Structure, function and pharmacology of neurotransmitter reuptake systems

神经递质再摄取系统的结构、功能和药理学

基本信息

批准号：
8940018
负责人：
Susan Amara
金额：
$ 143.91万
依托单位：
NATIONAL INSTITUTE OF MENTAL HEALTH
依托单位国家：
美国
项目类别：
财政年份：
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/8940018
关键词：
Adenylate Cyclase Alkalinization Amines Amino Acid Sequence Amino Acid Transporter Amino Acids Amphetamines Anions Antidepressive Agents Attention deficit hyperactivity disorder Back Behavioral Binding Binding Sites Biochemical Biogenic Amines Biological Brain Carrier Proteins Cell Line Cell membrane Cell physiology Cells Chemicals Chimeric Proteins Cocaine Cyclic AMP Cyclic AMP-Dependent Protein Kinases Cysteine Dopamine Dopamine Receptor Drug effect disorder EAAT3 Event Excitatory Amino Acids Family G-Protein-Coupled Receptors G-protein Beta gamma Glutamate Transporter Glutamates Glutathione Human Ion Channel Ion Transport Ions Kinetics Label Laboratories Light Link Measures Mediating Membrane Membrane Proteins Methylphenidate Midbrain structure Modeling Modification Molecular Conformation Molecular Genetics Monitor Monomeric GTP-Binding Proteins Movement Mutagenesis Mutation Neuroglia Neurons Neurotransmitters Oxidative Stress Pathway interactions Peptide Fragments Pharmaceutical Preparations Pharmacology Process Property Protein Isoforms Receptor Activation Regulation Research Role Selenocysteine Series Signal Transduction Site-Directed Mutagenesis Slice Sodium Structural Models Structure Surface System Therapeutic Transgenic Mice Variant Work crosslink dopamine transporter dopaminergic neuron extracellular inhibitor/antagonist insight neuronal excitability neurotoxic neurotoxicity neurotransmission neurotransmitter reuptake novel novel strategies prevent protonation psychostimulant receptor reuptake sensor uptake

Adenylate Cyclase Alkalinization Amines Amino Acid Sequence Amino Acid Transporter Amino Acids Amphetamines Anions Antidepressive Agents Attention deficit hyperactivity disorder Back Behavioral Binding Binding Sites Biochemical Biogenic Amines Biological Brain Carrier Proteins Cell Line Cell membrane Cell physiology Cells Chemicals Chimeric Proteins Cocaine Cyclic AMP Cyclic AMP-Dependent Protein Kinases Cysteine Dopamine Dopamine Receptor Drug effect disorder EAAT3 Event Excitatory Amino Acids Family G-Protein-Coupled Receptors G-protein Beta gamma Glutamate Transporter Glutamates Glutathione Human Ion Channel Ion Transport Ions Kinetics Label Laboratories Light Link Measures Mediating Membrane Membrane Proteins Methylphenidate Midbrain structure Modeling Modification Molecular Conformation Molecular Genetics Monitor Monomeric GTP-Binding Proteins Movement Mutagenesis Mutation Neuroglia Neurons Neurotransmitters Oxidative Stress Pathway interactions Peptide Fragments Pharmaceutical Preparations Pharmacology Process Property Protein Isoforms Receptor Activation Regulation Research Role Selenocysteine Series Signal Transduction Site-Directed Mutagenesis Slice Sodium Structural Models Structure Surface System Therapeutic Transgenic Mice Variant Work crosslink dopamine transporter dopaminergic neuron extracellular inhibitor/antagonist insight neuronal excitability neurotoxic neurotoxicity neurotransmission neurotransmitter reuptake novel novel strategies prevent protonation psychostimulant receptor reuptake sensor uptake

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项目摘要

Our laboratory has shown that amphetamines trigger the internalization of the dopamine transporter (DAT) by a series of intracellular events that are distinct from the generally established actions of amphetamines to inhibit dopamine (DA) uptake or to increase DA efflux. We have found that when applied to cell lines, cultured DA neurons or midbrain slices, amphetamine activates the small GTPases, RhoA and Rac-1 and triggers internalization of DAT by a specialized internalization pathway that requires the activation of the small GTPase, RhoA. Intriguingly, amphetamine must be transported into the cell to have these effects and its actions are actually blocked by cocaine, a drug that inhibits DAT and prevents amphetamine entry. We have also found that elevation of cAMP, via DA receptors or by amphetamine-induced adenylate cyclase activation, inactivates RhoA and serves as a break on carrier internalization, thus demonstrating an important interaction between PKA- and RhoA-dependent signaling in mediating the actions of amphetamines. These observations also imply the existence of a novel intracellular target for amphetamines and suggest new cellular pathways to target in order to disrupt amphetamine action. We have also observed that the same amphetamine-activated RhoA-dependent mechanism also downregulates a glutamate transporter, EAAT3, present on DA neurons. We have identified the EAAT3 amino acid sequence responsible for this regulation, generated a cell-permeant fusion protein that interferes with the process and have used it explore the effects of amphetamine on excitatory neurotransmission in brain slices. These findings provide a new context in which to consider the actions of amphetamine on dopaminergic and glutamatergic signaling, and should provide insight into the unique neurotoxic and behavioral properties of this class of psychostimulant drugs. Recent work in the laboratory has established the role of a trace amine receptor (TAAR1) a G-protein coupled receptor found in dopamine neurons, which may serve as a direct intracellular target for amphetamines. Using transgenic mouse lines lacking the TAAR1 receptor we have found that some of the intracellular effects of amphetamine on dopamine neurons can be linked to TAAR receptor activation. We have also identified a number of human TAAR1 variants that display altered function and have been using transfected cell lines to characterize the impact of these mutations on amphetamine sensitivity. G-protein beta-gamma subunits that are released following the activation of G-protein-coupled receptors have been shown to directly bind and inhibit the activity of the dopamine transporter. An additional line of research in the laboratory has used cell permeable peptide fragments and mutagenesis to define the domain on the transporter required for this interaction and developed structural models for how this interaction may facilitate dopamine efflux by the transporter. Glutamate transporters (also known as excitatory amino acid transporters or EAATs) present at the surface of neurons and supporting glial cells regulate the extracellular concentration of glutamate, the major excitatory neurotransmitter in the brain. By transporting glutamate back into the cell, these carrier proteins prevent glutamate from reaching toxic levels and also limit the extent and duration of transmitter signaling during glutamatergic neurotransmission. These carriers have an additional function in that they possess an anion channel activity that can regulate cellular excitability, which enables them to serve as sensors of glutamate levels outside the cell. Our laboratory has used site-directed mutagenesis, sulfhydryl modification, and chemical cross-linking approaches together with biochemical, and electrophysiological analyses of the mammalian carriers to examine the structural domains required for substrate transport and ion permeation. We have also developed new approaches that use chemical modifications of cytoplasmic cysteine residues to capture inward-facing conformations of these transporters. This work has shown that inward facing conformations of the carriers also mediate the anion channel activity associated with glutamate transporters. Overall, these results shed light on how the structure and conformation of these transporter proteins determine their functional dynamics and regulatory properties. The neuronal glutamate transporter isoform, EAAT3 has also been shown to transport the amino acid cysteine and has been proposed to be a primary mechanism used by neurons to obtain cysteine for the synthesis of glutathione, a molecule critical for preventing oxidative stress and neuronal toxicity. This year the laboratory also completed work examining the mechanism of cysteine transport by EAAT3. These studies showed that the transport of cysteine through EAAT3 requires formation of the thiolate form of cysteine in the binding site. These studies assessed the transport kinetics of different substrates and measured transporter-associated currents electrophysiologically. Using a membrane tethered pH sensor to monitor intracellular pH changes associated with transport activity, we observed that transport of acidic substrates such as L-glutamate or L-selenocysteine by EAAT3 decreased intracellular pH, whereas transport of cysteine resulted in cytoplasmic alkalinization. Under conditions that favor release of intracellular substrates through EAAT3 we observed release of labeled intracellular glutamate but did not detect cysteine release. Our results support a model whereby cysteine transport through EAAT3 is facilitated through cysteine de-protonation and that once inside, the thiolate is rapidly re-protonated. Moreover, these findings suggest that cysteine transport is predominantly unidirectional and that reverse transport does not contribute to depletion of intracellular cysteine pools.

我们的实验室表明，苯丙胺通过一系列细胞内事件触发了多巴胺转运蛋白（DAT）的内在化，这些事件与苯丙胺普遍确定的作用不同，以抑制多巴胺（DA）摄取或增加DA Efflux。我们发现，当应用于细胞系，培养的DA神经元或中脑切片时，苯丙胺会激活小的GTPase，RhoA和RAC-1，并通过一种需要激活小GTPase RhoA的专门内在化途径来激活DAT的内在化。有趣的是，必须将苯丙胺转运到细胞中，以产生这些作用，其作用实际上被可卡因阻塞，可卡因是一种抑制DAT并防止苯丙胺进入的药物。我们还发现，cAMP，通过DA受体或通过苯丙胺诱导的腺苷酸环化酶激活的升高，使RhoA失活并充当载体内在化的突破，从而证明了PKA-和RhoA依赖性信号在介导Amphetamines的作用中的重要相互作用。这些观察结果还意味着存在苯丙胺的新型细胞内靶标，并提出了新的细胞途径，以破坏苯丙胺作用。我们还观察到，同一苯丙胺激活的RhoA依赖性机制还下调了DA神经元上存在的谷氨酸转运蛋白EAAT3。我们已经确定了负责该调节的EAAT3氨基酸序列，生成了一种细胞融合蛋白，该蛋白会干扰该过程，并使用它探索了苯丙胺对脑切片中兴奋性神经传递的影响。这些发现提供了一种新的背景，可以在其中考虑苯丙胺对多巴胺能和谷氨酸能信号的作用，并应洞悉此类心理刺激药物的独特神经毒性和行为特性。实验室中的最新工作确定了痕量胺受体（TAAR1）在多巴胺神经元中发现的G蛋白偶联受体的作用，该受体可能是苯丙胺的直接细胞内靶标。使用缺乏TAAR1受体的转基因小鼠系，我们发现苯丙胺对多巴胺神经元的一些细胞内作用可以与TAAR受体激活有关。我们还确定了许多人的TAAR1变体，这些变体显示了变化的功能，并一直使用转染的细胞系来表征这些突变对苯丙胺敏感性的影响。激活G蛋白偶联受体后释放的G蛋白β-GAMMA亚基已被证明可以直接结合并抑制多巴胺转运蛋白的活性。实验室中的另一项研究线使用了可渗透的肽片段和诱变来定义这种相互作用所需的转运蛋白的结构域，并开发了这种相互作用如何促进转运蛋白的多巴胺外排的结构模型。存在于神经元表面的谷氨酸转运蛋白（也称为兴奋性氨基酸转运蛋白或EAATS），并支撑神经胶质细胞调节谷氨酸的细胞外浓度，谷氨酸谷氨酸，谷氨酸，这是大脑中主要的兴奋性神经递质。通过将谷氨酸转移回细胞，这些载体蛋白可以防止谷氨酸达到毒性水平，并限制谷氨酸能神经传递期间发射机信号传导的程度和持续时间。这些载体具有额外的功能，因为它们具有可以调节细胞兴奋性的阴离子通道活性，这使它们能够充当细胞外谷氨酸水平的传感器。我们的实验室使用了位置定向的诱变，磺胺修饰以及化学交联方法以及哺乳动物载体的生化分析，以检查底物运输和离子渗透所需的结构结构域。我们还开发了使用细胞质半胱氨酸残基的化学修饰来捕获这些转运蛋白的向内构象的新方法。这项工作表明，载体的内向构象还介导与谷氨酸转运蛋白相关的阴离子通道活性。总体而言，这些结果阐明了这些转运蛋白的结构和构象如何决定其功能动力学和调节性能。神经元谷氨酸转运蛋白同工型，EAAT3也已证明可以运输氨基酸半胱氨酸，并被认为是神经元用于获得半胱氨酸的主要机制，用于合成谷胱甘肽，这是一种用于预防氧化应激和神经元毒性的分子。今年，实验室还完成了研究EAAT3的半胱氨酸运输机制的工作。这些研究表明，半胱氨酸通过EAAT3的运输需要在结合位点形成半胱氨酸的硫醇酸盐形式。这些研究在电生理学上评估了不同底物的转运动力学和测量的转运蛋白相关电流。使用膜束缚pH传感器来监测与运输活性相关的细胞内pH变化，我们观察到，通过EAAT3通过EAAT3的酸性底物（如L-谷氨酸或L-甲状腺形成率）的转运降低了细胞内pH，而cySTEINE的转运会导致细胞质碱性碱化。在有利于细胞内底物通过EAAT3释放的条件下，我们观察到了标记的细胞内谷氨酸的释放，但未检测到半胱氨酸的释放。我们的结果支持了一个模型，通过半胱氨酸的去骨促进，半胱氨酸通过EAAT3进行了促进，并且一旦内部，硫醇酸酯就会迅速重新构成。此外，这些发现表明半胱氨酸的转运主要是单向的，并且反向转运不会导致细胞内半胱氨酸池的消耗。