Regulation of the Cardiac Na/Ca Exchanger

描述（由申请人提供）：心脏Na/Ca交换器（NCX）是心肌细胞中的主要Ca外排机制，在调节心肌收缩力中起重要作用。表征交换器的调节特性对于理解正常心脏生理学和在致病条件下发生的扰动是重要的。本项目的重点是细胞内钙离子对NCX活性的变构调节及其对细胞骨架的调节。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激活（持续Ca激活）后变得不依赖于Ca。我们提出滞后是由于（a）涉及NCX蛋白的“β重复区”的细胞骨架相互作用，其限制Ca接近调节结合位点，以及（B）PIP 2与NCX蛋白的所谓XIP区的相互作用。该项目有5个具体目标，旨在实现以下目标：（1）细胞溶质[Na]的升高诱导NCX活性的Ca非依赖性模式。我们将测量这种反应的[Na]依赖性，并研究细胞骨架相互作用、PIP 2和XIP突变在这种活性发展中的作用。(2)我们将确定是否刺激F-肌动蛋白动力学的蛋白激酶C，NO供体或激活的Rac或Cdc 42增强钙非依赖性活动的发展，并加快变构钙激活的速率。(3)我们将确定细胞骨架相互作用，胞质[Na]和PIP 2如何影响持续钙激活的发展和松弛。(4)将在交换剂的β重复区域中产生突变，并评估其对细胞骨架相互作用、变构Ca活化和Ca非依赖性NCX活性的影响。(5)我们将确定是否细胞骨架的相互作用调节NCX活性在大鼠新生儿和成人心肌细胞。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转运活性。

DESCRIPTION (provided by applicant): 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 NCX activation (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 NCX activity. 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 measure NCX transport activity.