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

• 批准号: 9361012
• 负责人: Susan Amara
• 金额: $ 235.32万
• 依托单位: NATIONAL INSTITUTE OF MENTAL HEALTH
• 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/9361012
• 关键词: Adenylate Cyclase; Amines; Amino Acid Transporter; Amino Acids; Amphetamines; Anions; Antidepressive Agents; Arginine; Attention deficit hyperactivity disorder; Back; Binding; Biochemical; Biogenic Amines; Biological; Brain; Carrier Proteins; Cell Line; Cell membrane; Cell physiology; Cell surface; Cells; Charge; Chemicals; Chimeric Proteins; Cocaine; Coupled; Cultured Cells; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Data; Dopamine; Dopamine Receptor; Drug effect disorder; EAAT3; Electrophysiology (science); Elements; Event; Excitatory Amino Acids; Family; G-Protein-Coupled Receptors; G-protein Beta gamma; GTP-Binding Protein alpha Subunits; GTP-Binding Proteins; Glutamate Transporter; Glutamates; Homology Modeling; Indium; Ion Channel; Ion Transport; Ions; Laboratories; Lead; Link; Mediating; Membrane Proteins; Methamphetamine; Methylphenidate; Midbrain structure; Modification; Molecular Conformation; Molecular Genetics; Monomeric GTP-Binding Proteins; Movement; Mutagenesis; Neuroglia; Neurons; Neurotransmitters; Pathway interactions; Peptide Fragments; Pharmaceutical Preparations; Pharmacology; Property; Receptor Activation; Regulation; Research; Role; Series; Signal Pathway; Signal Transduction; Site-Directed Mutagenesis; Slice; Sodium; Structural Models; Structure; Surface; System; Therapeutic; Transgenic Mice; Transmembrane Domain; Work; base; cell type; crosslink; dopamine transporter; dopaminergic neuron; extracellular; glutamatergic signaling; inhibitor/antagonist; methamphetamine effect; neuronal excitability; neurotransmission; neurotransmitter reuptake; novel; prevent; protein aminoacid sequence; receptor; reuptake; rho; sensor; tool; uptake

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 the dopamine transporter (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 limits carrier internalization, consistent with roles for PKA- and Rho-dependent signaling in mediating the actions of amphetamines in dopamine neurons. Our observations have also suggested the existence of a novel intracellular target for amphetamines and suggest new cellular pathways to target in order to disrupt amphetamine action. In recent studies we have established that a G-protein coupled trace amine receptor (TAAR1) serves as a direct intracellular target for amphetamines in dopamine neurons. Using transgenic mouse lines lacking the TAAR1 receptor we have shown that the intracellular effects of amphetamine, including both the elevation in cAMP and the increased RhoA activity, depend absolutely upon TAAR1 activation. We have shown that the when activated by amphetamine within the cell, TAAR1 couple through a G-protein alpha subunit, known as G13, and we have continued our efforts to identify the subcellular compartment where TAAR1 signaling takes place. In other studies within the group we have shown that G-protein beta-gamma subunits released when G-protein-coupled receptors are activated bind directly to the DAT and enhance dopamine efflux. Using cell permeable peptide fragments and mutagenesis of the DAT we have been able to define the transporter domains required for this interaction and to develop structural models for how this interaction may facilitate dopamine efflux by the transporter. Recent work has demonstrated that release of G-beta-gamma subunits triggered by endogenous receptor activation is sufficient to enhance DA efflux in cultured cells and neurons. We have also observed that the same amphetamine-activated RhoA-dependent mechanism downregulates a glutamate transporter, EAAT3, present on the surface of dopamine neurons. We have identified the EAAT3 peptide sequence responsible for this regulation, generated a cell-permeant fusion protein that blocks internalization and have used it to explore the effects of amphetamine on excitatory neurotransmission in brain slices. These studies have provided new tools to distinguish the effects of amphetamine on dopaminergic and glutamatergic signaling. We have also compared the effects of various amphetamine compounds on the activation of cellular signaling pathways. Comparison of the effects of methamphetamine on glutamate transport activity to those of amphetamine indicate that while both treatments lead to a loss of cell-surface EAAT3, the effects of methamphetamine are much broader and do not depend on the expression of the DAT. These findings provide an explanation for the broader, more devastating effects of methamphetamine: unlike amphetamine, methamphetamine has the capacity to alter EAAT3 surface expression and regulate excitatory neurotransmission, not only in dopamine neurons, but also in many other neuronal cell types within the brain. 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. Recent work has been directed at understanding the mechanism and structural basis of anion channel activation. We initially identified a positively-charged arginine residue in transmembrane domain 7 as an essential element in the anion channel gating mechanism. Substitution of this residue with a negatively-charged amino acid, eliminates sodium- and substrate-dependent anion channel gating, and drives the channel into a substrate-independent constitutively open state. Using a computationally-derived homology model we identified a larger network of charged residues that are required to stabilize the closed state of the channel and, when examined experimentally, appear to be intrinsic elements of the channel gating mechanism. Overall, our data suggest that anion channel gating occurs through a transition from intermediate conformations that are closely linked to transport.

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，苯丙胺会通过需要专门的内在化途径来激活多巴胺转运蛋白转运蛋白（DAT）的小GTPase，并触发了需要的特殊内在化途径，该途径需要小gtpase的活化。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受体或苯丙胺诱导的腺苷酸环化酶激活的升高，使RhoA失活和限制载体内在化，这与PKA-依赖PKA和Rho依赖性信号的作用一致，并在介导多巴胺神经元中苯丙胺的作用中。 我们的观察结果还表明，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。 在最近的研究中，我们确定G蛋白偶联的痕量胺受体（TAAR1）是多巴胺神经元中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，我们表明，苯丙胺的细胞内作用，包括CAMP的升高和RhoA活性增加，完全取决于TAAR1激活。我们已经表明，当细胞内的苯丙胺激活时，TAAR1夫妇通过G蛋白α亚基（称为G13），我们继续努力识别发生TAAR1信号传导的亚细胞隔室。 在小组内的其他研究中，我们已经表明，当G蛋白偶联受体被激活直接与DAT结合并增强多巴胺外排时，G蛋白β-gamma亚基释放出来。使用可渗透的肽片段和DAT的诱变，我们已经能够定义这种相互作用所需的转运蛋白结构域，并开发结构模型，以使这种相互作用如何促进转运蛋白的多巴胺外排。 最近的工作表明，由内源性受体激活触发的G-Beta-Gamma亚基的释放足以增强培养细胞和神经元的DA外排。 我们还观察到，相同的苯丙胺激活的RhoA依赖性机制下调了谷氨酸转运蛋白EAAT3，这是多巴胺神经元表面上的。我们已经确定了负责该调节的EAAT3肽序列，生成了一种细胞 - 佩里式融合蛋白，该融合蛋白可以阻止内在化，并使用它来探索苯丙胺对脑切片中兴奋性神经传递的影响。这些研究提供了区分苯丙胺对多巴胺能信号传导的影响的新工具。我们还比较了各种苯丙胺化合物对细胞信号通路激活的影响。甲基苯丙胺对谷氨酸转运活性与苯丙胺的作用的比较表明，两种处理导致了细胞表面EAAT3的丧失，但甲基苯丙胺的作用更广泛，并且不取决于DAT的表达。这些发现为甲基苯丙胺的更广泛，更具毁灭性的作用提供了解释：与苯丙胺不同，甲基苯丙胺具有改变EAAT3表达表达的能力并调节兴奋性神经传递，不仅在多巴胺神经元中，而且在大脑内许多其他神经元细胞中也是如此。 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。最近的工作旨在了解阴离子通道激活的机制和结构基础。我们最初将跨膜结构域7中带阳性的精氨酸残基确定为阴离子通道门控机制的重要元素。用负电荷的氨基酸取代该残基，消除钠和底物依赖性阴离子通道门控，并将通道驱动到独立的构成开放状态。使用计算衍生的同源模型，我们确定了一个较大的带电残基网络，该网络是稳定通道的封闭状态所需的，并且在经过实验检查时，似乎是通道门控机制的内在元素。总体而言，我们的数据表明，阴离子通道门控通过从与运输密切相关的中间构型的过渡中发生。