Trafficking and Regulation of Monoamine Transporters

描述（由申请人提供）：突触前单胺重摄取是影响神经元单胺水平并终止突触传递的主要因素。SL 6载体基因家族内的单胺转运蛋白介导再摄取，并且是成瘾性精神兴奋剂可卡因、安非他明和MDMA（“摇头丸”）以及治疗剂哌醋甲酯（“利他林”）和安非他酮（“Wellbutrin”、“Zyban”）的分子靶标。这些药物有效抑制单胺转运，显著提高细胞外单胺水平并增强下游信号传导。因此，转运细胞表面的可用性是至关重要的正常突触传递和精神药物的疗效。大量证据表明，单胺转运蛋白动态地往返于质膜。此外，安非他明暴露和蛋白激酶C（PKC）激活改变转运蛋白表面的可用性，通过调节转运蛋白运输动力学。尽管有大量文献记载了受调控的转运蛋白运输，但调控转运蛋白调控和运输的细胞和分子机制尚未明确定义。考虑到药理学转运蛋白阻断对突触传递的显著影响，转运蛋白隔离极有可能显著影响下游神经元信号传导。此外，转运蛋白可用性的调节肯定会对精神活性药物的疗效产生重大影响。本项目的主要目标是阐明介导急性转运蛋白调节和运输的细胞和分子机制。具体而言，我们假设：（1）制动机制控制组成和PKC刺激的多巴胺转运蛋白（DAT）内吞作用;（2）内吞后DAT运输不同于经典的运输途径;（3）安非他明和PKC诱导的DAT隔离机制不同。这些假设是基于强有力的初步数据，证明不同的内吞基序所需的组成和PKC调节的DAT内化，新的DAT后内吞运输性能和PKC独立的属性苯丙胺诱导的DAT螯合。我们将使用定点突变来定义组成型、PKC和安非他明调节的DAT内化和再循环信号。国家的最先进的活细胞成像方法将被用来定义基础和调节DAT内吞运输的机制。最后，分子和蛋白质组学的方法将确定DAT相互作用蛋白所需的DAT内吞运输。这些方法将全面介绍受管制运输者贩运的基本机制。我们希望我们的研究结果将对未来的成瘾和情感障碍治疗策略产生重大影响。此外，这些结果将大大提高我们对大脑中单胺可用性和信号传导的因素的理解。公共卫生相关性：在美国，精神疾病和精神兴奋剂成瘾问题日益严重，在过去几年中已接近流行病的程度。尽管对这些疾病的潜在机制进行了大量调查，但我们的知识仍然存在很大的差距。目前的项目将研究精神兴奋剂和抗抑郁药在大脑中的主要靶点，期望即将到来的信息可能会导致治疗这些疾病的新治疗方法。

DESCRIPTION (provided by applicant): Presynaptic monoamine reuptake is a major factor that influences extraneuronal monoamine levels and terminates synaptic transmission. Monoamine transporters within the SL6 carrier gene family mediate reuptake and are the molecular targets for the addictive psychostimulants cocaine, amphetamines, and MDMA ("ecstasy"), as well as for the therapeutic agents: methylphenidate ("Ritalin") and buproprion ("Wellbutrin", "Zyban"). These agents potently inhibit monoamine transport, significantly elevating extracellular monoamine levels and enhancing downstream signaling. Thus, transporter cell surface availability is paramount to both normal synaptic transmission and psychoactive drug efficacy. Abundant evidence demonstrates that monoamine transporters dynamically traffic to and from the plasma membrane. Moreover, both amphetamine exposure and protein kinase C (PKC) activation change transporter surface availability by modulating transporter trafficking kinetics. Although regulated transporter trafficking is well documented, the cellular and molecular mechanisms governing transporter regulation and trafficking are not clearly defined. Given the pronounced effect pharmacological transporter blockade exerts on synaptic transmission, transporter sequestration is highly likely to significantly effect downstream neuronal signaling. Moreover, modulation of transporter availability is certain to have significant impact on the efficacy of psychoactive drugs. The major goals of this project are to elucidate the cellular and molecular mechanisms mediating acute transporter regulation and trafficking. Specifically, we hypothesize that (1) A brake mechanism controls constitutive and PKC-stimulated dopamine transporter (DAT) endocytosis; (2) Post-endocytic DAT trafficking differs from classical trafficking pathways; and (3) Amphetamine- and PKC-induced DAT sequestration are mechanistically distinct. These hypotheses are based on strong preliminary data demonstrating distinct endocytic motifs required for constitutive and PKC-regulated DAT internalization, novel DAT post-endocytic trafficking properties and PKC-independent properties of amphetamine-induced DAT sequestration. We will use site- directed mutagenesis to define constitutive, PKC-, and amphetamine-regulated DAT internalization and recycling signals. State-of-the-art live cellular imaging approaches will be used to define the mechanisms governing basal and regulated DAT endocytic trafficking. Finally, molecular and proteonomic approaches will define DAT-interacting proteins required for DAT endocytic trafficking. These approaches will provide a comprehensive picture of the mechanisms underlying regulated transporter trafficking. We expect our results will significantly impact future addiction and affective disorder treatment strategies. Moreover, the outcomes will greatly improve our understanding of the factors contributing to monoamine availability and signaling in the brain. PUBLIC HEALTH RELEVANCE: Mental illness and psychostimulant addiction are growing problems in the United States, reaching near epidemic proportions over the past several years. Despite numerous investigations into the mechanisms underlying these conditions, there are still large gaps in our knowledge. The current project will investigate the major targets in the brain for both psychostimulants and antidepressants, with the expectation that the forthcoming information may lead to novel therapeutic approaches for treating these disorders.