TRANSMITTER SECRETION FROM OOCYTES, MYOCYTES AND NEURONS

这是一个新的建议，研究分子机制的基础 神经递质分泌 将采取两种互补的方法。 首先，在“自下而上”的方法中，我们将重构发射机 两种非神经元细胞类型，卵母细胞和 肌细胞，以确定（1）必要的成分 并足以有效地分泌钙依赖性递质， (2)各种突触前特异性蛋白质在突触后神经元中的功能 递质分泌 第二，在“自上而下”的方法中， 将操纵突触前神经元中的突触前蛋白， 将检查对突触传递的影响，以确定 这些蛋白质的功能。 两种突出的突触前蛋白， 突触素和突触素I，将是目前的主要焦点 项目，虽然其他突触前蛋白质将被研究时， 分子探针变得可用。 在第1部分中，我们将重新构建一个 有效的钙依赖性乙酰胆碱（ACh）分泌机制， 通过逐步注射ACh、纯化的含ACh的 突触囊泡和从胆碱能组织获得的mRNA（Torpedo 电叶）进入卵母细胞。 一个给定的突触前神经元 蛋白质在分泌过程中的作用将通过研究 共注射特定的反义寡核苷酸， 电鳐电叶总mRNA。 在第2部分中，我们将使用电压- 钳夹心肌细胞，以检测自发和 去极化诱发的乙酰胆碱分泌从卵母细胞，以更好地 描述卵母细胞分泌的特性，并鉴定 各种突触前蛋白的功能。 在第三部分，我们将 通过逐步重组非洲爪蟾肌细胞ACh分泌机制， 引入ACh、纯化的突触囊泡和突触前 proteins. 突触前蛋白质的功能将由 研究它们对自发性质和 去极化诱发的乙酰胆碱分泌，直接监测的 肌细胞自身ACh分泌诱导的膜电流。 在第四部分中， 我们将研究突触素和突触素I在ACh分泌中的作用 非洲爪蟾神经肌肉突触 这些蛋白质的状态在 突触前神经元将被过度或减少表达的 蛋白质和蛋白质磷酸化，以及对递质的影响 分泌将通过检查自发性的变化来揭示。 诱发突触电流 综合起来，拟议的研究将 有助于阐明突触前蛋白的功能作用， 特别是突触素和突触素I，在递质分泌中，和 提供深入了解突触传递的分子基础， 神经系统

This is a new proposal for studying the molecular mechanisms underlying neurotransmitter secretion. Two complementary approaches will be taken. First, in a "bottom-up" approach, we will reconstitute transmitter secretion mechanisms in two non-neuronal cell types, oocytes and myocytes, in order to determine (1) the components which are necessary and sufficient for efficient calcium-dependent transmitter secretion and (2) the functions served by various presynaptic specific proteins in transmitter secretion. Second, in a "top-down" approach, the status of presynaptic proteins in presynaptic neurons will be manipulated and the effects on synaptic transmission will be examined to determine the functions of these proteins. Two prominent presynaptic proteins, synaptophysin and synapsin I, will be the main focus of the present project, although other presynaptic proteins will be studied when molecular probes become available. In part 1, we will reconstitute an efficient calcium-dependent acetycholine (ACh) secretion mechanism in Xenopus oocytes by a stepwise injection of ACh, purified ACh-containing synaptic vesicles, and mRNA obtained from cholinergic tissue (Torpedo electric lobe) into the oocyte. The requirement of a given presynaptic protein in the secretion process will be examined by studying the effect of co-injection of specific antisense oligonucleotides together with total mRNA of Torpedo electric lobe. In part 2, we will use a voltage- clamped myocyte to detect electrophysiologically both spontaneous and depolarization-evoked ACh secretion from oocytes in order to better characterize the properties of oocyte secretion and to identify the functions of various presynaptic proteins. In part 3, we will reconstitute ACh secretion mechanisms in Xenopus myocytes by a stepwise introduction of ACh, purified synaptic vesicles, and presynaptic proteins. The functions of presynaptic proteins will be determined by examining their effects on the properties of spontaneous and depolarization-evoked ACh secretion, as monitored directly by the myocyte's membrane current induced by its own ACh secretion. In part 4, we will study the roles of synaptophysin and synapsin I in ACh secretion at Xenopus neuromuscular synapses. The status of these proteins in the presynaptic neuron will be altered by over- or reduced-expression of the protein and by protein phosphorylation, and the effect on transmitter secretion will be revealed by examining the changes in the spontaneous and evoked synaptic currents. Taken together, the proposed studies will help to elucidate the functional roles of presynaptic proteins, synaptophysin and synapsin I in particular, in transmitter secretion, and provide insights into the molecular basis of synaptic transmission in the nervous system.