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

心脏 NA /CA 交换器的结构和功能

• 批准号: 2378779
• 负责人: KENNETH PHILIPSON
• 金额: $ 34.14万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-04-01 至 1998-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2378779
• 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

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.

心肌细胞膜的Na+-Ca ~（2+）交换是心肌细胞内主要的钙外排途径 心肌中的机制。 因此，Na+-Ca 2+交换剂具有 在调节心脏收缩力中起关键作用。 进一步 对这种重要的心脏转运蛋白的分子知识将有助于 了解正常和患病的心脏。 长期目标是 项目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. 基因突变。 该实验室最近克隆了心脏Na+-Ca 2 + 交换器 现在将分析特定氨基酸的重要性 通过定点突变。 突变体将在卵母细胞中表达， 通过巨大切除贴片技术进行分析。 2. Topology. 免疫学方法将用于确定 Na+-Ca ~（2+）交换蛋白。 3.映射 结合位点。 Na+-Ca 2+交换剂能够结合钙调蛋白， 交换抑制肽、调节性Ca 2+和锚蛋白， 亲和力 参与这些配体结合的氨基酸 交换器将通过唯一的子库方法来确定。 4. 与细胞骨架的相互作用。 Na+-Ca 2+交换剂被限制为 通过与细胞骨架的相互作用，在肌膜的特定结构域中形成。 交换剂与细胞骨架成分的相互作用 将被定性。 5.蛋白质生物化学方法。 重组 交换蛋白将被纯化用于初步的结构研究， 交换蛋白