Non-vesicular GABA release via GABA transporter reversal

通过 GABA 转运蛋白逆转释放非囊泡 GABA

基本信息

批准号：
7388533
负责人：
GEORGE B RICHERSON
金额：
$ 33.06万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-04-01 至 2012-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7388533
关键词：
4-Aminobutyrate aminotransferase Action Potentials Address Affect Affinity Anions Anticonvulsants Astrocytes Behavior Biological Assay Brain Calcium Cells Chinese Hamster Ovary Cell Complex Condition Coupled Cultured Cells Data Dependence Disease Elements Epilepsy Equilibrium Extracellular Fluid FarGo Figs - dietary Fire - disasters Floor Frequencies GABA transporter Goals Health High Pressure Liquid Chromatography Hippocampus (Brain)Ischemia Knock-out Knockout Mice Lead Link Measurement Measures Mediating Membrane Membrane Potentials Methods Mus Neuroglia Neurons Neurotransmitters Personal Satisfaction Physiological Physiology Play Proteins Regulation Relative (related person)Rest Ringer&apos s solution Role Seizures Side Signal Transduction Simulate Slice Source Stimulus Stroke Swelling Synapses Synaptic Transmission Synaptic Vesicles System Testing Theoretical model Time Vacuum Vesicle Vigabatrin Wild Type Mouse Work design excitotoxicity extracellular gamma-Aminobutyric Acid in vivo insight mind control nervous system disorder neurotransmission neurotransmitter release neurotransmitter reuptake novel receptor research study response reuptake stoichiometry

项目摘要

DESCRIPTION (provided by applicant): The long-term goal of our work is to define the role of GABA transporters, which are a critical element of the GABAergic system that maintains brain excitability within normal limits. Many neuroscientists view GABA transporters simply as scavengers of GABA that has been released by vesicular fusion. However, new data suggest that the behavior of GABA transporters is much more complex, and that they play an active role in neuronal inhibition that goes far beyond simply reuptake of GABA. For example, among the neurotransmitter transporters they have a particularly low threshold for reversal, and when they reverse they release GABA into the extracellular fluid. Even when they don't reverse they play an important role in regulation of the amount of tonic inhibition, a newly discovered form of GABA signaling due to continuous activation of high affinity extrasynaptic GABAA receptors. Thus, accumulating evidence indicates that GABA transporters are not just GABA vacuum cleaners, but play a much more dynamic role in control of brain excitability. We have proposed the novel hypothesis that during neuronal firing the increase in membrane potential and rise in intracellular [Na+] leads to GABA transporter reversal, an increase in extracellular [GABA], and more tonic inhibition. We have further proposed that GABA transporters are one of the major determinants of extracellular [GABA] at rest, by virtue of the fact that they will only transport GABA into cells until they reach their equilibrium, and under normal conditions this equilibrium is reached when extracellular [GABA] is still relatively high. Thus, by establishing the "floor level" of extracellular GABA, they are responsible for maintaining a minimum amount of tonic inhibition. Here we plan experiments that test these hypotheses by: 1) Directly measuring how easily GAT1 and GAT3 reverse, using a novel, highly sensitive functional assay of transporter reversal; 2) Determining whether neurons can release GABA during action potentials via GAT1 reversal, 3) Measuring intracellular and extracellular [GABA] in response to treatment with the anticonvulsant vigabatrin, which selectively enhances tonic inhibition; 4) Determining the relative importance of GAT1 reversal compared to other forms of nonvesicular GABA release, and; 5) Defining the mechanism of a GAT1- independent nonvesicular form of GABA release that appears to come from glia. Loss of normal GABAergic inhibition can lead to seizures, and enhancement of inhibition may limit excitotoxicity during ischemia. Thus, the work proposed here will lead to better insight into normal synaptic physiology and control of inhibition during pathophysiological conditions such as epilepsy and strokes. The anticipated results may lead to new treatments for neurological disease aimed at enhancing nonvesicular GABA release and targeting the newly discovered form of tonic inhibition.

描述（由申请人提供）：我们工作的长期目标是确定 GABA 转运蛋白的作用，GABA 转运蛋白是维持大脑兴奋性在正常范围内的 GABA 能系统的关键要素。许多神经科学家将 GABA 转运蛋白简单地视为通过囊泡融合释放的 GABA 的清除剂。然而，新数据表明，GABA 转运蛋白的行为要复杂得多，它们在神经元抑制中发挥着积极作用，其作用远远超出了简单的 GABA 重摄取。例如，在神经递质转运蛋白中，它们的逆转阈值特别低，当它们逆转时，它们会将 GABA 释放到细胞外液中。即使它们没有逆转，它们在调节强直性抑制量方面也发挥着重要作用，强直性抑制是一种新发现的 GABA 信号传导形式，由于高亲和力突触外 GABAA 受体的持续激活而产生。因此，越来越多的证据表明，GABA 转运蛋白不仅仅是 GABA 真空吸尘器，而且在控制大脑兴奋性方面发挥着更加动态的作用。我们提出了新的假设，即在神经元放电过程中，膜电位的增加和细胞内 [Na+] 的增加导致 GABA 转运蛋白逆转、细胞外 [GABA] 增加和更多的强直性抑制。我们进一步提出，GABA 转运蛋白是静止状态下细胞外 [GABA] 的主要决定因素之一，因为它们只会将 GABA 转运到细胞内，直到达到平衡，而在正常条件下，当细胞外 [GABA] 达到平衡时，就会达到这种平衡。 [GABA]仍然较高。因此，通过建立细胞外 GABA 的“下限水平”，它们负责维持最小量的强直抑制。在这里，我们计划通过以下实验来测试这些假设：1）使用一种新颖的、高度灵敏的转运蛋白逆转功能测定法直接测量 GAT1 和 GAT3 逆转的难易程度； 2) 确定神经元是否可以通过 GAT1 逆转在动作电位期间释放 GABA，3) 测量细胞内和细胞外 [GABA] 以响应抗惊厥药氨己烯酸治疗，选择性增强强直抑制； 4) 确定 GAT1 逆转与其他形式的非囊泡 GABA 释放相比的相对重要性； 5) 定义似乎来自神经胶质细胞的 GAT1 独立非囊泡形式 GABA 释放的机制。正常 GABA 能抑制的丧失可能导致癫痫发作，而抑制的增强可能会限制缺血期间的兴奋性毒性。因此，这里提出的工作将有助于更好地了解癫痫和中风等病理生理条件下的正常突触生理学和抑制控制。预期结果可能会带来神经系统疾病的新疗法，旨在增强非囊泡 GABA 释放并针对新发现的强直抑制形式。