Regulation of the Cardiac Na/Ca Exchanger

描述（申请人提供）：心脏Na/Ca交换器（NCX）是心肌细胞中主要的Ca外排机制，在调节心脏收缩性中起重要作用。表征交换器的调节特性对于理解正常心脏生理和致病条件下发生的扰动都很重要。本项目主要研究细胞质钙对NCX活性的变构调节及细胞骨架对其的调节。指导这些研究的假设是，变构钙激活表现出滞后性，因此在NCX激活（持续钙激活）后，活性变得与钙无关。我们提出迟滞是由于(a)涉及NCX蛋白的“β重复区域”的细胞骨架相互作用，限制了Ca进入调节结合位点，以及(b) PIP2与NCX蛋白的所谓XIP区域的相互作用。该项目有5个具体目标，旨在实现以下目标：(1)细胞质[Na]的升高诱导NCX活性的钙独立模式。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、PIP2和XIP突变在这种活性发展中的作用。(2)我们将确定通过蛋白激酶C、NO供体或活化的Rac或Cdc42刺激F-actin动力学是否会增强Ca独立活性的发展并加速变构Ca活化的速度。(3)我们将确定细胞骨架相互作用、胞质[Na]和PIP2如何影响持续性Ca激活的发展和放松。(4)突变将在交换器的β重复区域产生，并将评估其对细胞骨架相互作用、变构Ca活化和Ca独立的NCX活性的影响。(5)我们将确定细胞骨架相互作用是否会调节大鼠新生儿和成年心肌细胞中的NCX活性。转染后的CHO细胞将用于Aims 1-4。使用荧光Ca探针的数字成像技术将用于测量NCX的运输活性。

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.