The dopamine transporter's lipid interactions: understanding transporter function

多巴胺转运蛋白的脂质相互作用：了解转运蛋白的功能

• 批准号: 10545756
• 负责人: AURELIO GALLI
• 金额: $ 42.27万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10545756
• 关键词: Action Potentials; American; Amphetamines; Animal Model; Attention Deficit Disorder; Attention deficit hyperactivity disorder; Award; Behavior; Behavioral; Behavioral Assay; Biochemical; Biochemistry; Biological Assay; Biological Models; Biophysics; Brain; Cell membrane; Cells; Cessation of life; Charge; Complex; Computer Models; Cytoplasm; Data; Distal; Dopamine; Drosophila genus; Drosophila melanogaster; Electrophysiology (science); Electrostatics; Elements; Event; Family member; Human; Impairment; Lipids; Locomotion; Measures; Mediating; Medical; Membrane; Membrane Lipids; Membrane Proteins; Modification; Molecular; Molecular Conformation; Motor Activity; Movement; Mutation; N-terminal; Neurons; Outcome; Pharmaceutical Preparations; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Phosphorylation; Play; Probability; Process; Property; Reporting; Rewards; Role; Stimulant; System; Testing; Translating; United States National Institutes of Health; United States Substance Abuse and Mental Health Services Administration; abuse liability; behavioral phenotyping; behavioral study; biological preparation; dopamine transporter; dopaminergic neuron; extracellular; fly; in vivo; molecular dynamics; neuropsychiatric disorder; pharmacologic; preference; psychostimulant; uptake

Amphetamines (AMPHs) are psychostimulants commonly used for the treatment of neuropsychiatric disorders (e.g. attention deficit disorders). They are also abused, with devastating outcomes. The abuse potential of AMPHs has been associated with their ability to cause mobilization of cytoplasmic dopamine (DA), which leads to an increase in extracellular DA levels. This increase is mediated by the reversal of the DA transporter (DAT) function that causes non-vesicular DA release, herein defined as DA efflux. However, the molecular events underlying DA efflux and how these events translate to specific AMPH behaviors is not well understood and is the focus of this proposal. We have shown that the DAT N-terminus (NT) is a structural domain that upon phosphorylation supports AMPH-induced DA efflux, but does not regulate DA uptake. Also, our preliminary data indicate that this phosphorylation event regulates DA-associated behaviors. Previously, using a combination of biochemistry, electrophysiology, and atomistic molecular dynamics simulations, as well as behavioral assays, we have shown that the DAT NT contains structural elements (Lys) that interact with plasma membrane lipids, specifically, phosphatidylinositol (4,5)-bisphosphate (PIP2). Impairing the interaction of the DAT NT with PIP2, either pharmacologically or molecularly, inhibits both DA efflux and AMPH hyperlocomotion. This was the first demonstration that the interaction of a plasma membrane protein with PIP2 is essential for psychostimulant behaviors. It also raised the possibility, that this interaction is essential for AMPH to cause DAT NT phosphorylation. DA efflux also requires the NT to be present and highly dynamic, since either anchoring the DAT NT to the plasma membrane or deleting the NT impairs DA efflux, but not DA uptake. Our mechanistic hypothesis is that the interaction between the NT and PIP2 is pivotal for AMPH to cause NT phosphorylation. Upon phosphorylation, the DAT NT uncouples from PIP2 and disengages from the membrane, forming new interactions with a specific motif of intracellular loop 4 (IL4) as predicted by our preliminary data. These new interactions, facilitated by NT phosphorylation, are essential for AMPH actions. We propose to test this hypothesis through the following specific aims: 1) To determine the role of hDAT-plasma membrane interactions in regulating NT phosphorylation; 2) To determine how hDAT NT phosphorylation supports DA efflux and the involvement of IL4. Our molecular discoveries will be then translated in vivo using Drosophila melanogaster as an animal model in which we express the human DAT (hDAT) in DA neurons of flies lacking the Drosophila DAT (“humanized flies”). In this animal model, we developed the ability to study hDAT function in isolated brains, both biochemically and biophysically, and to determine whether molecular manipulations of hDAT impairing DA efflux, but not uptake, impair complex behaviors associated with AMPH, including reward/preference. Therefore, in specific aim 3) we will determine the requirement of hDAT IL4-PIP2 interactions for AMPH-induced behaviors and the role played by NT phosphorylation.

苯丙胺（AMPHS）是通常用于治疗神经精神疾病（例如注意力缺陷障碍）的精神刺激因素。他们也受到虐待，结果造成了毁灭性的结果。 AMPH的滥用潜力与它们引起动员细胞质多巴胺（DA）的能力有关，这导致细胞外DA水平的升高。这种增加是由导致非挥发性DA释放的DA转运蛋白（DAT）函数的反转介导的，此处被定义为DA外排。但是，DA外排的基础事件以及这些事件如何转化为特定的AMPH行为尚不清楚，这是该建议的重点。我们已经表明，DAT N末端（NT）是一个结构域，磷酸化后支持AMPH诱导的DA外排，但不调节DA的吸收。同样，我们的初步数据表明该磷酸化事件调节与DA相关的行为。以前，使用生物化学，电生理学和原子分子动力学模拟以及行为测定的组合，我们已经表明，DAT NT包含与质膜脂质相互作用的结构元素（LYS），具体来说，具体来说受损 DAT NT与PIP2的相互作用是在药理或分子上，抑制了DA外排和AMPH的超塑料。这是第一次证明质膜蛋白与PIP2的相互作用对于精神刺激行为至关重要。它还提出了这种相互作用对AMPH引起DAT NT磷酸化至关重要的可能性。 DA外排还需要NT存在并且高度动态，因为这两个污水都会存在。将DAT NT锚定在质膜上或删除NT损害DA外排，但不会摄取DA。我们的机械假设是，NT和PIP2之间的相互作用对于AMPH引起NT磷酸化是关键的。磷酸化后，DAT NT脱离了PIP2并脱离膜，形成了新的相互作用，该相互作用与我们的初步数据所预测的细胞内环4（IL4）的特定基序相互作用。这些由NT磷酸化制备的新相互作用对于AMPH作用至关重要。我们建议通过以下特定目的检验这一假设：1）确定HDAT - 铂膜相互作用在确定NT磷酸化中的作用； 2）确定HDAT NT磷酸化如何支持DA外排和IL4的参与。然后，我们的分子发现将在体内使用果蝇Melanogaster在体内翻译为一种动物模型，在该模型中，我们在缺乏果蝇DAT（“人性化的果蝇”）的苍蝇中表达人DAT（HDAT）。在这种动物模型中，我们发展了在生物化学和生物物理上研究HDAT功能的能力，并确定HDAT的分子操作是否会损害DA外排的分子操作，但不损害与AMPH相关的复杂行为，包括奖励/偏好。因此，在特定目标中3）我们将确定HDAT IL4-PIP2相互作用对AMPH诱导的行为的要求以及NT磷酸化的作用。

项目成果

Glucagon-like peptide 1 receptor activation regulates cocaine actions and dopamine homeostasis in the lateral septum by decreasing arachidonic acid levels.

• DOI: 10.1038/tp.2016.86
• 发表时间: 2016-05-17
• 期刊: Translational psychiatry
• 影响因子: 6.8
• 作者: Reddy IA;Pino JA;Weikop P;Osses N;Sørensen G;Bering T;Valle C;Bluett RJ;Erreger K;Wortwein G;Reyes JG;Graham D;Stanwood GD;Hackett TA;Patel S;Fink-Jensen A;Torres GE;Galli A
• 通讯作者: Galli A

Gβγ subunit activation promotes dopamine efflux through the dopamine transporter.

• DOI: 10.1038/mp.2017.176
• 发表时间: 2017-12
• 期刊: Molecular psychiatry
• 影响因子: 11
• 作者: Garcia-Olivares J;Baust T;Harris S;Hamilton P;Galli A;Amara SG;Torres GE
• 通讯作者: Torres GE

The dopamine transporter gene SLC6A3: multidisease risks.

• DOI: 10.1038/s41380-021-01341-5
• 发表时间: 2022-03
• 期刊: Molecular psychiatry
• 影响因子: 11
• 作者: Reith MEA;Kortagere S;Wiers CE;Sun H;Kurian MA;Galli A;Volkow ND;Lin Z
• 通讯作者: Lin Z

PIP2 regulates psychostimulant behaviors through its interaction with a membrane protein.

PIP2 通过与膜蛋白相互作用来调节精神兴奋剂行为。

• DOI: 10.1038/nchembio.1545
• 发表时间: 2014-07
• 期刊: NATURE CHEMICAL BIOLOGY
• 影响因子: 14.8
• 作者: Hamilton, Peter J.;Belovich, Andrea N.;Khelashvili, George;Saunders, Christine;Erreger, Kevin;Javitch, Jonathan A.;Sitte, Harald H.;Weinstein, Harel;Matthies, Heinrich J. G.;Galli, Aurelio
• 通讯作者: Galli, Aurelio

Computational modeling of the N-terminus of the human dopamine transporter and its interaction with PIP2 -containing membranes.

• DOI: 10.1002/prot.24792
• 发表时间: 2015-05
• 期刊: PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS
• 影响因子: 2.9
• 作者: Khelashvili, George;Doktorova, Milka;Sahai, Michelle A.;Johner, Niklaus;Shi, Lei;Weinstein, Harel
• 通讯作者: Weinstein, Harel