Molecular basis of GPCR-mediated presynaptic inhibition

描述（由申请人提供）： 在突触前末梢，中枢神经系统可改变疗效 在很大的时间范围内的突触传递。G蛋白偶联 受体（GPCR）通常介导这种可塑性。我们已经表明，在一个中央 在突触前末梢，注射γ-氨基丁酸模拟5-HT抑制 神经传递。γ射线对于这种效应至关重要，因为γ射线 清除剂阻断5-HT的抑制作用。伽马射线对快速，动作没有影响 电位诱发细胞内Ca 2+瞬变，触发神经传递。 因此，γ阻断Ca 2+进入下游的胞吐作用，以抑制 神经递质释放5-HT介导的突触前抑制仍然可见 在阻断经典的γ-效应子途径后。我们还表明 Gbetagamma在体外与SNARE蛋白相互作用， 第二次（PC 12细胞）分泌测定。我们的初步数据显示 假设γ射线直接靶向融合机器， 突触前末梢抑制释放。我们将研究这个假设。 是γ射线信号细胞溶质和相对缓慢，涉及中间 磷酸化或去磷酸化事件，或者这种快速涉及直接 γ-射线与膜内蛋白质的结合，例如SNARE？我们 将探测GPCR介导的突触前抑制的时间， 研究胞质信使的可能作用。γ射线结合两个 核心复合物蛋白组分和组装的t-SNARE。我们将探索 核心复合体形成的状态是什么？ 囊泡融合我们将注射蛋白质，缩氨酸和毒素到 功能巨大的突触或将其应用于破裂的PC 12细胞。我们将使用 肉毒杆菌毒素，以确定GPCR的激活是否抑制神经递质 在对接或启动融合机器之前或之后释放。我们将 使用γ-射线突变体，其导致针对 经典的G β γ效应子，并鉴定G β突变， 核心复合物组分的性质发生了变化。这些Gbetagamma 然后将使用突变体来筛选抑制的获得或丧失， 胞吐作用我们将鉴定SNARE蛋白上的Gbeta γ结合位点， 负责β-γ相互作用，然后转染PC 12细胞系 这些蛋白质中的这些位点已经被改变，以确定是否 这些位点的改变可导致功能丧失， γ射线介导的神经递质释放抑制。这些研究将 有助于阐明突触调制的分子机制， 有助于我们理解信息编码的规则， 被认为是学习和记忆的基础的大脑。

DESCRIPTION (provided by applicant): At the presynaptic terminal, the central nervous system can change the efficacy of synaptic transmission over a large temporal range. G protein coupled receptors (GPCRs) commonly mediate this plasticity. We have shown, in a central presynaptic terminal, that Gbetagamma microinjection mimics 5-HT's inhibition of neurotransmission. Gbetagamma is critical for this effect since a Gbetagamma scavenger blocks inhibition by 5-HT. Gbetagamma has no effect on fast, action potential evoked intracellular Ca2+ transients that trigger neurotransmission. Thus, Gbetagamma blocks the exocytosis downstream of Ca2+ entry to inhibit neurotransmitter release. 5-HT-mediated presynaptic inhibition is still seen after blockade of classical Gbetagamma effector pathways. We have also shown that Gbetagamma interacts with SNARE proteins in vitro and inhibits release in a second (PC 12 cell) assay of secretion. Our preliminary data lead us to hypothesize that Gbetagamma directly targets the fusion machinery at the presynaptic terminal to inhibit release. We will investigate this hypothesis. Is Gbetagamma signalling cytosolic and relatively slow, involving intermediate phosphorylation or dephosphorylation events, or is this rapid involving direct binding of Gbetagamma to proteins within the membrane, for example SNARE? We will probe the timing of GPCR-mediated presynaptic inhibition and will also investigate the possible role of cytosolic messengers. Gbetagamma binds both to core complex protein components and to the assembled t-SNARE. We will probe at what state of core complex formation Gbetagamma acts to prevent synaptic vesicular fusion. We will inject proteins, peptides and toxins into a functioning giant synapse or apply these to cracked PC12 cells. We will use botulinum toxins to determine if activation of GPCRs inhibits neurotransmitter release prior to or after docking or priming of the fusion machinery. We will use Gbetagamma mutants which result in both gain and loss of functions against classical Gbetagamma effectors and identify Gbeta mutations whose binding properties to components of the core complex have changed. These Gbetagamma mutants will then be used to screen for gain or loss of inhibition of exocytosis. We will identify Gbetagamma binding sites on the SNARE proteins responsible for Gbetagamma interactions and then transfect PC 12 cell lines with proteins in which these sites have been altered to identify whether alterations in these sites can lead to a loss of function of Gbetagamma-mediated inhibition of neurotransmitter release. These studies will serve to elucidate the molecular mechanisms of synaptic modulation and thus contribute to our understanding of the regulation of information encoding in the brain thought to underlie learning and memory.