Dopamine Transporter Cell Surface Dynamics

项目总结 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的可用性受到突触前的限制 再摄取，由质膜DA转运体(DAT)介导。DAT是SLC6运营商的成员 基因家族，是安非他明等成瘾性和治疗性精神刺激剂的主要靶点， 可卡因和哌醋甲酯(利他林)以及安非他酮(威布曲林)等抗抑郁药。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 并显著调节DA相关的行为。因此，DAT活动和可用性至关重要 测定正常多巴胺神经传递和精神活性药物疗效。DAT质膜呈现 不是静态的。相反，DAT是通过构成内吞作用动态地往返于质膜 贩卖人口。蛋白激酶C(PKC)激活和苯丙胺(AMPH)暴露对DAT的调节作用 内部化和循环率，最终降低了DAT的表面利用率。ADHD的最新研究进展 家系已经发现了一个改变了DAT转运动力学的DAT突变体，在基础和长寿两种情况下都是如此。 刺激条件，并改变靶向膜筏微域，从而涉及改变的DAT 贩卖人类疾病。然而，研究DAT内吞机制的研究已经取得了成果 相互冲突的结果和DAT细胞表面行为没有很好地定义。这些研究的主要目标是直接 用TIRF显微镜检测活细胞DAT的表面动力学并阐明其内吞机制 它调节基础的和调节的DAT内化。具体地说，我们的目标是(1)表征表面 野生型和运输突变DAT的动态，(2)检验基础、PKC和AMP刺激的假设 DAT内吞作用是不依赖于笼蛋白和依赖于镉的，以及(3)检验DAT的假设 内部化是一个不依赖于动力的过程。这些假设源于强劲的初步数据 论证1)DAT表面主要定位于不依赖于分子筛蛋白的病灶，2)不依赖动力蛋白的DAT 贩运和3)依赖于镉的DAT内吞作用。对于实时TIRF成像研究，我们利用了一种新的 直接将荧光团与细胞表面DAT偶联的化学生物学方法。这项创新的技术将 便于首次直接检查DAT表面动力学，无需与体积较大的抗体或抑制物结合 配基。补充生化研究将在神经细胞系和小鼠纹状体中进行。 切片，利用小分子网状蛋白、动力素和CdC42抑制剂来强烈扰乱膜转运， 以及标准的shRNA和GTPase突变方法。从这些研究中收集的信息将 更清楚地了解DAT表面动力学和介导DAT内吞作用的机制， 无论是野生型还是贩卖变种人。我们预计，我们的发现将极大地影响未来的战略。 旨在治疗情感障碍和药物成瘾。此外，结果无疑将增强我们的 了解影响脑内DA利用度的分子因素。

PROJECT SUMMARY 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 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.