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

心肌细胞膜Na+-Ca~(2+)交换器是主要的Ca~(2+)外流 心肌中的作用机制。因此，Na+-Ca2+交换器具有 在心脏收缩能力调节中的关键作用。进一步 对这种重要的心脏转运蛋白的分子知识将有所帮助 了解正常和患病的心脏。这样做的长期目标是 该项目的目的是了解参与该过程的分子机制。 运输过程。 针对这一目标，项目的具体目标如下：1. 诱变。本实验室最近克隆了心肌细胞的钠-钙离子 交易所。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，并 采用巨型切除补片技术进行分析。2.拓扑学。 将使用免疫学方法来确定 肌膜上的Na+-Ca2+交换蛋白。3.地图绘制 结合部位。所述Na+-Ca~(2+)交换器能够结合钙调蛋白， 高水平的交换抑制肽、调节性钙离子和锚蛋白 亲和力。参与与这些配体结合的氨基酸 交易所将通过一种独特的子库方法确定。4. 与细胞骨架的相互作用。Na+-Ca~(2+)交换器被限制为 通过与细胞骨架的相互作用，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 将被描述为。5.蛋白质生化方法。重组 交换蛋白将被提纯，用于初步结构研究 交换蛋白。

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.