STRUCTURE AND FUNCTION OF THE CARDIAC NA+/CA++ EXCHANGER

心脏 NA /CA 交换器的结构和功能

基本信息

批准号：
2225198
负责人：
KENNETH PHILIPSON
金额：
$ 30.36万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
1993
资助国家：
美国
起止时间：
1993-04-01 至 1998-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2225198
关键词：
aminoacid ankyrins calcium channel calmodulin chemical binding cytoskeleton dogs glycosylation heart contraction laboratory rabbit membrane transport proteins molecular site sarcolemma site directed mutagenesis sodium channel

项目摘要

The Na+-Ca2+ exchanger of cardiac sarcolemma is the dominant Ca2+ efflux mechanism in the myocardium. As such, the Na+-Ca2+ exchanger has a critical role in the regulation of cardiac contractility. Further molecular knowledge of this essential cardiac transporter will help understand both normal and diseased hearts. The long-term goal of this project is to understand the molecular mechanisms involved in the transport process. Towards this goal, the specific aims of the project are as follows: 1. Mutagenesis. This laboratory has recently cloned the cardiac Na+-Ca2+ exchanger. The importance of specific amino acids will now be analyzed by site-directed mutagenesis. Mutants will be expressed in oocytes and analyzed by the giant excised patch technique. 2. Topology. Immunological approaches will be used to determine the arrangement of the Na+-Ca2+ exchange protein in the sarcolemmal membrane. 3. Mapping of binding sites. The Na+-Ca2+ exchanger is capable of binding calmodulin, exchanger inhibitory peptide, regulatory Ca2+, and ankyrin with high affinity. The amino acids involved in the binding of these ligands to the exchanger will be determined by a unique sublibrary approach. 4. Interaction with cytoskeleton. the Na+-Ca2+ exchanger is constrained to specific domains of the sarcolemma by interactions with the cytoskeleton. These interactions of the exchanger with components of the cytoskeleton will be characterized. 5. Protein biochemical approaches. Recombinant exchanger protein will be purified for initial structural studies of the exchanger protein.

心脏肌膜的 Na+-Ca2+ 交换器是主要的 Ca2+ 流出心肌中的机制。因此，Na+-Ca2+交换器具有在心肌收缩力的调节中发挥着重要作用。更远这种重要的心脏转运蛋白的分子知识将有助于了解正常心脏和患病心脏。本次活动的长远目标该项目的目的是了解参与的分子机制运输过程。为实现这一目标，本项目的具体目标如下： 1. 诱变。该实验室最近克隆了心脏Na+-Ca2+ 交换器。现在将分析特定氨基酸的重要性通过定点诱变。突变体将在卵母细胞中表达通过巨型切除补丁技术进行分析。 2. 拓扑结构。将使用免疫学方法来确定肌膜膜中的 Na+-Ca2+ 交换蛋白。 3. 映射结合位点。 Na+-Ca2+交换器能够结合钙调蛋白，交换抑制肽、调节性 Ca2+ 和锚蛋白，具有高亲和力。参与这些配体结合的氨基酸交换器将通过独特的子库方法确定。 4. 与细胞骨架的相互作用。 Na+-Ca2+交换器被限制为通过与细胞骨架的相互作用来形成肌膜的特定结构域。交换器与细胞骨架成分的这些相互作用将被表征。 5.蛋白质生化方法。重组交换蛋白将被纯化用于初始结构研究交换蛋白。