Molecular Physiology of y-Aminobutyric Acid Transporters

γ-氨基丁酸转运蛋白的分子生理学

基本信息

批准号：
8209038
负责人：
SEPEHR ESKANDARI
金额：
$ 31.63万
依托单位：
CALIFORNIA STATE POLY U POMONA
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-01-01 至 2013-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8209038
关键词：
Address Affinity Aminobutyric Acids Antiepileptic Agents Applications Grants Binding Binding Sites Biological Assay Brain Cell membrane Cells Charge Chemosensitization Computer Simulation Dependence Development Docking Educational workshop Electron Microscopy Electrons Ensure Epilepsy Event Exhibits Experimental Designs Experimental Models Family Family member Freeze Fracturing Funding Future GABA Transporter 1 GAT3 transporter Glean Goals Grant Heart Homology Modeling Individual Kinetics Knowledge Lead Leucine Measurement Measures Mediating Membrane Methods Microscopic Modeling Molecular Molecular Models Mutagenesis National Institute of General Medical Sciences National Institute of Neurological Disorders and Stroke Nerve Nervous system structure Neuraxis Neurotransmitters Oocytes Pharmaceutical Preparations Pharmacology Phenotype Physiological Physiology Play Principal Investigator Property Protein Engineering Protein Isoforms Publications Research Research Personnel Resolution Role Screening procedure Shapes Signal Transduction Site Site-Directed Mutagenesis Sodium Specificity Stroke Structure Synapses System Techniques Temperature Therapeutic Time Tissues Work Writing Xenopus laevis analog design extracellular gamma-Aminobutyric Acid inhibitor/antagonist insight meetings molecular modeling molecular site mutant neurotransmission novel pharmacophore poly(L-glutamic acid(60)-L-alanine(30)-L-tyrosine(10))prevent protein transport receptor research study symporter tiagabine voltage

项目摘要

DESCRIPTION (provided by applicant): 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 transport GABA into cells after its release from nerve terminals and, thus, they regulate the concentration profile and lifetime of GABA in synaptic and extra-synaptic regions of the nervous system. 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. Our understanding of the function and pharmacology of GAT2, GAT3, and GAT4 lags far behind that of GAT1. The GABA transporters belong to the neurotransmitter:Na+ symporter family, and a recent high-resolution crystal structure of a bacterial family member (leucine transporter, LeuTAa) has dramatically enhanced our understanding of structure and function of these transporters. The crystal structure has also paved the way for detailed structure-function characterization of the GABA transporters. We will express the GABA transporters in Xenopus laevis oocytes in order to address the following Specific Aims: (1) To combine electrophysiological and electron microscopic measurements in order to determine the physiological turnover rate of GAT2, GAT3, and GAT4. (2) To use site-directed mutagenesis in order to identify functional sites that distinguish the kinetic phenotypes of GABA transporter isoforms GAT3 and GAT4. (3) To use molecular modeling techniques in conjunction with experimental screening assays in order to gain mechanistic insight regarding substrate coordination in the binding site of the GABA transporters GAT3 and GAT4. Gamma-Aminobutyric acid (GABA) is the most abundant inhibitory neurotransmitter in the brain. The brain extracellular concentration of GABA is regulated by four GABA transporter isoforms, only one of which is the target of the antiepileptic drug tiagabine. The proposed research examines the mechanism of function of GABA transporters and will pave the way for the development of novel isoform-specific antiepileptic drugs.

描述（由申请人提供）：我们的长期目标是对γ-氨基丁酸（GABA）转运蛋白实现Na+/Cl-/gaba cotransport的分子机制进行全面了解。 GABA转运蛋白从神经末端释放后将GABA转运到细胞中，因此，它们在神经系统的突触和突触外区域中调节了GABA的浓度和寿命。 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。我们对GAT2，GAT3和GAT4的功能和药理学的理解远远落后于GAT1。 GABA转运蛋白属于神经递质：Na+ Somporter家族，而细菌家族成员（Leucine Transporter，Leutaa）最近的高分辨率晶体结构显着增强了我们对这些转运蛋白结构和功能的理解。晶体结构还为GABA转运蛋白的详细结构 - 功能表征铺平了道路。我们将在爪蟾卵母细胞中表达GABA转运蛋白，以解决以下特定目的：（1）结合电生理和电子显微镜测量，以确定GAT2，GAT3和GAT4的生理转移率。（2）使用位置定向的诱变以识别区分GABA转运蛋白同工型GAT3和GAT4的动力学表型的功能位点。（3）将分子建模技术与实验筛选分析结合使用，以获取有关GABA转运蛋白GAT3和GAT4结合位点底物协调的机械洞察力。 γ-氨基丁酸（GABA）是大脑中最丰富的抑制性神经递质。 GABA的大脑细胞外浓度受四个GABA转运蛋白同工型调节，其中只有一个是抗癫痫药的靶标。拟议的研究研究了GABA转运蛋白功能的机理，并将为开发新型同工型特异性抗癫痫药铺平道路。