Dopamine Transporter Interactions: Understanding Transporter Function

描述(申请人提供)：多巴胺转运体(DAT)通过重新摄取来调节释放的多巴胺(DA)的失活。精神刺激剂苯丙胺(AMPH)通过作为DAT底物，促进DA转运的逆转(DA外流)并增加细胞外DA水平，这对AMPH的兴奋特性具有重要意义。我们实验室先前已经证明，富含在膜筏中的SNARE蛋白合成素1(STX1)与DAT N末端结合，并且这种结合调节AMPH诱导的DA外流。我们还表明，膜筏相关蛋白Flotillin-1(Flot1)对于DAT在这些膜微区的定位是必要的。Flot1被敲除，取代了膜筏上的DAT，钝化了Amph诱导的DA外流。这项建议旨在揭示DAT翻译后修饰和DAT蛋白质/脂相互作用调节DAT功能的机制，更重要的是，调节AMPH诱导的DA外流。磷脂酰肌醇-4，5-二磷酸(PIP2)是一种重要的磷脂，主要集中在质膜内叶，富含在脂筏结构域中。除了其他功能外，PIP2还作为一种重要的辅因子来调节蛋白质的募集和定位。这包括STX1，它在Ser14被酪蛋白激酶2(CK2)磷酸化。我们已经证明STX1与DAT的N末端结合，AMPH促进了这种相互作用。初步结果表明，PIP2协调STX1磷酸化、DAT/STX1关联以及DA外流。这些结果还表明，AMPH刺激CK2介导的STX1磷酸化，我们推测这是DAT/STX1相互作用和DA外流所必需的分子事件。与这一假设一致，我们的证据表明，抑制CK2减少了STX1的磷酸化和DA的外流。这项研究的长期目标是了解AMPH诱导的DA外流是如何由DAT与STX1和PIP2的相互作用和/或DAT和DAT相关蛋白的翻译后修饰所决定的。这些假说将(首次)在突触释放点进行检验。我们建议通过以下具体目标来检验我们的总体假设：S.A.#1)定义PIP2如何与DAT N末端相互作用并协调DAT/STX1关联。S.A.#2)，以确定DAT相互作用分子在Amph诱导的DA外流中的作用及其磷酸化状态。

DESCRIPTION (provided by applicant): The dopamine transporter (DAT) mediates the inactivation of released dopamine (DA) through its reuptake. The psychostimulant amphetamine (AMPH), by acting as a DAT substrate, promotes reversal of DA transport (DA efflux) and increases extracellular DA levels, an event of importance for the stimulant properties of AMPH. Our laboratory has previously demonstrated that the SNARE protein syntaxin 1 (STX1) that is enriched in membrane rafts, associates with the DAT N-terminus, and that this association regulates AMPH-induced DA efflux. We have also shown that the membrane raft-associated protein Flotillin-1 (Flot1) is necessary for the localization of DAT to these membrane microdomains. Flot1 knockdown, which displaces DAT from membrane rafts, blunts AMPH-induced DA efflux. This proposal aims to uncover the mechanisms by which DAT posttranslational modifications and DAT protein/lipid interactions regulate DAT function, and importantly, AMPH-induced DA efflux. Phosphatidylinositol-4,5-bisphosphate (PIP2) is a key phospholipid that is mainly concentrated in the inner leaflet of the plasma membrane and is enriched in lipid raft domains. In addition to other functions, PIP2 acts as an essential cofactor to mediate protein recruitment and localization. This includes STX1, which is phosphorylated at Ser14 by casein kinase 2 (CK2). We have shown that STX1 binds the DAT N-terminus and that AMPH promotes this interaction. Preliminary results suggest that PIP2 coordinates STX1 phosphorylation, DAT/STX1 associations, as well as DA efflux. These results also suggest that AMPH stimulates CK2-mediated STX1 phosphorylation, a molecular event that we hypothesized to be required for DAT/STX1 interactions as well as DA efflux. Consistent with this hypothesis, our evidence suggest that inhibition of CK2 reduces STX1 phosphorylation and DA efflux. The long-term goals of this research are to understand how AMPH-induced DA efflux is dictated by DAT interactions with STX1 and PIP2, and/or by post-translational modifications of DAT and DAT-associated proteins. These hypotheses will be tested (for the first time) at synaptic release sites. We propose to test our overarching hypothesis through the following specific aims: S.A. #1) To define how PIP2 interacts with the DAT N-terminus and coordinates DAT/STX1 associations. S.A. #2) To determine the role of DAT interacting molecules and their phosphorylation status in AMPH-induced DA efflux.