PHYSIOLOGICAL STUDIES OF NERVE TERMINAL PHOSPHORYLATION

大脑的计算和认知能力源于 其内部通信链路的数量和种类繁多。这个 这些通信链路的结构和功能单位是 Synapse。调节突触强度是ALL的基本属性 神经元回路，被认为是一种基本的机制 隐藏在中枢神经系统的可塑性之下。近几年基本面 多种神经的分离和鉴定研究进展 末端浓缩的蛋白质已经被制造出来了。本研究将审查 协调突触小泡对接和 核聚变。我们将直接比较它们对神经递质释放的影响 的翻译后修饰(蛋白质磷酸化) 释放机械的不同部件。其中的第一部分 研究将确定哪些选定的蛋白质是底物 一组与神经末梢功能有关的蛋白激酶。神经 末端蛋白VAMP、突触素、突触素和SNAP-25将是 作为钙/钙调素依赖的生理底物 蛋白激酶II、蛋白激酶C、cAMP依赖的蛋白激酶和 酪蛋白激酶II的体外和体内检测。在每种情况下， 将确定磷酸化的位置。然后，我们将使用直接 将这些蛋白质注射到突触前元件中作为一种方式 剖析它们在突触小泡调节中的具体作用 贩卖和神经递质释放。使用电子显微镜 我们将比较神经末梢的超微结构变化 由这些注射引起的。每种蛋白的磷酸化作用 这些蛋白质对特定的蛋白质-蛋白质相互作用已经 显示为生理相关的，将使用量化评估 体外结合试验。磷酸化状态特异性抗体将 也可用于超微结构研究，以定位这些 突触内的磷酸化事件。最后，荧光标记的 注射中将使用磷酸化状态特异性抗体。 活体轴突监测磷酸化驱动的实验 对正常运作的突触的调节。这些研究将揭示 选定神经末梢磷酸化的生理作用 蛋白质在神经递质的释放和释放调节中起作用。

The computational and cognitive abilities of the brain arise from the sheer number and variety of its internal communication links. The structural and functional unit of these communication links is the synapse. Modulation of synaptic strength is the basic property of all neuronal circuits and is believed to be a fundamental mechanism underlying the plasticity of the CNS. In recent years fundamental progress in the isolation and characterization of a variety of nerve terminal-enriched proteins has been made. The present study will examine the biochemical machinery that orchestrates synaptic vesicle docking and fusion. We will directly compare the effects on neurotransmitter release of the post-translational modification (protein phosphorylation) of different components of the release machinery. The first part of these studies will determine which of the selected proteins are substrates for a set of protein kinases implicated in nerve terminal function. The nerve terminal proteins VAMP, synaptotagmin, synaptophysin and SNAP-25 will be characterized as physiological substrates for Ca2+/ calmodulin-dependent protein kinase II, protein kinase C, cAMP-dependent protein kinase and casein kinase II using in vitro and in vivo assays. In each case, the sites of phosphorylation will be identified. We will then use direct injections of these proteins into the presynaptic element as a way to dissect their specific roles in the regulation of synaptic vesicles trafficking and neurotransmitter release. Using electron microscopy techniques we will compare ultrastructural changes in the nerve terminal resulting from these injections. Effects of phosphorylation of each of these proteins on specific protein-protein interactions that have been shown as physiologically relevant, will be assessed using quantitative in vitro binding assays. Phosphorylation state-specific antibodies will also be used for ultrastructural studies to localize these phosphorylation events within the synapse. Finally, fluorescently-labeled phosphorylation state-specific antibodies will be employed in injection experiments into living axons to monitor phosphorylation driven regulation in a functioning synapse. These studies will reveal the physiological role for phosphorylation of selected nerve terminal proteins in neurotransmitter release and release modulation.