Dopamine Transporter Cell Surface Dynamics

描述（由申请人提供）：大脑中的细胞外多巴胺（DA）水平直接影响各种生理功能，包括运动、认知和奖励。突触释放后，DA的可用性受到突触前再摄取的限制，由质膜DA转运蛋白（DAT）介导。DAT是SLC6载体基因家族的成员，并且是成瘾性和治疗性精神兴奋剂的主要靶点，如安非他明，可卡因和哌甲酯（利他林）以及抗抑郁药，如安非他酮（Wellbutrin）。这些药物有效地抑制DA摄取，从而增加细胞外DA浓度，增强神经元信号传导并显著调节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质膜呈递不是静态的。相反，DAT通过组成性内吞运输动态地穿梭于质膜和从质膜穿梭。蛋白激酶C（PKC）激活和安非他明（AMPH）暴露调节DAT内化和再循环速率，最终降低DAT表面可用性。最近在ADHD家系中的研究已经确定了DAT突变体，其在基础和AMPH刺激条件下改变了DAT运输动力学，并且改变了对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，研究DAT内吞机制的研究产生了相互矛盾的结果，DAT细胞表面行为没有得到很好的定义。这些研究的主要目标是直接检查DAT表面动态TIRF显微镜在活细胞中，并阐明介导的基础和调节DAT内化的内吞机制。具体而言，我们的目标是（1）野生型和贩运突变DAT的表面动力学特征，（2）测试的假设，即基础，PKC和AMP刺激的DAT内吞是网格蛋白独立和Cdc42依赖，（3）测试的假设，DAT内化是一个动力蛋白独立的过程。这些假设源于强有力的初步数据，表明1）DAT表面定位主要是网格蛋白非依赖性病灶，2）动力蛋白非依赖性DAT运输和3）Cdc42依赖性DAT内吞作用。对于现场TIRF成像研究，我们利用一种新的化学生物学方法，直接耦合荧光团细胞表面DAT。这种创新的技术将有利于DAT表面动力学的第一次直接检查，而不结合庞大的抗体或抑制配体。将在神经元细胞系和小鼠纹状体切片中进行补充生物化学研究，利用小分子网格蛋白、发动蛋白和Cdc42抑制剂急性干扰膜运输，以及标准shRNA和GTdR突变体方法。从这些研究中收集的信息将提供一个更清晰的理解DAT表面动力学和介导DAT内吞作用的机制，无论是野生型和贩运突变体。我们预计，我们的研究结果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，这些结果无疑将增强我们对影响大脑中DA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.