proteins, ultrastructural characterization, and determination of phosphorylation effects on protein conformation and function. We will determine whether purified phospholamban behaves as a channel and/or interacts directly with the SR Ca pump to modulate Ca transport, and whether the HMW proteins and calsequestrin (and copurifying 110- to 160-kDa proteins) interact with each other, other proteins, and/or the lipid bilayer to alter SR ionic permeability. We will also determine whether the HMW proteins are integral protein components of the SR feet. Experiments will focus on defining potential protein-protein and protein-lipid interactions in purified-reconstituted systems. In this way the functions of the three proteins may be better understood at the molecular level. Important techniques to be utilized in the studies include fusion of the cardiac SR vesicles with planar lipid bilayers to measure single Ca channel activity; gas-phase protein sequencing and recombinant DNA methods to determine primary structure; spectrophotometric, covalent probe, monoclonal antibody and hydrodynamic techniques to examine higher order structure and membrane-protein topology; and membrane-protein reconstitution to examine protein function. Completion of the studies described will increase our understanding of the role of the SR in basic mechanisms of excitation-contraction coupling in cardiac muscle.