TRANSMITTER SECRETION FROM OOCYTES, MYOCYTES AND NEURONS

卵母细胞、肌细胞和神经元分泌递质

基本信息

批准号：
2269880
负责人：
MU-MING POO
金额：
$ 22.55万
依托单位：
COLUMBIA UNIV NEW YORK MORNINGSIDE
依托单位国家：
美国
项目类别：
财政年份：
1993
资助国家：
美国
起止时间：
1993-08-01 至 1997-07-31
项目状态：
已结题

项目摘要

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.

这是研究潜在分子机制的新提议神经递质的分泌。将采取两种互补的方法。首先，以“自下而上”的方式，我们将重构发射机两种非神经元细胞类型（卵母细胞和）的分泌机制肌细胞，以确定（1）必需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的功能递质分泌。其次，以“自上而下”的方式，突触前神经元中的突触前蛋白将被操纵，并且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 将是本次研究的重点项目，尽管其他突触前蛋白将被研究分子探针变得可用。在第 1 部分中，我们将重构一个有效的钙依赖性乙酰胆碱（ACh）分泌机制通过逐步注射乙酰胆碱（含有纯化的乙酰胆碱）的爪蟾卵母细胞突触小泡和从胆碱能组织（Torpedo 电叶）进入卵母细胞。给定突触前的要求通过研究蛋白质在分泌过程中的作用来检查与特定反义寡核苷酸一起共注射鱼雷电叶总 mRNA。在第 2 部分中，我们将使用电压夹住肌细胞以检测自发和电生理学去极化诱发卵母细胞分泌乙酰胆碱，以便更好地表征卵母细胞分泌的特性并确定各种突触前蛋白的功能。在第 3 部分中，我们将通过逐步重建非洲爪蟾肌细胞中的 ACh 分泌机制引入 ACh、纯化的突触小泡和突触前蛋白质。突触前蛋白的功能由以下因素决定检查它们对自发和自然特性的影响去极化诱发的 ACh 分泌，直接由肌细胞由其自身的ACh分泌引起的膜电流。在第 4 部分中，我们将研究突触素和突触素 I 在乙酰胆碱分泌中的作用在非洲爪蟾的神经肌肉突触处。这些蛋白质的状态突触前神经元的过度或减少表达会改变蛋白质和蛋白质磷酸化，以及对递质的影响通过检查自发性的变化可以揭示分泌情况并诱发突触电流。总而言之，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，以及提供对突触传递的分子基础的见解神经系统。