MODULATION OF CA FLUXES IN HEART SARCOPLASMIC RETICULUM

该提案的目标是根据其作用来表征心脏SR 心脏的生理和功能。 最近，我们开发了一种 高纯度和稳定的制备肌浆网（SR）， 心 这种准备的可用性鼓励我们进行一项 心脏SR的详细分子生物学方法类似于目前 在我们的实验室进行骨骼肌SR。具体来说，我们 目的是确定心脏Ca ++摄取过程的调节性质 通过cAMP和Ca ++-钙调蛋白依赖性蛋白激酶。 第二个重点是 在定义Ca ++释放机制的分子方面， 调变 这两种方法都将使我们的决议和重建 方法论 这样我们计划：1）直接测试 受磷蛋白及其对钙离子的磷酸化和去磷酸化作用 2）检测心脏受磷蛋白对心肌SR的影响， 骨骼肌SR钙泵; 3）靶向失活 分析心脏SR以测量钙泵蛋白的大小， 受磷蛋白在泵循环的不同状态下以及在调节后的 受磷蛋白（受磷蛋白的磷酸化/去磷酸化）; 4） 分离部分可与心脏SR的终末和侧池相连 并就Ca ++的吸收和释放及其 调制; 5）尝试隔离和表征所涉及的通道 在Ca~（++）释放方面，6）研究Ca~（++）-钙调素和cAMP依赖性 磷酸化对Ca ++释放的影响。 第三个重点是定义其他 心脏SR执行的功能。这些功能的意义 这些研究提供了心脏调节的基本知识， 肌肉收缩和舒张，其部分由心脏SR介导。

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.