Dopamine Transporter Cell Surface Dynamics

描述(申请人提供)：大脑中的细胞外多巴胺(DA)水平直接影响各种生理功能，包括运动、认知和奖励。在突触释放后，DA的可用性受到由质膜DA转运体(DAT)介导的突触前再摄取的限制。DAT是SLC6携带者基因家族的成员，是成瘾和治疗性精神刺激剂(如苯丙胺、可卡因和哌甲酯(利他林))以及抗抑郁药(如安非他酮(Wellbutrin))的主要靶点。这些药物有效地抑制DA摄取，从而增加细胞外DA浓度，增强神经元信号转导，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质膜呈现不是静态的。相反，DAT通过结构性的内吞运输动态地往返于质膜。蛋白激酶C(PKC)的激活和苯丙胺(AMPH)的暴露调节了DAT的内化和循环速率，最终降低了DAT的表面利用率。最近在一个ADHD家系中的研究已经发现了一个DAT突变体，它改变了基础条件和AMH刺激条件下的DAT转运动力学，并改变了对膜筏微域的靶向，从而暗示DAT转运在人类疾病中发生了改变。然而，研究DAT内吞机制的研究产生了相互矛盾的结果，DAT细胞的表面行为还没有很好的定义。这些研究的主要目的是通过TIRF显微镜直接观察活细胞中DAT的表面动力学，并阐明介导基础和调节DAT内化的内吞机制。具体地说，我们的目标是(1)表征野生型和运输突变的DAT的表面动力学，(2)检验基础的、PKC和AMP刺激的DAT内吞是笼蛋白非依赖和CDC42依赖的假说，以及(3)检验DAT内化是动力非依赖性过程的假说。这些假说源于强大的初步数据，这些数据表明：1)DAT表面主要定位于网状蛋白非依赖性的病灶，2)动力蛋白非依赖性的DAT运输和3)依赖于Cdc42的DAT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，将荧光团直接连接到细胞表面DAT。这项创新的技术将有助于首次直接检查DAT表面动力学，而无需与笨重的抗体或抑制性配体结合。补充生化研究将在神经细胞系和小鼠纹状体切片上进行，利用小分子网状蛋白、动力素和Cdc42抑制剂来强烈干扰膜转运，以及标准的shRNA和GTP酶突变方法。从这些研究中收集的信息将为更清楚地了解DAT表面动力学和介导DAT内吞作用的机制，无论是野生型突变还是运输突变体。我们预计，我们的发现将极大地影响未来旨在治疗情感障碍和药物成瘾的策略。此外，这些结果无疑将加深我们对影响大脑中DA利用度的分子因素的理解。

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.