NEURONAL PACEMAKERS--ANTIBODIES AS PROBES

本申请中描述的研究集中在 起搏器机制及其在神经元中的调节。 最终目标是 为了确定介导这一过程的一组细胞质和膜蛋白， 重要的功能，并确定调制的性质所造成的 环核苷酸依赖性蛋白磷酸化。 特别是， 将在几种细胞中产生针对膜蛋白的单克隆抗体， 在软体动物中的起搏器系统。 这些抗体将 通过免疫印迹和免疫细胞化学筛选，以选择那些 唯一识别起搏器系统的抗原。 这样的“起搏器 将检测“特异性”抗体的药理学干扰， 通过细胞内注射实现起搏器功能。 最后，我们希望 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， 确定起搏器功能的所有基本成分 被隔离了 这最后一个目标在第一个目标期间可能无法实现。 项目期间。 对于这些研究，我们将集中在三种不同的起搏器系统 在一种生物体中，失智症：1）一种短暂的起搏器-袋细胞， 一组肽能神经元，释放激素信使， 产生大约30分钟的突发起搏器活动。 2)一 恒定起搏器--可识别的神经元R15，L2，L3，L4和L 6， 腹神经节，产生称为“脉冲群”的脉冲群， 由持续数秒到数分钟的无声脉冲串间隔隔开。 第三章 昼夜节律起搏系统--眼睛的一部分， 开启和调节起搏器活动，在光学中发射 在24小时内， 计划开展以下具体项目 第一阶段：A）。 产生单克隆抗体， 上述三种起搏器系统的粗膜部分。 B）。 特异性识别抗原的单克隆抗体的筛选 免疫细胞化学法检测三种起搏系统的决定因素， 免疫印迹（Western印迹）。 C）。 试图干扰起搏器 功能通过细胞内注射“起搏器特异性”的完整的 腹神经节 D）。 全细胞电压和膜膜片钳 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蛋白调节 磷酸化

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.