PHOSPHATASES IN EXCITATION/CONTRACTION COUPLING

心脏收缩是由电压门控的Ca 2+内流触发的， 并受随后的细胞调节 [Ca2+] I瞬态。这种[Ca 2 +] I瞬变取决于 通过SR Ca 2+释放放大触发Ca 2+内流， 是兴奋-收缩（EC）偶联的中心特征。 使用 分子和细胞工具，申请人已经开始研究这一点， 过程中，并确定了定量的一个重要因素：蛋白质 磷酸酶调节 初步结果显示， [Ca 2 +] I瞬变的减少，这可以完全归因于 SR蛋白的磷酸化状态。 申请人建议延长 这项初步工作与拟议的工作，确定具体的 心脏细胞的细胞和分子特征。 该项目将侧重于以下相互关联的具体问题。 (1)蛋白磷酸酶如何影响成人和成人的EC偶联， 新生大鼠心脏细胞 PI将使用隔离式膜片钳单通道 细胞和indo-1来检测丝氨酸/苏氨酸蛋白磷酸酶 (PPs)改变EC偶联。 (2)什么是活性细胞内的身份 成人和新生儿心脏细胞中的磷酸酶 该系列利用了 灵敏的测定法，以鉴定特异性蛋白磷酸酶活性， 确定被研究细胞中的亚细胞位置。 (3)如何 抑制特异性细胞内磷酸酶影响EC偶联？ 一 基因转染技术将用于靶向抑制 具体的PP来解决这个问题。 (4)怎么样~ Ca 2+火花~， 基本SR Ca 2+释放事件，通过改变 具体PP？ 负载Ca ~（2+）的膜片钳单个心脏细胞 指示剂Fluo-3将使用共聚焦显微镜成像。 将根据上述研究对PP水平进行操纵 以调节EC偶联。

Cardiac contraction is triggered by voltage-gated Ca2+-influx across the sarcolemmal membrane and regulated by the subsequent cellular [Ca2+] I transient. This [Ca2+] I transient depends on the amplification of the triggering Ca2+ influx by SR Ca2+ release and is a central feature in excitation-contraction (EC) coupling. Using molecular and cellular tools, the applicant has started to examine this process and has identified quantitatively an important element: protein phosphatase modulation. Preliminary results show a nearly three-fold decrease in the [Ca2+] I transient that can be attributable solely to the phosphorylation-state of SR proteins. The applicant proposes to extend this preliminary work with the proposed work, identifying specific cellular and molecular features of the heart cell that are responsible. The project will focus on the following specific inter-related questions. (1) How do protein phosphatases affect EC coupling in adult and neonatal rat heart cells? The PI will use isolated patch-clamped single cells and indo-1 to examine how serine/threonine protein phosphatases (PPs) alter EC coupling. (2) What is the identity of active intracellular phosphatases in adult and neonatal heart cells? This series exploits a sensitive assay to identify specific protein phosphatase activity and determine subcellular location in cells being studies. (3) How does inhibition of specific intracellular phosphatases affect EC coupling? A gene transfection technique will be used to target the inhibition of specific PPs to address this question. (4) How are ~Ca2+ sparks~, the elementary SR Ca2+ release events, altered by changing levels of specific PPs? Patch clamped single heart cells loaded with Ca2+ indicator fluo-3 will be imaged using confocal microscopy. Manipulations of PP levels, based on the above studies, will be used to modulate EC coupling.