DIHYDROPYRIDINE BINDING SITES AND CALCIUM CHANNELS

二氢吡啶结合位点和钙通道

• 批准号: 3074260
• 负责人: SUSAN L HAMILTON
• 金额: $ 6.64万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-03-01 至 1993-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3074260
• 关键词: binding proteins; calcium transporting ATPase; chemical structure function; high performance liquid chromatography; hormone receptor; laboratory mouse; laboratory rabbit; membrane channels; membrane potentials; protein purification

Increased permeability to Ca2+ in response to a depolarizing membrane potential occurs in a wide variety of excitable cell types. In some cells this permeability increase is either blocked or enhanced by dihydropyridines, leading to the hypothesis that the dihydropyridine binding site is part of or associated with the voltage-dependent calcium channel. We propose to purify the DHP binding protein from bovine ventricular tissue and to characterize its subunit composition. This will be accomplished using the binding radioactive DHPS to follow the purification. Purification procedures will utilize conventional column chromatography, HPLC and FPLC techniques, affinity chromatography, and ultracentrifugation. In a collaborative effort, we will use reconstitution techniques to establish whether the DHP binding protein is also the voltage- dependent calcium channel. Important to the purification and characterization of the cardiac DHP binding protein will be the production of antibody and toxin probes specific for this protein. One approach to the preparation of specific antibodies will be to use the subunits of the skeletal muscle DHP binding protein as antigens. We will also use the partially-purified cardiac preparation. Probes of the DHP binding protein will be used to purify it further, to determine the subunit composition, and to analyze the arrangement of the subunits in the membrane.

对去极化的响应增加了对钙的通透性 膜电位存在于多种可兴奋细胞中 类型。在一些细胞中，这种通透性增加要么被阻止 或被二氢吡啶增强，导致假设 二氢吡啶结合部位是部分或与之相关的 电压依赖性钙通道。 我们提议从牛中提纯DHP结合蛋白 并对其亚单位组成进行表征。 这将使用结合的放射性DHP来完成 遵循净化程序。净化程序将利用 常规柱层析、高效液相色谱和高效毛细管电泳法 技术、亲和层析和超速离心法。在……里面 作为一项合作努力，我们将使用重建技术来 确定DHP结合蛋白是否也是电压- 依赖性钙通道。 对心脏的纯化和特性的重要作用 DHP结合蛋白将产生抗体和毒素 这种蛋白质的特异探针。准备工作的一种方法 将使用骨骼的亚单位 肌肉DHP结合蛋白作为抗原。我们还将使用 部分纯化的心脏制剂。 将使用DHP结合蛋白的探针来纯化它 进一步，确定亚基组成，并分析 膜上亚基的排列。