Regulation of the Cardiac Na/Ca Exchanger

心肌Na/Ca交换器（NCX）是心肌细胞中主要的Ca外排机制， 在调节心脏收缩力中的重要作用。表征交换器的调节特性是 这对于理解正常心脏生理学和致病性心脏病中发生的扰动都很重要。 条件本项目的重点是细胞内钙离子对NCX活性的变构调节， 通过细胞骨架进行调节。指导这些研究的假设是，变构Ca激活显示 滞后，使得活性在NCX活化后变得不依赖于Ca（持续Ca活化）。我们 提出滞后是由于（a）细胞骨架的相互作用，涉及“β重复区”的 （B）限制Ca进入调节结合位点的NCX蛋白，以及（b）PIP 2与所述NCX蛋白的相互作用。 称为NCX蛋白的XIP区域。该项目有5个具体目标，旨在实现以下目标：（1） 胞浆[Na]升高诱导了非Ca依赖的NCX活性模式。我们将以[a]来衡量。 依赖于这种反应，并研究细胞骨架相互作用，PIP 2和XIP突变的作用， 这项活动的发展。(2)我们将确定蛋白质刺激F-肌动蛋白动力学是否 激酶C、NO供体或活化的Rac或Cdc 42增强Ca非依赖性活性的发展， 加速别构Ca激活的速率。(3)我们将确定如何细胞骨架相互作用，胞质 [Na]和PIP 2影响持续性Ca激活的发展和松弛。(4)突变将被 在交换器的β重复区域中产生，以及它们对细胞骨架相互作用、变构 将评估Ca激活和Ca非依赖性NCX活性。(5)我们将确定细胞骨架 相互作用调节新生大鼠和成年大鼠心肌细胞的NCX活性。转染的CHO细胞将 用于目标1-4。使用荧光Ca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.