G Protein Coupled Receptor Modulation of Synaptic Transmission

描述（由申请人提供）：调节性神经递质如多巴胺和5-HT涉及严重的神经病理学，包括偏头痛、抑郁症、精神分裂症、记忆障碍、癫痫以及感觉和运动过程缺陷。受体，特别是G蛋白偶联受体（GPCR），是这些神经递质的突触前和突触后靶点。GPCR是细胞表面受体的最大家族。因此，了解这些受体如何表现出其无数的影响至关重要。特别是涉及5-HT 1B受体的信号传导缺陷在上述所有病理中值得注意。该受体位于突触前并抑制递质释放。这种抑制的潜在分子机制已经在原始脊椎动物七鳃鳗的轴突中确定。在七鳃鳗中，5-HT通过Gpy对基本囊泡融合机制（SNARE复合物）的直接作用来抑制递质释放，从而引起突触囊泡融合模式的改变并降低诱发的谷氨酸间隙浓度。然而，尚不清楚这种机制是否在哺乳动物中保守。了解哺乳动物中枢神经系统中的生化级联反应可以为药物干预提供模型。在海马体中， 5-HT 1B受体在CA 1锥体细胞的突触前末梢上表达，所述锥体细胞局部突触并且还投射到下托和内嗅皮层。5-HT 1B受体减少突触间隙 谷氨酸浓度而不影响释放概率，这表明融合模式的转变。经典模型的发射器释放预测囊泡融合发生通过完全融合，但最近的进展表明，另一种模式，吻和运行，可能发生在中枢神经系统。我打算确定减少谷氨酸浓度对突触后活动的影响，并假设NMDA和AMPA受体反应不受5-HT的影响。突触前末梢的不可接近性使得对单个突触的检查变得困难。为了克服这一限制，我将分析Ca 2+瞬变在单个树突棘。此外，GPCR介导的抑制作用最常见的突触前机制是突触前钙离子内流衰减。因此，我将类似地调查突触前钙离子进入，以确定介导的5-HT介导的神经递质释放减少的过程。最后，为了理解5-HT 1B受体降低裂谷谷氨酸浓度的分子机制，我们将使用肉毒杆菌毒素A探测囊泡融合机制，肉毒杆菌毒素A切割形成SNARE复合物的蛋白质的一部分，其中G| ty已知与Gpy-SNARE相互作用，最后将使用载体技术放置肽抑制剂以干扰Gpy-SNARE复合物相互作用。

DESCRIPTION (provided by applicant): Modulatory neurotransmitters such as dopamine and 5-HT are implicated in serious neuropathologies that range from migraine, depression, schizophrenia, memory impairment, epilepsy, and defects in sensory and motor processes. Receptors, particularly G protein coupled receptors (GPCRs), are the presynaptic and postsynaptic targets of these neurotransmitters. GPCRs are the largest family of cell surface receptors. Therefore, understanding how these receptors exhibit their myriad effects is vital. Defects in signaling involving 5-HT1B receptors, in particular, are of note in all the pathologies above. This receptor is presynaptically localized and inhibits transmitter release. An underlying molecular mechanism of this inhibition has been determined in axons of a primitive vertebrate, the lamprey. In lamprey, 5-HT inhibits transmitter release by a direct action of Gpy on the essential vesicle fusion machinery, the SNARE complex, to cause a change in mode of synaptic vesicle fusion and reduce the evoked cleft glutamate concentration. However, it is unknown whether this mechanism is conserved in mammals. Understanding of the biochemical cascade in the mammalian CNS could provide a model for drug intervention. In hippocampus, the 5-HT1B receptor is expressed on presynaptic terminals of CA1 pyramidal cells that synapse locally and also project to the subiculum and entorhinal cortex. The 5-HT1B receptor reduces the synaptic cleft glutamate concentration without affecting release probability, which suggests a shift in mode of fusion. The classical model of transmitter release predicts vesicle fusion occurs through full fusion, but recent advances indicate an alternative mode, kiss-and-run, may occur in the CNS. I intend to determine the effect of reduced cleft glutamate concentrations on postsynaptic activity and hypothesize that NMDA and AMPA receptor responses are not impacted equally by 5-HT. The inaccessibility of the presynaptic terminal makes examination of individual synapses difficult. To overcome this limitation, I will analyze Ca2+ transients in single dendritic spines. Furthermore, the presynaptic mechanism most commonly attributed to GPCR mediated inhibition is presynaptic attenuation of Ca2+ entry. Thus, I will similarly investigate presynaptic Ca2+ entry to determine processes that mediate the 5-HT-mediated reduction in neurotransmitter release. Finally, to understand the molecular mechanism by which 5-HT1B receptor reduces cleft glutamate concentration, we will probe the vesicle fusion machinery using botulinum toxin A, which cleaves a part of the protein that makes the SNARE complex, where G|ty is known to interact with and finally a carrier technique will be used to place peptide inhibitors to interfere with Gpy-SNARE complex interactions.