The dopamine transporter's lipid interactions: understanding transporter function

安非他明（AMPH）是通常用于治疗神经精神障碍（例如注意力缺陷障碍）的精神兴奋剂。它们也被滥用，造成毁灭性的后果。AMPH的滥用潜力与其引起细胞质多巴胺（DA）动员的能力有关，这导致细胞外DA水平增加。这种增加是由DA转运蛋白（DAT）功能的逆转介导的，该功能引起非囊泡DA释放，本文定义为DA流出。然而，DA外排的分子事件以及这些事件如何转化为特定的AMPH行为还没有很好的理解，这是本提案的重点。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流出，但不调节DA摄取。此外，我们的初步数据表明，这种磷酸化事件调节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的组合，以及行为测定，我们已经表明，DAT NT包含与质膜脂质，特别是磷脂酰肌醇（4，5）-二磷酸（PIP 2）相互作用的结构元件（赖氨酸）。损害 DAT NT与PIP 2的相互作用，无论是直接的还是分子的，都抑制DA流出和AMPH超运动。这是第一次证明质膜蛋白与PIP 2的相互作用对精神兴奋剂行为至关重要。这也提出了这种相互作用是AMPH引起DAT NT磷酸化所必需的可能性。DA外排也需要NT的存在和高度动态的，因为无论是锚定DAT NT质膜或删除NT损害DA外排，但不DA摄取。我们的机制假设是NT和PIP 2之间的相互作用是AMPH引起NT磷酸化的关键。磷酸化后，DAT NT与PIP 2解偶联并与膜脱离，与细胞内环4（IL 4）的特定基序形成新的相互作用，如我们的初步数据所预测的。这些新的相互作用，促进NT磷酸化，是必不可少的AMPH行动。我们提出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IL 4的参与。然后，我们的分子发现将使用黑腹果蝇作为动物模型在体内转化，其中我们在缺乏果蝇DAT的果蝇（“人源化果蝇”）的DA神经元中表达人DAT（hDAT）。在这种动物模型中，我们开发了在分离的大脑中研究hDAT功能的能力，包括生物化学和生物药理学，并确定hDAT的分子操作是否损害DA流出，但不摄取，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诱导的行为对hDAT IL 4-PIP 2相互作用的需求以及NT磷酸化所起的作用。

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.