Dopamine Transporter Cell Surface Dynamics

描述（由申请人提供）：大脑中的细胞外多巴胺（DA）水平直接影响多种生理功能，包括运动，认知和奖励。突触释放后，由质膜DA转运蛋白（DAT）介导的突触前再摄取限制了DA的有效性。DAT是SLC6载体基因家族的一员，是成瘾性和治疗性精神兴奋剂的主要靶点，如安非他明、可卡因和哌醋甲酯（利他林），以及抗抑郁药，如安非他酮（安非他酮）。这些药物能有效抑制DA摄取，从而增加细胞外DA浓度，增强神经元信号传导，显著调节DA相关行为。因此，DAT的活性和可用性决定了正常的DA神经传递和精神活性药物的疗效。DAT质膜的表现不是静态的。相反，DAT通过本构内吞运输动态穿梭于质膜之间。蛋白激酶C （PKC）的激活和安非他明（AMPH）的暴露调节了DAT的内在化和再循环率，最终降低了DAT表面的可用性。最近对ADHD谱系的研究发现，在基础和amph刺激条件下，一个DAT突变体改变了DAT运输动力学，并改变了膜筏微结构域的靶向，从而暗示了人类疾病中DAT运输的改变。然而，调查DAT内吞机制的研究产生了相互矛盾的结果，并且DAT细胞表面行为没有很好地定义。这些研究的主要目的是通过TIRF显微镜直接检查活细胞中的DAT表面动力学，并阐明介导基础和受调节的DAT内化的内吞机制。具体来说，我们的目标是(1)表征野生型和运输突变型DAT的表面动力学特征，(2)验证基础、PKC和amp刺激的DAT内吞作用是网格蛋白独立和cdc42依赖的假设，以及(3)验证DAT内化是一个动力独立过程的假设。这些假设源于强有力的初步数据，这些数据表明：1)数据表面定位主要是与网格蛋白无关的病灶，2)与动力无关的数据运输，以及3)依赖cdc42的数据内吞作用。对于实时TIRF成像研究，我们利用了一种新的化学生物学方法，直接将荧光团与细胞表面DAT偶联。这种创新技术将有助于首次直接检查DAT表面动力学，而不结合大体积抗体或抑制性配体。补充生化研究将在神经细胞系和小鼠纹状体切片中进行，利用小分子网格蛋白、动力蛋白和Cdc42抑制剂来剧烈扰乱膜运输，以及标准shRNA和GTPase突变方法。从这些研究中收集的信息将提供对DAT表面动力学和介导DAT内吞作用的机制的更清晰的理解，无论是对野生型还是贩运突变体。我们预计我们的发现将极大地影响未来治疗情感障碍和药物成瘾的策略。此外，该结果无疑将增强我们对影响DA在大脑中可用性的分子因素的理解。

DESCRIPTION (provided by applicant): Extracellular dopamine (DA) levels in the brain directly impact a variety of physiological functions, including movement, cognition and reward. Following synaptic release, DA availability is limited by presynaptic reuptake, mediated by the plasma membrane DA transporter (DAT). DAT is a member of the SLC6 carrier gene family and is the primary target for addictive and therapeutic psychostimulants, such as amphetamine, cocaine and methylphenidate (Ritalin) as well as for antidepressants, such as bupropion (Wellbutrin). These drugs potently inhibit DA uptake, and thereby increase extracellular DA concentrations, enhance neuronal signaling and significantly modulate DA-related behaviors. Thus, DAT activity and availability critically determine normal DA neurotransmission and psychoactive drug efficacy. DAT plasma membrane presentation is not static. Rather, DAT is dynamically shuttled to and from the plasma membrane by constitutive endocytic trafficking. Protein kinase C (PKC) activation and amphetamine (AMPH) exposure modulate DAT internalization and recycling rates, ultimately decreasing DAT surface availability. Recent studies in an ADHD pedigree have identified a DAT mutant that has altered DAT trafficking kinetics, both under basal and AMPH-stimulated conditions, and altered targeting to membrane raft microdomains, thereby implicating altered DAT trafficking in human disease. However, studies investigating DAT endocytic mechanisms have yielded conflicting results and DAT cell surface behavior is not well defined. The major goal of these studies is to directly examine DAT surface dynamics by TIRF microscopy in living cells and to elucidate the endocytic mechanisms that mediate basal and regulated DAT internalization. Specifically, we aim to (1) characterize the surface dynamics of wildtype and trafficking mutant DATs, (2) test the hypothesis that basal, PKC- and AMP-stimulated DAT endocytosis are clathrin-independent and Cdc42-dependent, and (3) test the hypothesis that DAT internalization is a dynamin-independent process. These hypotheses stem from strong preliminary data that demonstrate 1) DAT surface localization primarily to clathrin-independent foci, 2) dynamin-independent DAT trafficking and 3) Cdc42-dependent DAT endocytosis. For live TIRF imaging studies, we capitalize on a novel chemical biology approach that directly couples fluorophore to cell surface DAT. This innovative technique will facilitate the first direct examination of DAT surface dynamics without binding to bulky antibodies or inhibitory ligands. Complementary biochemical studies will be performed both in neuronal cell lines and mouse striatal slices, utilizing small molecule clathrin, dynamin and Cdc42 inhibitors to acutely perturb membrane trafficking, as well as standard shRNA and GTPase mutant approaches. The information gleaned from these studies will provide a clearer understanding of DAT surface dynamics and the mechanisms mediating DAT endocytosis, both for wildtype and trafficking mutants. We anticipate that our findings will greatly impact future strategies aimed at treating affective disorders and drug addiction. Moreover, the results will undoubtedly enhance our understanding of the molecular factors influencing DA availability in the brain.