MODULATION OF CA FLUXES IN HEART SARCOPLASMIC RETICULUM

这项建议的目标是根据心脏SR的作用来描述其特征 在心脏的生理和功能方面。最近，我们开发了一种 高纯度、高稳定性肌浆网的制备 心。这种制剂的提供鼓励我们进行一项 类似于目前心脏SR的详细分子生物学研究方法 我们实验室的骨骼肌SR正在进行中。具体来说，我们 目的明确心脏钙摄取过程的调控性质 通过cAMP和钙调素依赖的蛋白激酶。第二个焦点是 关于确定钙释放机制及其分子方面的问题 调制。这两种方法都将使我们得到解决和重组 方法论。通过这种方式，我们计划：1)直接测试 磷蛋白及其对钙的磷酸化和去磷酸化 心脏肌浆网泵入；2)检测心脏磷蛋白对心肌细胞的影响。 骨骼肌肌浆网钙泵；3)靶向失活 心脏肌浆网测定钙泵蛋白大小的分析 在泵周期的不同状态下的磷蛋白以及在调节 磷蛋白(磷蛋白的磷酸化/去磷酸化)；4) 分离与心脏SR末端池和侧池有关的组分 并对它们的钙吸收和释放以及它们的 调制；5)尝试分离和表征所涉及的频道(S) 在钙释放方面，6)研究钙调素和cAMP依赖的作用 磷酸化对钙释放的影响。第三个重点是定义其他 心脏SR执行的功能。这些因素的意义 研究提供了心脏调节的基本知识 肌肉收缩和松弛，部分是由心脏SR调节的。

The goal of this proposal is to characterize heart SR in terms of its role in the physiology and function of the heart. Recently, we have developed a highly purified and stable preparation of sarcoplasmic reticulum (SR) from heart. The availability of such a preparation encourages us to carry out a detailed molecular biology approach on heart SR analogous to that currently in progress in our laboratory with skeletal muscle SR. Specifically, we aim to define the nature of modulation of the Ca++ uptake process in heart by cAMP and Ca++ -calmodulin dependent protein kinases. A second focus is on defining molecular aspects of the Ca++ release mechanism and its modulation. Both approaches will make us of resolution and reconstitution methodology. In this way we plan to: 1) test directly the influence of phospholamban and its phosphorylation and dephosphorylation on calcium pumping in heart SR; 2) test the influence of phospholamban from heart on the calcium pump from skeletal muscle SR; 3) use target inactivation analysis on heart SR to measure the size of calcium pump protein and phospholamban in different states of the pump cycle and after modulation of phospholamban (phosphorylation/dephosphorylation of phospholamban); 4) isolate fractions referable to terminal and lateral cisternae of heart SR and characterize them with regard to Ca++ uptake and release and their modulation; 5) attempt to isolate and characterize the channel(s) involved in Ca++ release, 6) study the effect of Ca++ -calmodulin and cAMP dependent phosphorylation on Ca++ release. A third focus is to define other functions which are carried out by heart SR. The significance of these studies is that they provide basic knowledge of the regulation of heart muscle contraction and relaxation which in part are mediated by heart SR.