Dopamine Transporter Interactions: Understanding Transporter Function

描述（由申请人提供）：多巴胺转运蛋白（DAT）通过其再摄取介导释放的多巴胺（DA）的失活。精神兴奋剂安非他明（AMPH）作为DAT底物，促进DA转运逆转（DA外排）并增加细胞外DA水平，这是AMPH兴奋剂特性的重要事件。我们的实验室先前已经证明，在膜筏中富集的SNARE蛋白syntaxin 1 （STX1）与DAT n端相关，并且这种关联调节amph诱导的DA外排。我们还表明，膜筏相关蛋白flotilin -1 （Flot1）对于DAT定位到这些膜微域是必要的。Flot1敲低，从膜筏中置换了DAT，减弱了amph诱导的DA外排。本研究旨在揭示DAT翻译后修饰和DAT蛋白/脂质相互作用调节DAT功能的机制，重要的是，amph诱导的DA外排。磷脂酰肌醇-4,5-二磷酸（PIP2）是一种关键的磷脂，主要集中在质膜的内小叶中，并富集于脂筏结构域。除了其他功能外，PIP2还作为介导蛋白质募集和定位的重要辅助因子。这包括STX1，它在Ser14位点被酪蛋白激酶2 （CK2）磷酸化。我们已经证明STX1结合DAT n端，AMPH促进这种相互作用。初步结果表明，PIP2协调STX1磷酸化、DAT/STX1关联以及DA外排。这些结果还表明，AMPH刺激ck2介导的STX1磷酸化，我们假设这是DAT/STX1相互作用以及DA外排所必需的分子事件。与这一假设一致，我们的证据表明，抑制CK2可减少STX1磷酸化和DA外排。本研究的长期目标是了解amph诱导的DA外排是如何由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。这些假设将（首次）在突触释放位点得到检验。我们建议通过以下具体目标来验证我们的总体假设：S.A. #1)定义PIP2如何与DAT n端相互作用并协调DAT/STX1关联。sa #2)确定DAT相互作用分子及其磷酸化状态在amph诱导的DA外排中的作用。

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.