DIHYDROPYRIDINE BINDING SITES AND CALCIUM CHANNELS

二氢吡啶结合位点和钙通道

• 批准号: 3074259
• 负责人: SUSAN L HAMILTON
• 金额: $ 6.56万
• 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/3074259
• 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.

对去极化反应的Ca 2+渗透性增加 膜电位存在于多种可兴奋细胞中 类型 在某些细胞中，这种渗透性的增加要么被阻断， 或被二氢吡啶类化合物增强，这导致了一种假设， 二氢吡啶结合位点是二氢吡啶的一部分或与二氢吡啶结合位点缔合， 电压依赖性钙通道 我们建议从牛中纯化DHP结合蛋白 心室组织并表征其亚基组成。 这将使用结合放射性DHPS来实现， 遵循净化。 纯化程序将利用 常规柱色谱法、HPLC和FPLC 技术、亲和层析和超离心。 在 一个合作的努力，我们将使用重组技术， 确定DHP结合蛋白是否也是电压- 依赖性钙通道 对心脏的纯化和表征很重要 DHP结合蛋白会产生抗体和毒素 针对这种蛋白质的探针 准备方法之一 将利用骨骼肌的亚单位， 肌肉DHP结合蛋白作为抗原。 我们还将使用 部分纯化的心脏标本 DHP结合蛋白的探针将用于纯化它 此外，为了确定亚基组成，并分析 细胞膜中亚基的排列。