NEURONAL PACEMAKERS--ANTIBODIES AS PROBES

神经起搏器——抗体作为探针

• 批准号: 3401790
• 负责人: FELIX STRUMWASSER
• 金额: $ 13.02万
• 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 1987-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3401790
• 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集中于以下性质： 起搏器机制及其在神经元中的调节。 最终目标是 定义介导这一过程的一组细胞质和膜蛋白 重要功能并确定由引起的调制的性质 环核苷酸依赖性蛋白质磷酸化。 尤其， 将在多种情况下产生针对膜蛋白的单克隆抗体 软体动物海兔的起搏器系统。 这些抗体将 通过免疫印迹和免疫细胞化学筛选，选择那些 独特地识别起搏器系统的抗原。 这样的“起搏器 将测试特定“抗体的药理干扰 通过细胞内注射发挥起搏器功能。 最终，我们希望 通过注射将非起搏细胞转化为起搏细胞 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重要组成部分是否 已被隔离。 最后一个目标可能无法在第一个目标中实现 项目期。 对于这些研究，我们将重点关注三种不同的起搏器系统 在一种生物体海兔中：1）一种短暂的起搏器——袋细胞， 一组肽能神经元，通过释放激素信使 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 腹部神经节，产生称为“爆发”的脉冲组， 由持续几秒到几分钟的无声间歇间隔分开。 3） 昼夜节律起搏器系统——眼睛的一部分，表现为 起搏器活动的开启和调制，在光学器件中发射 神经，24小时内。 规划了以下具体项目 在第一个项目期间：A). 生成单克隆抗体 上述三个起搏器系统的粗膜部分。 B）。 筛选特异性识别抗原的单克隆抗体 通过免疫细胞化学分析三种起搏器系统的决定因素 免疫印迹（蛋白质印迹）。 C）。 试图干扰起搏器 通过细胞内注射完整的“起搏器特异性”来发挥功能 腹部神经节。 D）。 全细胞电压膜片钳 研究后两个起搏器系统以确定什么离子 通道由环核苷酸依赖性蛋白调节 磷酸化。