PHOSPHATASES IN EXCITATION/CONTRACTION COUPLING

电压门控钙离子跨膜内流触发心肌收缩 肌膜并受随后的细胞调节 [Ca~(2+)]i瞬变。这种[Ca~(2+)]i瞬变取决于 肌质网钙释放和钙离子释放对触发钙内流的放大作用 是兴奋-收缩(EC)耦合的中心特征。vbl.使用 分子和细胞工具，申请人已经开始检查这一点 并定量确定了一种重要的元素：蛋白质 磷酸酶调节。初步结果显示，几乎三倍于 [Ca~(2+)]i瞬变的降低，这完全可以归因于 SR蛋白的磷酸化状态。申请人建议延长 这项前期工作与拟开展的工作，确定了具体的 负责的心脏细胞的细胞和分子特征。 该项目将侧重于下列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 新生大鼠心肌细胞？PI将使用隔离的膜片钳单次 细胞和Indo-1检测丝氨酸/苏氨酸蛋白磷酸酶 (PPS)更改EC联轴器。(2)细胞内活性的身份是什么？ 成人和新生儿心肌细胞中的磷酸酶？本系列利用了一个 鉴定特定蛋白磷酸酶活性的灵敏方法和 确定正在研究的细胞中的亚细胞位置。(3)如何 特异性细胞内磷酸酶抑制会影响EC偶联吗？一个 基因转染技术将被用来靶向抑制 解决这一问题的具体PPS。(4)~Ca~(2+)火花如何？ 基础肌质网钙释放事件，通过改变水平改变 具体的PPS？膜片钳内载钙离子的单个心肌细胞 指示剂Fluo-3将使用共聚焦显微镜进行成像。 基于上述研究，将使用PP水平的操作 来调节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.