Elucidating the structual biomechanics of glutamate transporter proteins

阐明谷氨酸转运蛋白的结构生物力学

基本信息

批准号：
7408910
负责人：
Paul J Focke
金额：
$ 4.1万
依托单位：
OREGON HEALTH & SCIENCE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-12-01 至 2009-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7408910
关键词：
Alzheimer&apos s Disease Amino Acid Transporter Amphipathic Alpha Helix Amyotrophic Lateral Sclerosis Anxiety Area Bathing Binding Binding Sites Biomechanics C-terminal Carrier Proteins Cell membrane Cerebral Ischemia Complementary DNA Complex Condition Cysteine Development Disease Drug usage EAAT3 Electrodes Ensure Epilepsy Excitatory Amino Acids Face Glutamate Receptor Glutamate Transporter Glutamates Helix (Snails)Homeostasis Homologous Gene Human Individual Lead Length Link Measures Membrane Mental Depression Messenger RNA Methods Modeling Molecular Mutagenesis Mutate Neuraxis Neurodegenerative Disorders Neuroglia Neurons Neurotransmitters Numbers Obesity Oocytes Parkinson Disease Pathway interactions Pharmaceutical Preparations Process Property Publications Publishing Reagent Research Scanning Side Signal Transduction Site-Directed Mutagenesis Solutions Spider Venoms Structure Sulfhydryl Reagents Synapses Techniques Testing Transmembrane Domain Water Xenopus oocyte aqueous base clinical application day ear helix extracellular insight methanethiosulfonate neurotoxic novel novel strategies relating to nervous system research study small molecule success uptake voltage clamp

项目摘要

DESCRIPTION (provided by applicant): In the central nervous system (CMS), the extracellular concentration of the excitatory amino acid L- glutamate must be tightly regulated to ensure proper neural signaling. This is achieved mainly by excitatory amino acid transporters (EAATs) located in the plasma membranes of both glial cells and neurons. Many neurodegenerative diseases such as Parkinson's, Alzheimer's, cerebral ischemia, epilepsy, and amyotrophic lateral sclerosis (ALS) are linked to abnormal glutamate homeostasis. Previous efforts to treat these diseases focused on glutamate receptors, rather than glutamate transporter proteins, and have yielded little success. This suggests the need for alternative approaches to treat the large number of neuropathological conditions associated with abnormal glutamate homeostasis. To find novel approaches for treating these various diseases, we have focused our research on elucidating the biophysical properties of glutamate transporters. This is a credible alternative based on the broad spectrum of drugs which target various neurotransmitter transporter proteins, including drugs used to treat depression, anxiety, obesity, and epilepsy. Recent publication of bacterial glutamate transporter crystal structures has provided tantalizing clues as to how these transporters function. We aim to experimentally test predictions made from analyzing the crystal structures in order to define the precise glutamate transporter gating mechanism. This information will provide insight toward the development of novel compounds with clinical applications for the treatment of neurodegenerative disorders. To conduct our experiments, mRNA transcribed from cDNA encoding wild-type (WT) and mutated versions of the human glutamate transporter EAATS will be injected into Xenopus oocytes. 3-6 days later, glutamate-induced membrane currents will be recorded by two- electrode voltage clamp (TEVC). The mutated versions of EAATS will be made using site-directed mutagenesis to introduce cysteine residues which can react with methanethiosulfonate reagents. We aim to investigate the hypothesis that the extracellular and putative intracellular gates undergo conformational changes subsequent to the binding of glutamate and that these conformational changes are important for the translocation step of the glutamate transport cycle. We will also determine the pore-like region of the glutamate transporter that functions to provide a pathway for glutamate uptake. All of our experiments will utilize the technique of MTS accessibility under conditions that favor different states of the glutamate transporter cycle. These experiments will allow us to define the glutamate transporter gating mechanism and will lead to new avenues of research to treat neurodegenerative disease.

描述（由申请人提供）：在中枢神经系统（CMS）中，必须严格调节兴奋性氨基酸L-谷氨酸的细胞外浓度，以确保正确的神经信号传导。这主要是通过位于神经胶质细胞和神经元质膜中的兴奋性氨基酸转运蛋白（EAAT）来实现的。许多神经退行性疾病，例如帕金森氏症，阿尔茨海默氏症，脑缺血，癫痫和肌萎缩性侧面硬化症（ALS）与异常的谷氨酸稳态有关。以前的治疗这些疾病的努力集中在谷氨酸受体上，而不是谷氨酸转运蛋白，几乎没有成功。这表明需要采用替代方法来治疗与异常谷氨酸稳态相关的大量神经病理疾病。为了找到治疗这些各种疾病的新方法，我们将研究重点放在阐明谷氨酸转运蛋白的生物物理特性上。这是基于针对各种神经递质转运蛋白的广泛药物的可靠替代品，包括用于治疗抑郁症，焦虑，肥胖和癫痫的药物。细菌谷氨酸转运蛋白晶体结构的最新发表提供了有关这些转运蛋白如何发挥作用的诱人线索。我们的目的是通过分析晶体结构进行实验测试预测，以定义精确的谷氨酸转运蛋白输控机制。该信息将为开发新型化合物提供临床应用来治疗神经退行性疾病。为了进行我们的实验，将从编码野生型（WT）和突变版本的人谷氨酸转运蛋白EAAT的cDNA转录的mRNA被注入Xenopus卵母细胞。 3-6天后，将通过两电极电压夹（TEVC）记录谷氨酸诱导的膜电流。 EAATS的突变版本将使用定位的诱变制作，以引入半胱氨酸残基，这些残基可以与甲烷硫酸盐试剂反应。我们的目的是研究以下假设：谷氨酸结合后的细胞外和假定的细胞内门经历构象变化，并且这些构象变化对于谷氨酸转运循环的易位步骤很重要。我们还将确定谷氨酸转运蛋白的孔状区域，该区域功能为谷氨酸摄取提供途径。我们所有的实验都将在有利于谷氨酸转运蛋白周期不同状态的条件下利用MTS可及性的技术。这些实验将使我们能够定义谷氨酸转运蛋白门控机制，并导致研究的新途径以治疗神经退行性疾病。