Molecular Physiology of gamma-Aminobutyric Acid Transporters

γ-氨基丁酸转运蛋白的分子生理学

• 批准号: 7478641
• 负责人: SEPEHR ESKANDARI
• 金额: $ 18.06万
• 依托单位: CALIFORNIA STATE POLY U POMONA
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7478641
• 关键词: Acids; Address; Affinity; Amino Acids; Aminobutyric Acid; Aminobutyric Acids; Antiepileptic Agents; Binding; Binding Sites; Brain; Carbon; Cell membrane; Cells; Charge; Chemosensitization; Chromosome Pairing; Complement component C1s; Data Set; Dissociation; Ensure; Epilepsy; Event; Exhibits; Flufenamic Acid; Future; GAT3 transporter; Ions; Kinetics; Laboratories; Lead; Ligands; Methods; Modeling; Molecular; Molecular Conformation; Movement; Nature; Nerve; Nervous system structure; Neuraxis; Neurotransmitters; Nipecotic Acids; Numbers; Oocytes; Operative Surgical Procedures; Patch-Clamp Techniques; Perfusion; Pharmaceutical Preparations; Pharmacology; Phase; Physiological; Physiology; Preclinical Drug Evaluation; Probability; Process; Protein Isoforms; Rate; Reaction; Research; Role; Rotation; Screening procedure; Solutions; Specificity; Stroke; Structure; Surface; Synapses; System; Therapeutic; Valproic Acid; Xenopus laevis; analog; aqueous; base; channel blockers; computer studies; design; extracellular; gamma-Aminobutyric Acid; inhibitor/antagonist; insight; molecular mechanics; neurotransmission; nipecotic acid; novel; patch clamp; pharmacophore; prevent; research study; single bond; tiagabine; valproate; voltage; voltage clamp

Our long-term objective is to gain a comprehensive understanding of the molecular mechanism by which gamma-aminobutyric acid (GABA) transporters accomplish Na+/Cl-/GABA cotransport across the plasma membrane. The GABA transporters use the Na + electrochemical gradient to transport GABA into cells after its release from nerve terminals and, thus, they regulate the concentration and lifetime of GABA in synaptic and extra-synaptic regions in the nervous system. In addition, they prevent spillover of GABA to surrounding synapses and, therefore, they ensure synaptic specificity. GABA is the most abundant inhibitory neurotransmitter in the central nervous system and, therefore, potentiation of GABAergic neurotransmission via inhibition or reversal of the GABA transporters is believed to have therapeutic value in treating epileptic seizures and stroke. Four GABA transporter isoforms are present in the mammalian brain (GAT1, GAT2, GAT3, and GAT4), and exhibit significant differences in function, pharmacology, and localization. Indeed, the GABA transporters have been implicated in epilepsy, and one isoform (GAT1) is the target of the anti-epileptic drug tiagabine. Unfortunately, no drug exists which specifically targets GAT2 or GAT3, but we have recently identified a GAT4-specific inhibitor. We will express the GABA transporters in Xenopus laevis oocytes in order to address the following Specific Aims: (1) To use rapid concentration jumps at the GABA transporter binding pocket in order to gain mechanistic insight about ion and substrate binding and translocation across the plasma membrane. These experiments will use a novel rapid perfusion system developed in this laboratory, and will perform Na +, CI-, and GABA jumps at the transporter to gain a detailed understating of ligand interaction with the transporter. (2) To fully examine a novel Cl- channel mode identified in GAT4. These experiments will illuminate the significance of a novel Cl- channel mode, which we have recently identified in GAT4. (3) To fully characterize the action of a recently identified inhibitor with selectivity for GAT4. We have succeeded in identifying a novel specific inhibitor for GAT4. The experiments of this aim will fully characterize the inhibitory action of this agent, and will pave the way for determining the contribution of GAT4 to GABAergic inhibitory neurotransmission. (4) To formulate a substrate pharmacophore for the GABA transporters GAT3 and GAT4. As most studies have focused on GAT1, very little is known about the substrate binding pocket of GAT3 and GAT4. The experiments of this aim will identify lead compounds for future structure-guided design of specific inhibitors of GAT3 and GAT4.

我们的长期目标是对γ-氨基丁酸（GABA）转运蛋白的分子机制进行全面了解，从而使Na+/cl-/gaba cotransport跨越了质膜。 GABA转运蛋白使用Na +电化学梯度从神经末端释放后将GABA转运到细胞中，因此，它们调节了神经系统中突触和突触外区域中GABA的浓度和寿命。此外，它们可以防止GABA溢出到周围的突触，因此可以确保突触特异性。 GABA是中枢神经系统中最丰富的抑制性神经递质，因此，通过抑制或逆转GABA转运蛋白可以增强GABA能神经传递的增强，在治疗癫痫发作和中风方面具有治疗价值。哺乳动物脑（GAT1，GAT2，GAT3和GAT4）中存在四种GABA转运蛋白同工型，并且在功能，药理学和定位方面表现出显着差异。实际上，GABA转运蛋白已经与癫痫有关，一种同工型（GAT1）是抗癫痫药的靶标。不幸的是，不存在专门针对GAT2或GAT3的药物，但是我们最近确定了GAT4特异性抑制剂。我们将在Xenopus laevis卵母细胞中表达GABA转运蛋白，以解决以下特定目的：（1）在GABA上使用快速浓度跳跃 转运蛋白结合袋，以获得有关跨质膜的离子和底物结合和易位的机械洞察力。这些实验将使用该实验室中开发的新型快速灌注系统，并将在转运蛋白上进行NA +，CI-和GABA跳跃，以获得与转运蛋白配体相互作用的详细低估。 （2）充分检查GAT4中确定的新型CL-通道模式。这些实验将阐明 新型Cl-通道模式的重要性，我们最近在GAT4中确定了该模式。 （3）充分表征了最近鉴定的抑制剂对GAT4的选择性的作用。我们成功地识别了一种新型的GAT4特异性抑制剂。该目标的实验将充分表征该药物的抑制作用，并为确定GAT4对GABA能抑制性神经传递的贡献铺平道路。 （4）制定 GABA转运蛋白GAT3和GAT4的底物药效团。由于大多数研究都集中在GAT1上，因此对GAT3和GAT4的底物结合袋知之甚少。该目标的实验将识别铅化合物，以实现GAT3和GAT4的特定抑制剂的未来结构引导设计。