G Protein Coupled Receptor Modulation of Synaptic Transmission

描述(申请人提供)：调节性神经递质，如多巴胺和5-羟色胺，与严重的神经病理有关，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(GPCRs)，是这些神经递质的突触前和突触后靶。GPCRs是细胞表面最大的受体家族。因此，了解这些受体如何发挥其众多作用是至关重要的。尤其是涉及5-HT1B受体的信号缺陷，在上述所有病理中都是值得注意的。这种受体在突触前定位，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被确定。在七鳃鳗中，5-羟色胺通过直接作用于必需的囊泡融合机制SNARE复合体来抑制递质的释放，从而导致突触囊泡融合模式的改变，并降低诱发的裂隙谷氨酸浓度。然而，目前尚不清楚这种机制在哺乳动物中是否保守。了解哺乳动物中枢神经系统的生化级联反应可以为药物干预提供模型。在海马体中， 5-HT1B受体表达于CA1锥体细胞的突触前终末，这些细胞在局部突触，也投射到下丘脑和内嗅觉皮质。5-HT1B受体减少突触裂隙 谷氨酸浓度不影响释放概率，这表明融合模式发生了变化。经典的递质释放模型预测囊泡融合是通过完全融合发生的，但最近的研究表明，另一种模式，即接吻和逃跑，可能发生在中枢神经系统。我打算确定裂隙谷氨酸浓度降低对突触后活性的影响，并假设5-羟色胺对NMDA和AMPA受体反应的影响不是一样的。突触前终末的不可达性使得对单个突触的检查变得困难。为了克服这一限制，我将分析单个树突棘中的钙瞬变。此外，突触前机制最常见的归因于GPCR介导的抑制是突触前钙离子进入的减弱。因此，我将类似地研究突触前钙离子进入，以确定介导5-羟色胺介导的神经递质释放减少的过程。最后，为了了解5-HT1B受体降低裂隙谷氨酸浓度的分子机制，我们将使用肉毒杆菌毒素A来探索囊泡融合机制，它切割使SNARE复合体的蛋白质的一部分，其中G|ty已知与之相互作用，最后将使用载体技术放置肽抑制剂来干扰GPY-SNARE复合体的相互作用。

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.