The dopamine transporter's lipid interactions: understanding transporter function

苯丙胺(AMPH)是一种精神刺激剂，通常用于治疗神经精神障碍(如注意力缺陷障碍)。他们也被滥用，造成了毁灭性的后果。AMPHs的滥用潜力与它们引起胞浆多巴胺(DA)动员的能力有关，这导致细胞外DA水平的增加。这种增加是通过DA转运体(DAT)功能的逆转而调节的，该功能导致非囊泡性DA释放，这里定义为DA外流。然而，DA外流背后的分子事件以及这些事件如何转化为特定的amph行为并不是很好的理解，这是本提案的重点。我们已经证明，DAT的N末端(NT)是一个结构域，它在磷酸化后支持AMPH诱导的DA外流，但不调节DA的摄取。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，结合生物化学、电生理学和原子分子动力学模拟以及行为分析，我们已经证明DAT NT含有与质膜脂类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，无论从药理上还是分子上，都能抑制DA外流和AMPH超速运动。这是第一次证明质膜蛋白与PIP2的相互作用对精神刺激行为是必不可少的。这也提出了这样一种可能性，即这种相互作用对于amph引起DAT NT磷酸化是必不可少的。DA外流还要求NT存在并高度动态，因为将DAT NT锚定在质膜上或删除NT都会损害DA外流，但不会损害DA摄取。我们的机制假设是，NT和PIP2之间的相互作用是AMPH导致NT磷酸化的关键。在磷酸化后，DAT NT从PIP2解偶联并从膜上脱离，与我们初步数据预测的细胞内环4(IL4)的特定基序形成新的相互作用。这些新的相互作用是由NT磷酸化促进的，对amph的作用是必不可少的。我们建议通过以下特定目的来验证这一假说：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外流和IL4的参与。我们的分子发现将被用黑腹果蝇作为动物模型进行体内翻译，在该动物模型中，我们在缺乏果蝇DAT的果蝇的DA神经元中表达人DAT(HDAT)。在这个动物模型中，我们发展了在分离的大脑中从生化和生物物理的角度研究HDAT功能的能力，并确定HDAT的分子操作是否会损害与AMPH相关的复杂行为，包括奖赏/偏好。因此，在特定的目标3)，我们将确定HDAT IL4-PIP2相互作用对AMPH诱导行为的要求以及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.