TRANSMITTER SECRETION FROM OOCYTES, MYOCYTES AND NEURONS

这是一项研究分子机制的新建议。 神经递质分泌。将采取两种相辅相成的方法。 首先，在“自下而上”的方法中，我们将重建发射机 两种非神经细胞的分泌机制，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的递质和 (2)各种突触前特异性蛋白在脑内的作用 递质分泌。第二，在“自上而下”的方法中， 突触前神经元中的突触前蛋白将被操纵， 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，尽管其他突触前蛋白将在何时研究 分子探针问世了。在第1部分中，我们将重新构建 钙依赖性乙酰胆碱(ACh)的高效分泌机制 非洲爪哇卵母细胞分步注射ACh，纯化含ACh的卵母细胞 突触小泡，以及从胆碱能组织中获得的mRNA(鱼雷 电叶)进入卵母细胞。给定突触前的要求 将通过研究分泌过程中的蛋白质的影响来检测 将特定的反义寡核苷酸与 鱼雷电叶的总mRNA。在第2部分中，我们将使用电压- 钳制肌细胞以电生理检测自发性和 去极化诱导的卵母细胞分泌ACh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3部分，我们将 分步重建非洲爪哇肌细胞的ACh分泌机制 ACh、纯化突触小泡和突触前的介绍 蛋白质。突触前蛋白的功能将由 考察它们对自发性和自发性的影响 去极化诱发的ACh分泌，直接由 心肌细胞的膜电流是由其自身分泌的ACh引起的。在第四部分， 我们将研究突触素和突触素I在ACh分泌中的作用 在非洲爪哇的神经肌肉突触。这些蛋白质在细胞内的地位 突触前神经元将被过度表达或减少表达所改变 蛋白质和蛋白质磷酸化及其对递质的影响 分泌物将通过检查自发的变化来揭示 并激发出突触电流。综上所述，拟议研究将 有助于阐明突触前蛋白的功能作用， 尤其是突触素和突触素I，在递质分泌中，以及 提供对脑内突触传递的分子基础的见解 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.