SYNAPTIC VESICLE CATECHOLAMINE/SEROTONIN PORTER

突触小泡儿茶酚胺/血清素 PORTER

• 批准号: 2263976
• 负责人: JOSEPH A NEAR
• 金额: $ 17.04万
• 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/2263976
• 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 运输系统（搬运工）直接参与了 突触囊泡和储存颗粒中的儿茶酚胺和5-羟色胺。 我们建议制备小鼠单克隆抗体，以两种形式的 porter最近使用合成肽从脑和PC 12细胞中克隆 作为免疫原。 针对搬运工的单克隆抗体还没有被 如前所述，并将用于广泛的应用。 涉及检测、纯化和 转运蛋白同种型的表征。 抗体将用于识别 并描述转运蛋白的亚型，首先在肾上腺， 在其它含有儿茶酚胺和血清素的组织中。 差异不 将使用凝集素和 免疫亲和层析，特异性去糖基化方法，和 等电聚焦，沿着免疫化学检测方法。 在异源细胞中表达的转运蛋白同种型的功能异质性 将通过检查抑制剂结合参数对系统进行评估， 主要亚型的底物特异性和动力学参数。 一种新的放射性同位素标记的类似物--氚化利血平的结合作用 利血平，将被表征。 初步研究表明 可逆地和特异性地结合到所述抗体的初始底物结合位点。 波特，并且具有这些期望性质的配体尚未被发现。 先前描述的。 使用氚化形式的甲基利血平和 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，各种化学计量学 网站将被确定。 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。 在存在的情况下， 并且四级结构上不存在底物或抑制剂， 搬运工的构象和取向将使用 可逆交联剂，疏水和膜表面探针， 和免疫化学方法。 底物的电化学测量 在平面脂质双层中重组的转运蛋白的转运将是 企图。 这将允许测试各种动力学模型， 运输，并可以作为一种通用的方法，研究其他 儿茶酚胺运输系统也是如此。 Dr. R.马克·怀特曼是一位 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有选择性的 涉及儿茶酚胺的中枢神经系统疾病的治疗 或者血清素 此外，囊泡转运蛋白在这种 疾病未知。 彻底的分子形式表征 各种组织中的搬运工，以及任何解释 相关的功能差异，是解决这一问题的关键步骤 问题.