Trafficking and Regulation of Monoamine Transporters

描述（由申请人提供）：突触前再摄取是限制细胞外单胺水平和终止突触信号传导的主要手段。SLC6载体基因家族中的生物胺转运体促进再摄取，是成瘾性和治疗性精神兴奋剂以及抗抑郁药物的主要靶点。这些药物能有效抑制单胺再摄取，从而增加细胞外单胺浓度，增强神经元信号传导，显著调节单胺相关行为。因此，转运体的活性和有效性是正常生物胺神经传递和精神活性药物疗效的关键决定因素。大量的数据支持生物胺转运体的表面呈现不是静态的。更确切地说，转运蛋白受到强大的本构内吞运输的影响。多巴胺转运体（DAT）受到蛋白激酶C （PKC）激活和暴露于精神兴奋剂可卡因和安非他明（AMPH）的急性调节，后者调节了DAT的内化和再循环率，最终降低了DAT表面的可用性。这些研究的主要目的是阐明控制基础和调节生物胺贩运的机制。在这个更新应用中，我们建立在我们之前在细胞和分子方法方面的优势，以及我们之前的发现，即Rin GTPase与DAT结合，并且是pkc介导的DAT内在化所必需的。具体来说，我们的目标是：(1)阐明控制Rin依赖性DAT内吞作用的分子决定因素，并确定在神经元细胞系中amph介导的DAT运输是否需要Rin GTPase；(2)确定PKC和amph介导的DAT运输是否具有区域依赖性，以及PKC和amph介导的DAT原位运输是否需要Rin GTPase；(3)测试Rin依赖性DAT运输是否需要精神兴奋剂奖励。这些假设是基于强有力的初步数据，证明了特定的Rin与DAT相互作用，而不是其他SLC6转运蛋白，以及钙调蛋白在Rin下游信号传导中的潜在作用。我们将使用嵌合蛋白来定义赋予DAT/Rin相互作用特异性的DAT结构域。我们将使用生化和药理学方法结合GTPase突变体和shrna介导的Rin敲低来确定Rin下游的信号通路，这些信号通路对于神经元细胞系和小鼠纹状体中受调节的DAT内化是必要和充分的。我们将进一步测试PKC和amph介导的DAT转运是否以特定区域的方式发生在纹状体中，并使用体内的Rin敲低来测试Rin在原位调节DAT内吞作用中的作用。最后，我们将使用体内shRNA方法敲低小鼠VTA中的Rin GTPase，以测试DAT运输是否需要精神兴奋剂奖励。从这些努力中获得的结果将为控制转运体表面表达的机制和DAT贩运在奖励行为中的作用提供更清晰的理解。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，这些结果无疑将增强我们对影响单胺在大脑中可用性的分子因素的理解。

DESCRIPTION (provided by applicant): Presynaptic reuptake is the primary means to limit extracellular monoamine levels and terminate synaptic signaling. Biogenic amine transporters in SLC6 carrier gene family facilitate reuptake and are the major targets for addictive and therapeutic psychostimulants, as well as for antidepressants. These drugs potently inhibit monoamine reuptake, and thereby increase extracellular monoamine concentrations, enhance neuronal signaling and significantly modulate monoamine-related behaviors. Thus, transporter activity and availability are critical determinants of normal biogenic amine neurotransmission and psychoactive drug efficacy. A wealth of data supports that biogenic amine transporter surface presentation is not static. Rather, transporters are subject to robust constitutive endocytic trafficking. The dopamine transporter (DAT) is acutely modulated by protein kinase C (PKC) activation and exposure to psychostimulants cocaine and amphetamine (AMPH), which modulate DAT internalization and recycling rates, ultimately decreasing DAT surface availability. The major goal of these studies is to elucidate the mechanisms that control both basal and regulated biogenic amine trafficking. In this renewal application we build on our previous strengths in cellular and molecular approaches and our previous findings that Rin GTPase binds to DAT and is required for PKC-mediated DAT internalization. Specifically, we aim to (1) Elucidate the molecular determinants governing Rin-dependent DAT endocytosis and determine whether Rin GTPase is required for AMPH-mediated DAT trafficking in neuronal cell lines, (2) Determine whether PKC- and AMPH-mediated DAT trafficking are region-dependent and whether Rin GTPase is required for PKC- and AMPH-mediated DAT trafficking in situ, and (3) test whether Rin-dependent DAT trafficking is required for psychostimulant reward. These hypotheses are based on strong preliminary data that demonstrate specific Rin interactions with DAT, but not other SLC6 transporters, and a potential role for calmodulin in Rin downstream signaling. We will use chimeric proteins to define the DAT domains that confer specificity of the DAT/Rin interaction. We will use biochemical and pharmacological approaches in combination with GTPase mutants and shRNA-mediated Rin knockdown to determine the signaling pathways downstream of Rin that are necessary and sufficient for regulated DAT internalization in neuronal cell lines and mouse striatum. We will further test whether PKC- and AMPH-mediated DAT trafficking occur in a region-specific manner in the striatum, and use in vivo Rin knockdown to test the role Rin in regulated DAT endocytosis in situ. Finally, we will use in vivo shRNA approaches to knockdown Rin GTPase in mouse VTA to test whether DAT trafficking is required for psychostimulant reward. The results obtained from these endeavors will provide a clearer understanding of the mechanisms controlling transporter surface expression and the role of DAT trafficking in rewarding behaviors. 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 monoamine availability in the brain.