Regulation of the Cardiac Na/Ca Exchanger

心肌Na/Ca交换器(NCX)是心肌细胞内钙外流的主要机制，在心肌细胞内起重要作用。 在调节心脏收缩能力方面的重要作用。交易所的监管属性的特征是 对于理解正常的心脏生理和发生在致病性心脏疾病中的扰动很重要 条件。本项目主要研究细胞内钙及其受体对NCX活性的变构调节。 由细胞骨架进行调制。指导这些研究的假设是变构钙激活表现为 滞后，使活性在NCX激活后变得不依赖于钙(持久的钙激活)。我们 提出迟滞是由于：(A)细胞骨架相互作用涉及到 限制钙进入调节结合位点的NCX蛋白以及(B)PIP2与SO-2的相互作用。 称为NCX蛋白的XIP区。该项目有5个具体目标，旨在实现以下目标：(1) 胞浆[Na]升高可诱导NCX活性的钙非依赖性模式。我们将测量[Na] 这种反应的依赖性，并研究细胞骨架相互作用、PIP2和XIP突变的作用 这项活动的开展。(2)我们将确定蛋白质对F-肌动蛋白动力学的刺激作用 KC、NO供体或激活的RAC或CDC42促进钙非依赖性活性的发展 加快变构钙激活的速度。(3)我们将确定细胞骨架如何相互作用，细胞内 [NA]和PIP2影响持续性钙激活的发展和松弛。(4)突变将是 在交换器的β重复区域中产生及其对细胞骨架相互作用的影响，变构 将评估钙激活和钙非依赖性NCX活性。(5)我们将确定细胞骨架是否 相互作用调节新生和成年大鼠心肌细胞中Ncx的活性。转基因的CHO细胞将 在目标1-4中使用。使用荧光钙探针的数字成像技术将用于测量NCX 运输活动。

The cardiac Na/Ca exchanger (NCX) is the principal Ca efflux mechanism in cardiac myocytes and plays an essential role in regulating cardiac contractility. Characterizing the exchanger's regulatory properties is important for understanding both normal cardiac physiology and the perturbations that occur in pathogenic conditions. This project focuses on the allosteric regulation of NCX activity by cytosolic Ca and its modulation by the cytoskeleton. The hypothesis guiding these studies is that allosteric Ca activation displays hysteresis so that activity becomes Ca-independent following NCXactivation (persistent Ca activation). We propose that the hysteresis is due to (a) cytoskeletal interactions involving the "beta repeat regions" of the NCX protein that restrict access of Ca to the regulatory binding sites and (b) interaction of PIP2 with the so- called XIP region of the NCX protein. The project has 5 specific aims directed toward the following goals: (1) Elevations in cytosolic [Na] induce a Ca-independent mode of NCXactivity. We will measure the [Na] dependence of this response and investigate the role of cytoskeletal interactions, PIP2 and XIP mutations in the development of this activity. (2) We will determine whether stimulation of F-actin dynamics by protein kinase C, NO donors or activated Rac or Cdc42 enhances the development of Ca-independent activity and accelerates the rate of allosteric Ca activation. (3) We will determine how cytoskeletal interactions, cytosolic [Na] and PIP2 influence the development and relaxation of persistent Ca activation. (4) Mutations will be created in the beta repeat regions of the exchanger and their influence on cytoskeletal interactions, allosteric Ca activation and Ca-independent NCX activity will be assessed. (5) We will determine whether cytoskeletal interactions modulate NCX activity In rat neonatal and adult cardiac myocytes. Transfected CHO cells will be used in Aims 1-4. Digital imaging techniques using fluorescent Ca probes will be used to measureNCX transport activity.