Modulation of Serotonergic Transporters

描述(申请人提供)：5-羟色胺[5-羟色胺，5-羟色胺]是一种神经递质，其功能障碍与许多精神疾病的发生有关，包括情绪障碍。突触前5-羟色胺转运体(SERT)是5-羟色胺在突触中重新摄取和终止信号的主要机制，是MDMA、可卡因和三环类成瘾药物的高亲和力靶点，SERT抑制剂是治疗抑郁症的最大类药物之一。关于SERT功能的调节方式，人们知之甚少。我们的初步数据已经确定α-突触核蛋白，一种功能未知的突触前囊泡相关蛋白，是SERT功能的调节器，导致正常转运蛋白活性的减弱，通过减少[~3H]5-羟色胺摄取试验来指示。SERT和α-突触核蛋白之间的相互作用通过形成稳定的蛋白质：蛋白质异构体复合体进行，并导致SERT从质膜的细胞表面运输出去。在这项建议中，我们将使用与α-突触核蛋白及其亚型共转染的细胞、SERT基因以及培养的原代神经元，详细研究这种转运蛋白活性调节的机制。特别是，通过一系列研究，包括免疫学、转运蛋白分析、免疫细胞化学和FRET，将彻底分析促进和逆转α-突触核蛋白对SERT功能和运输的影响的结构成分的同一性。我们还将研究α-突触核蛋白在蛋白激酶C介导的SERT磷酸化以及暴露于5-羟色胺时转运体脱敏中的作用。我们以前的研究表明，α-突触核蛋白也可以调节多巴胺转运体的功能，这表明该蛋白的主要功能可能是调节单胺类神经递质的动态平衡。因此，我们对SERT的研究可能为理解与精神疾病和药物滥用有关的其他神经递质转运蛋白增加了启发式和实用的好处。α-突触核蛋白作为SERT的调节蛋白伙伴的发现有望在抑郁症和药物成瘾的治疗中开发新的治疗策略。

DESCRIPTION (provided by applicant): Serotonin [5-hydroxytryptamine, 5-HT] is a neurotransmitter, whose dysfunction is linked to the genesis of numerous psychiatric diseases, including mood disorders. The presynaptic serotonin transporters (SERTs) constitute the primary mechanism for re-uptake of 5-HT and termination of its signal in the synapse, and are high-affinity targets for addictive drugs including MDMA, cocaine and tricyclics, and SERT inhibitors constitute one of the largest groups of drugs used in the therapeutic treatment of depression. Little is known about the mode by which SERT function is regulated. Our preliminary data has identified a-synuclein, a presynaptic vesicle-associated protein of unknown function, as a modulator of SERT function, causing attenuation of normal transporter activity, indexed by diminished [3H]5HT uptake assays. The interaction between SERT and a-synuclein proceeds through the formation of stable protein:protein heteromeric complexes, and results in trafficking of SERT away from the cell surface of the plasma membrane. In this proposal we will study in detail the mechanisms which underline such regulation of transporter activity, using cells co-transfected with alpha-synuclein, and its subtypes, and the SERT cDNA, as well as in cultured primary neurons. In particular, the identity of the structural components which both facilitate and reverse a-synuclein effects on SERT function and trafficking, will be analyzed thoroughly, through a battery of studies to include, immunology, transporter assays, immunocytochemistry and FRET. We will also examine the participation of a-synuclein in both protein kinase C-mediated phosphorylation of SERT, and in transporter desensitization upon exposure to 5-HT. Our previous studies showing that a-synuclein can also modulate the function of the dopamine transporter, suggests that a primary function of this protein may be the regulation of monamine neurotransmitter homeostasis. Thus, our studies on SERT may have added heuristic and practical benefits for the understanding of other neurotransmitter transporters linked to mental illness and drug abuse. The identification of a-synuclein as a regulatory protein partner of SERT holds promise in the development of novel therapeutic strategies in the treatment of depression and drug addiction.