GABA transporter type 1 (GAT1) function in epilepsy

GABA 转运蛋白 1 型 (GAT1) 在癫痫中的功能

• 批准号: 8422868
• 负责人: Christopher Bruce Ransom
• 金额: --
• 依托单位: VA PUGET SOUND HEALTHCARE SYSTEM
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-01-01 至 2014-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8422868
• 关键词: 3-aminobutyric acid; Acute; Affect; Afghanistan; Aging; American; Animals; Anions; Anticonvulsants; Antiepileptic Agents; Biological Assay; Brain; Caring; Cell physiology; Characteristics; Chemicals; Conflict (Psychology); Connexins; Data; Disease; Dose; Down-Regulation; Drug effect disorder; Electric Stimulation; Electroconvulsive Shock; Epilepsy; Extracellular Space; Failure; Frequencies; GABA Transporter 1; GABA transporter; Genetic; High Pressure Liquid Chromatography; Hippocampus (Brain); Iraq; Kainic Acid; Knock-out; Measurement; Mediating; Membrane Transport Proteins; Mentorship; Modeling; Molecular; Mus; Neurons; Neurotransmitters; Patients; Pentylenetetrazole; Pharmaceutical Preparations; Process; Publishing; Recurrence; Refractory; Research; Research Personnel; Residual state; Seizures; Severities; Signal Transduction; Slice; Status Epilepticus; Stroke; Surface; Synapses; Synaptic Vesicles; Testing; Therapeutic; Traumatic Brain Injury; Veratridine; Veterans; Vigabatrin; base; design; extracellular; gabapentin; gamma-Aminobutyric Acid; improved; in vivo; neuronal excitability; postsynaptic; receptor; relating to nervous system; research study; response; skills; synaptic inhibition; tiagabine; uptake

DESCRIPTION (provided by applicant): Epilepsy affects nearly 3 million Americans and approximately 30% of these patients continue to have seizures despite treatment with multiple antiepileptic drugs. Improved understanding of the cellular and molecular changes underlying seizures, and antiepileptic drug action, is essential to advance the care of veterans suffering from medically refractory epilepsy. Among the changes seen in epilepsy is a reduction of neuronal membrane transporters for the inhibitory neurotransmitter 3-aminobutyric acid (GABA) [GABA transporter type 1 (GAT1)] (During et al., 1995; Patrylo et al., 2001). Classically, GABA transporters were thought to function by removing GABA from the extracellular space, thereby terminating GABA actions. It is now clear that GABA transporters can also operate in the reverse direction to release GABA into the extracellular space, a process termed nonvesicular release. Nonvesicular GABA release is augmented by neuronal depolarization and alterations of ionic gradients, conditions that exist during epileptic seizures. Nonvesicular GABA release may be particularly important during prolonged seizures when vesicular GABA release at synapses is compromised by reductions in extracellular [Ca2+], GABA packaging into synaptic vesicles is limited by energy failure, and GABA synthesis is stimulated by low [ATP], causing an increase in cytoplasmic [GABA] that favors nonvesicular release (Erecinska et al., 1984; Hablitz and Heinemann, 1987; Allen et al., 2004). This proposal will test the hypothesis that nonvesicular GABA release has an important antiepileptic action and GAT1 downregulation in epilepsy is maladaptive. Specifically, it is hypothesized that the loss of nonvesicular GABA release mediated by GAT1 will exacerbate seizures despite elevated basal concentrations of ambient GABA. Consistent with this, studies on animals with genetic deletion of GABA transporter type 1 [GAT1 knockout (GAT1 KO)] have identified a heightened sensitivity to chemoconvulsants (Chiu et al., 2005). GAT1 function in epilepsy will be evaluated by three specific aims: Specific Aim 1 will test the hypothesis that nonvesicular GABA release in brain slices from GAT1 KO animals is reduced compared to wild type littermates. Specific Aim 2 will test the hypothesis that AEDs that increase GABA concentration or GAT1 surface expression will enhance nonvesicular release from wild type but not GAT1 KO neurons. Specific Aim 3 will test the hypothesis that nonvesicular release mediated by GAT1 affects seizure threshold in vivo and limits seizure frequency/severity in epileptic mice. Identification of changes in nonvesicular release in GAT1 knockout neurons and determination of AED effects on nonvesicular release in Specific Aims 1-2 will provide a basis for interpretation of in vivo data.

描述（由申请人提供）： 癫痫症影响着近 300 万美国人，其中约 30% 的患者在接受多种抗癫痫药物治疗后仍持续癫痫发作。更好地了解癫痫发作背后的细胞和分子变化以及抗癫痫药物的作用，对于促进患有难治性癫痫的退伍军人的护理至关重要。癫痫中观察到的变化之一是抑制性神经递质 3-氨基丁酸 (GABA) [GABA 转运蛋白 1 型 (GAT1)] 的神经元膜转运蛋白减少（During 等人，1995 年；Patrylo 等人，2001 年）。传统上，GABA 转运蛋白被认为是通过从细胞外空间去除 GABA 来发挥作用，从而终止 GABA 的作用。现在很清楚，GABA 转运蛋白也可以反向操作，将 GABA 释放到细胞外空间，这一过程称为非囊泡释放。非囊泡 GABA 的释放会因​​神经元去极化和离子梯度的改变（癫痫发作期间存在的条件）而增强。非囊泡 GABA 释放在长时间癫痫发作期间可能特别重要，此时突触处的囊泡 GABA 释放受到细胞外 [Ca2+] 减少的影响，GABA 包装到突触囊泡中受到能量衰竭的限制，并且 GABA 合成受​​到低 [ATP] 的刺激，导致细胞质 [GABA] 的增加有利于非囊泡释放（Erecinska 等，1984；Hablitz 和海涅曼，1987；艾伦等人，2004）。该提案将检验以下假设：非囊泡 GABA 释放具有重要的抗癫痫作用，并且癫痫中 GAT1 下调会导致适应不良。具体来说，假设 GAT1 介导的非囊泡 GABA 释放的丧失将加剧癫痫发作，尽管环境 GABA 的基础浓度升高。与此相一致的是，对 1 型 GABA 转运蛋白基因缺失 [GAT1 敲除 (GAT1 KO)] 的动物进行的研究发现，动物对化学惊厥药的敏感性更高（Chiu 等，2005）。 GAT1 在癫痫中的功能将通过三个具体目标进行评估： 具体目标 1 将测试以下假设：与野生型同窝动物相比，GAT1 KO 动物脑切片中的非囊泡 GABA 释放减少。具体目标 2 将检验以下假设：增加 GABA 浓度或 GAT1 表面表达的 AED 将增强野生型而非 GAT1 KO 神经元的非囊泡释放。具体目标 3 将测试以下假设：GAT1 介导的非囊泡释放影响体内癫痫阈值并限制癫痫小鼠的癫痫发作频率/严重程度。鉴定 GAT1 敲除神经元中非囊泡释放的变化以及确定特定目标 1-2 中 AED 对非囊泡释放的影响将为体内数据的解释提供基础。