SYNAPTIC VESICLE CATECHOLAMINE/SEROTONIN PORTER

突触小泡儿茶酚胺/血清素 PORTER

• 批准号: 2263975
• 负责人: JOSEPH A NEAR
• 金额: $ 17.28万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-04-01 至 1996-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2263975
• 关键词: CHO cells; catecholamine inhibitor; catecholamines; chemical binding; conformation; electrofocusing; granule; immunoaffinity chromatography; laboratory mouse; membrane transport proteins; molecular site; monoclonal antibody; neurotransmitter transport; protein isoforms; protein reconstitution; protein structure function; reserpine; serotonin; serotonin inhibitor; stoichiometry; synaptic vesicles; tetrabenazine; voltage /patch clamp

The long-term objective of the proposed research is characterization of the transport systems (porter) directly involved in the accumulation of catecholamines and serotonin in synaptic vesicles and storage granules. We propose to prepare mouse monoclonal antibodies to two forms of the porter recently clone from brain and PC12 cells using synthetic peptides as immunogens. Monoclonal antibodies to the porter have not been described previously, and will be useful for a wide range of investigations involving the detection, purification, and characterization of porter isoforms. Antibodies will be used to identify and characterize isoforms of the porter, first in the adrenal and then in other catecholamine and serotonin containing tissues. Differences not attributable to primary sequence will be determined using lectin and immunoaffinity chromatography, specific deglycosylation methods, and isoelectric focusing, along with immunochemical detection methods. Functional heterogeneity of porter isoforms expressed in a heterologous system will be assessed by examination of inhibitor binding parameters, substrate specificities and kinetic parameters for the major isoforms. The binding of tritiated methyl reserpate, a new radiolabeled analog of reserpine, will be characterized. Preliminary studies suggest it binds reversibly and specifically to the initial substrate binding site of the porter, and a ligand with these desirable properties has not been described previously. Using the tritiated forms of methyl reserpate and the noncompetitive transport inhibitor dihydrotetrabenazine, the relationships between binding sites for substrates and various inhibitors will be examined in competition studies, and the stoichiometry of various sites will be determined. The topological orientation of these sites will be assessed by use of impermeant hydrophilic analogs of tetrabenazine and reserpine. The effects of energization in the presence and absence of substrate or inhibitors on the quaternary structure, conformation and orientation of the porter will be evaluated using reversible crosslinking agents, hydrophobic and membrane surface probes, and immunochemical methods. Electrochemical measurements of substrate transport by the porter reconstituted in a planar lipid bilayer will be attempted. This would allow testing of various kinetic models for transport, and could serve as a general method for the study of other catecholamine transport systems as well. Dr. R. Mark Wightman, an expert in the field of analytical electrochemistry, will act as a consultant in these studies. The proposed investigations could lead to more selective treatments for central nervous system disorders involving catecholamines or serotonin. Furthermore, the role of the vesicular porter in such disorders is unknown. A thorough characterization of the molecular forms of the porter present in various tissue, as well as elucidation of any related functional differences, are crucial steps in resolving this questions.

拟议研究的长期目标是表征 直接参与积累的运输系统（搬运工） 突触小泡和储存颗粒中的儿茶酚胺和血清素。 我们建议制备针对两种形式的小鼠单克隆抗体 波特最近使用合成肽从大脑和 PC12 细胞中克隆 作为免疫原。 尚未发现针对搬运工的单克隆抗体 前面已经描述过，并且将用于广泛的范围 涉及检测、纯化和 波特异构体的表征。 抗体将用于鉴定 并表征搬运工的亚型，首先在肾上腺，然后 在其他含有儿茶酚胺和血清素的组织中。 差异不 归因于一级序列将使用凝集素和 免疫亲和层析、特定的去糖基化方法，以及 等电聚焦以及免疫化学检测方法。 异源表达的波特异构体的功能异质性 将通过检查抑制剂结合参数来评估系统， 主要亚型的底物特异性和动力学参数。 氚化甲基利血酸（一种新的放射性标记类似物）的结合 利血平，将被表征。 初步研究表明它结合 可逆地且特异性地结合到初始底物结合位点 波特，并且具有这些理想特性的配体尚未被发现 前面已经描述过。 使用利血酸甲酯的氚化形式和 非竞争性转运抑制剂二氢丁苯那嗪 底物和各种抑制剂的结合位点之间的关系 将在竞争研究中进行检查，以及各种化学计量 地点将被确定。 这些站点的拓扑方向 将通过使用不渗透的亲水类似物进行评估 丁苯那嗪和利血平。 存在时通电的影响 并且四级结构上不存在底物或抑制剂， 将使用以下方法评估搬运工的构象和方向 可逆交联剂、疏水性和膜表面探针、 和免疫化学方法。 底物的电化学测量 由平面脂质双层中重构的搬运工进行的运输将是 尝试过。 这将允许测试各种动力学模型 运输，并可以作为研究其他运输的通用方法 儿茶酚胺运输系统也是如此。 R. Mark Wightman 博士，专家 在分析电化学领域，将担任顾问 这些研究。 拟议的调查可能会导致更具选择性 涉及儿茶酚胺的中枢神经系统疾病的治疗 或血清素。 此外，囊泡搬运工在这种情况下的作用 疾病未知。 分子形式的彻底表征 存在于各种组织中的搬运工，以及任何 相关的功能差异，是解决此问题的关键步骤 问题。