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

基本信息

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
项目类别：
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

项目摘要

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.

安非他明 (AMPH) 是常用于治疗神经精神疾病（例如注意力缺陷障碍）的精神兴奋剂，它们也被滥用，并造成破坏性后果。 AMPH 的滥用潜力与其引起细胞质多巴胺 (DA) 动员的能力有关。，导致细胞外 DA 水平增加，这种增加是由 DA 转运蛋白 (DAT) 功能逆转介导的，从而导致非囊泡 DA。然而，DA 流出的分子事件以及这些事件如何转化为特定的 AMPH 行为尚未得到很好的理解，并且我们已经表明 DAT N 末端（NT）是该提案的重点。磷酸化后支持 AMPH 诱导的 DA 流出，但不调节 DA 摄取的结构域。此外，我们的初步数据表明，该磷酸化事件表明以前使用以下组合来调节 DA 相关行为。通过生物化学、电生理学和原子分子动力学模拟以及行为分析，我们证明 DAT NT 含有与膜质脂质相互作用的结构元件 (Lys)，特别是磷脂酰肌醇 (4,5)-二磷酸 (PIP2)。损害 DAT NT 与 PIP2 的相互作用，无论是在药理学上还是在分子上，都会抑制 DA 外流和 AMPH 过度运动。这是首次证明质膜蛋白与 PIP2 的相互作用对于精神兴奋行为至关重要。这种相互作用对于 AMPH 引起 DAT NT 磷酸化至关重要，也需要 NT 存在且高度动态，因为要么将 DAT NT 锚定在血浆上。膜或删除 NT 会损害 DA 流出，但不会损害 DA 摄取。我们的机制假设是 NT 和 PIP2 之间的相互作用对于 AMPH 引起 NT 磷酸化至关重要。磷酸化后，DAT NT 与 PIP2 解偶联并脱离膜。正如我们的初步数据预测的那样，与细胞内环 4 (IL4) 的特定基序形成新的相互作用，这些新的相互作用由 NT 促进。我们建议通过以下具体目标来检验这一假设：1) 确定 hDAT-质膜相互作用在调节 NT 磷酸化中的作用；2) 确定 hDAT NT 磷酸化如何支持 DA 流出和我们的分子发现随后将使用黑腹果蝇作为动物模型进行体内转化，其中我们在 DA 神经元中表达人类 DAT (hDAT)。缺乏果蝇 DAT 的果蝇（“人源化果蝇”）在该动物模型中，我们开发了从生物化学和生物物理角度研究离体大脑中 hDAT 功能的能力，并确定 hDAT 的分子操作是否会损害 DA 流出，但不会损害摄取。损害与 AMPH 相关的复杂行为，包括奖励/偏好，因此，在具体目标 3) 中，我们将确定 AMPH 诱导行为的 hDAT IL4-PIP2 相互作用的要求。 NT磷酸化发挥的作用。

项目成果

期刊论文数量（14）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

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