Molecular basis of GPCR-mediated presynaptic inhibition

描述(由申请人提供)： 在突触前终末，中枢神经系统可以改变疗效 在很大的时间范围内的突触传输。G蛋白偶联 受体(GPCRs)通常介导这种可塑性。我们已经展示了，在中央 突触前终末Gbetagamma微量注射模拟5-羟色胺的抑制 神经传递的影响。Gbetagamma对这种效果至关重要，因为Gbetagamma 清道夫可阻断5-羟色胺的抑制作用。Gbetagamma对快速、动作没有影响 电位诱发细胞内钙瞬变，从而触发神经传递。 因此，Gbetagamma阻止钙离子进入下游的胞吐作用以抑制 神经递质释放。5-羟色胺介导的突触前抑制仍可见 在阻断经典Gbetagamma效应器通路后。我们还展示了 Gbetagamma在体外与SNARE蛋白相互作用并抑制 第二次(PC 12细胞)分泌物检测。我们的初步数据引导我们 假设Gbetagamma将聚变机器直接对准 突触前终末抑制释放。我们将调查这一假设。 Gbetagamma是否胞浆信号传递较慢，涉及中间体 磷酸化或去磷酸化事件，或者这种快速涉及直接 Gbetagamma与膜内蛋白质的结合，例如SNARE？我们 将探索GPCR介导的突触前抑制的时间，还将 研究胞浆信使的可能作用。Gbetagamma将两者绑定到 核心复杂蛋白质组分和组装的T-SNARE。我们将探讨 Gbetagamma以什么状态形成核心复合体来防止突触 囊泡融合。我们将把蛋白质、多肽和毒素注入一个 正常运作的巨型突触或将这些应用于破裂的PC12细胞。我们将使用 肉毒杆菌毒素确定GPCRs激活是否抑制神经递质 在聚变机械对接或引爆之前或之后释放。我们会 使用Gbetagamma突变体，它会同时导致功能的获得和丧失 经典的Gbetagamma效应器并鉴定其结合的Gbeta突变 核心复合体的组件的属性发生了变化。这些Gbetagamma 然后将使用突变体来筛选抑制作用的获得或丧失 胞吐。我们将确定SNARE蛋白上的Gbetagamma结合位点 负责Gbetagamma相互作用，然后转染PC 12细胞系 这些位点已经被改变的蛋白质来识别 这些部位的改变可能会导致功能丧失 Gbetagamma介导的神经递质释放抑制。这些研究将 有助于阐明突触调节的分子机制，从而 有助于我们理解信息编码的规则 大脑被认为是学习和记忆的基础。

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.