NEURONAL PACEMAKERS--ANTIBODIES AS PROBES

神经起搏器——抗体作为探针

• 批准号: 3401791
• 负责人: FELIX STRUMWASSER
• 金额: $ 11.6万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-08-01 至 1987-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3401791
• 关键词: circadian rhythms; cyclic nucleoside monophosphate; electrophysiology; ganglions; histochemistry /cytochemistry; immunochemistry; immunologic techniques; messenger RNA; monoclonal antibody; neurochemistry; neuroendocrine system; neurons; phosphorylation; saltwater environment; tissue /cell culture

The research described in this application centers on the nature of pacemaker mechanisms and their modulation in neurons. The ultimate goal is to define the set of cytoplasmic and membrane proteins that mediate this important function and to determine the nature of the modulation caused by cyclic nucleotide-dependent protein phosphorylation. In particular, monoclonal antibodies will be generated to membrane proteins in several pacemaker systems in the mollusk Aplysia. These antibodies will be screened, by immonoblots and immunocytochemistry, to select for those that uniquely recognize antigens of pacemakers systems. Such "pacemaker specific" antibodies will be tested for pharmacological interference of pacemaker function by intracellular injection. Eventually, we would like to convert non-pacemaker cells into pacemaker cells by injecting the appropriate messenger RNAs (mRNAs) into non-pacemaker cells in order to determine whether all the essential constituents for pacemaker function have been isolated. This last goal may not be achieved during the first project period. For these studies, we will concentrate on three different pacemaker systems in one organism, Aplysia: 1) A transient pacemaker -- the bag cells, a group of peptidergic neurons, which release their hormonal messenger, by producing bursting pacemaker activity for approximately 30 minutes. 2) A constant pacemaker -- the identifiable neurons R15, L2, L3, L4 and L6 in the abdominal ganglion, which produce groups of impulses termed "bursts", separated by a silent interburst interval lasting seconds to minutes. 3) A circadian pacemaker system -- part of the eye, which is expressed as a turning on and modulation of pacemaker activity , emitted in the optic nerve, over a 24-hour period. The following specific projects are planned in the first project period: A). Generating monoclonal antibodies to crude membrane fractions of the above three pacemaker systems. B). Screening the monoclonal antibodies for specific recognition of antigenic determinants of the three pacemaker systems by immunocytochemistry and immunoblots (Western blots). C). Attempting to interfere with pacemaker function by intracellular injection of "pacemaker-specific" of the intact abdominal ganglion. D). Whole-cell voltage- and membrane patch-clamp studies of the latter two pacemaker systems in order to determine what ion channels are modulated by cyclic nucleotide-dependent protein phosphorylation.

本申请中描述的研究的中心是 神经元中的起搏机制及其调制。最终目标是 为了确定一组介导这一过程的细胞质和细胞膜蛋白 重要功能和确定调制的性质所引起的 环核苷酸依赖的蛋白质磷酸化。特别是， 将在几种情况下产生抗膜蛋白的单抗 软体动物海兔的起搏系统。这些抗体将是 通过免疫印迹和免疫细胞化学进行筛选，以选择那些 唯一识别起搏器系统的抗原。这样的“起搏器” 将对特定的“抗体”进行药理干扰测试 起搏器功能是通过细胞内注射实现的。最终，我们希望 将非起搏器细胞注射到起搏器细胞中 适当的信使RNA(MRNA)进入非起搏细胞，以便 确定起搏器功能的所有必要成分 已经被隔离了。这最后一个目标可能不会在第一个目标实现 项目期。 在这些研究中，我们将集中研究三种不同的起搏器系统 在一种生物体中，海兔：1)一种瞬时起搏器--袋状细胞，一种 一组肽能神经元，释放它们的激素信使，通过 产生爆发性起搏器活动约30分钟。2)A 恒定起搏器--可识别的神经元R15、L2、L3、L4和L6 腹神经节产生一组称为“爆发”的冲动， 由持续几秒到几分钟的静默猝发间隔隔开。3) 昼夜节律起搏器系统--眼睛的一部分，表示为 开启和调制在光学系统中发射的起搏器活动 神经，在24小时内。计划开展以下具体项目 在第一个项目期：a)。产生抗病毒的单抗 上述三种起搏器系统的粗膜部分。b)。 特异性识别抗原的单抗的筛选 三种起搏器系统决定因素的免疫细胞化学和 免疫印迹(免疫印迹)。c)。企图干扰起搏器 功能通过细胞内注射“起搏器特异性”的完整 腹神经节。d)。全细胞电压膜片钳 后两种起搏器系统的研究，以确定什么离子 通道受环核苷酸依赖蛋白的调节 磷酸化。