Non-vesicular GABA release via GABA transporter reversal

描述(由申请人提供)：我们工作的长期目标是确定GABA转运体的作用，这是GABA能系统的一个关键元素，将大脑的兴奋性保持在正常范围内。许多神经学家认为GABA转运体仅仅是囊泡融合释放的GABA的清道夫。然而，新的数据表明，GABA转运体的行为要复杂得多，它们在神经元抑制中发挥着积极的作用，远远超出了简单地重新摄取GABA的范围。例如，在神经递质转运体中，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。即使它们不逆转，它们也在调节紧张性抑制量方面发挥着重要作用，紧张性抑制量是一种新发现的GABA信号形式，其原因是高亲和力突触外GABAA受体的持续激活。因此，越来越多的证据表明，GABA转运体不仅是GABA真空吸尘器，而且在控制大脑兴奋性方面发挥着更具活力的作用。我们提出了一个新的假设，即在神经元放电过程中，膜电位的增加和细胞内[Na+]的升高导致GABA转运体逆转，细胞外[GABA]增加，更多的紧张性抑制。我们进一步提出，GABA转运体是静息状态下细胞外[GABA]的主要决定因素之一，因为它们只会将GABA转运到细胞内，直到细胞达到平衡，而在正常条件下，当细胞外[GABA]仍然相对较高时，才会达到这种平衡。因此，通过建立细胞外GABA的“底限水平”，它们负责维持最低限度的紧张性抑制。在这里，我们计划通过以下方式来测试这些假设：1)使用一种新颖的、高灵敏的转运体逆转功能分析来直接测量GAT1和GAT3逆转的容易程度；2)确定神经元是否可以通过GAT1逆转在动作电位中释放GABA；3)测量抗惊厥剂Vigabatrin治疗后细胞内和细胞外的[GABA]，它选择性地增强紧张性抑制；4)确定GAT1逆转与其他形式的非囊泡性GABA释放相比的相对重要性；以及5)确定GAT1不依赖于GAT1的非囊状形式的GABA释放的机制。失去正常的GABA能抑制可导致癫痫发作，而加强抑制可限制缺血时的兴奋性毒性。因此，这里提出的工作将导致更好地洞察正常的突触生理和控制在癫痫和中风等病理生理条件下的抑制。预期的结果可能会导致神经疾病的新疗法，旨在增强非囊泡性GABA的释放，并针对新发现的紧张性抑制形式。

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.