Alpha-synuclein Regulates Dopamine Transporter Functions

α-突触核蛋白调节多巴胺转运蛋白功能

• 批准号: 8463261
• 负责人: Habibeh Khoshbouei
• 金额: $ 36.96万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-15 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8463261
• 关键词: Address; Affect; Binding; Binding Sites; Biochemical; Ca(2+)-Calmodulin Dependent Protein Kinase; Calcium; Cell surface; Cells; Conflict (Psychology); Coupling; Data; Dimensions; Disease; Disease model; Dopamine; Electrophysiology (science); Fibroblasts; Fluorescence Resonance Energy Transfer; Goals; Health; Homeostasis; Human; Imaging Techniques; Immunoprecipitation; In Vitro; Individual; Knowledge; Lead; Measurement; Mediating; Membrane; Membrane Potentials; Microscopy; Midbrain structure; Molecular; Molecular Biology; Mus; Neurodegenerative Disorders; Neuronal Injury; Neurons; Parkinson Disease; Patients; Permeability; Phosphorylation; Presynaptic Terminals; Property; Regulation; Role; Surface; Synapses; Synaptic Transmission; Techniques; Testing; Work; alpha synuclein; base; calmodulin-dependent protein kinase II; dopamine transporter; dopaminergic neuron; drug discovery; human subject; induced pluripotent stem cell; innovation; insight; molecular imaging; novel; novel strategies; patch clamp; protein protein interaction; research study; stem; synuclein; trafficking; transmission process; uptake

DESCRIPTION (provided by applicant): The overall goal of this proposal is to understand the mechanism and functional consequence of regulation of the dopamine transporter by a-synuclein. The work described in this application is focused on the problem of whether a-synuclein over-expression affects the dopamine transporter activity, leading to alterations in dopaminergic transmission. The proposed studies will potentially define the molecular mechanisms of dopamine transporter regulation and thus dopaminergic transmission upstream of neuronal injury when a-synuclein is over-expressed. We hypothesize that a-synuclein interacts with the C-terminus domain of the dopamine transporter to alter the ionic permeability of the transporter, thus increasing dopamine efflux and decreasing substrate uptake without an effect on dopamine transporter surface levels. The project will address this hypothesis with the following specific aims: 1) to determine whether a-synuclein modulates the biophysical properties of the dopamine transporter, and therefore its functions such as dopamine transporter-mediated whole cell currents, forward and reverse transport of the substrate, and whether these functions are mediated by alterations in surface levels of the transporter; 2) to determine whether a-synuclein regulation of dopamine transporter function is through a physical interaction with the dopamine transporter via a shared binding domain with calcium calmodulin kinase II alpha (CaMKIIa) on the C-terminus domain of the dopamine transporter; and 3) to determine the impact of a-synuclein over-expression on dopamine transporter function in human pleuripotent cells differentiated to midbrain dopaminergic neurons derived from fibroblasts obtained from Parkinson's disease patients and normal subjects. We will use simultaneous whole cell patch clamp electrophysiology with amperometric quantification of released dopamine via the dopamine transporter, and measurement of substrate uptake to study a-synuclein regulation of the dopamine transporter in primary cultures of mouse midbrain dopaminergic neurons. Using Fluorescence Resonance Energy Transfer, biochemical, and immunoprecipitation strategies, we will determine the regulatory role of a-synuclein over-expression on association of the dopamine transporter with CaMKIIa, in addition to the phosphorylation state of the transporter under these conditions. Furthermore, these innovative approaches will be deployed to determine the consequences of a-synuclein regulation of dopamine transporter function in human midbrain dopaminergic neurons obtained from normal human subjects and individuals with idiopathic Parkinson's disease and Parkinson's disease with a-synuclein triplication. As both a-synuclein and the dopamine transporter have been implicated in neurodegenerative diseases such as Parkinson's disease, results from our studies will provide important insight into our mechanistic knowledge of these disease states and could be used to develop novel strategies in disease modeling and targeted drug discovery.

描述（由申请人提供）：该提案的总体目标是了解α-突触核蛋白调节多巴胺转运蛋白的机制和功能结果。本申请中描述的工作重点关注α-突触核蛋白过度表达是否影响多巴胺转运蛋白活性，从而导致多巴胺能传递改变的问题。拟议的研究将有可能确定多巴胺转运蛋白调节的分子机制，从而确定当α-突触核蛋白过度表达时神经元损伤上游的多巴胺能传递。我们假设α-突触核蛋白与多巴胺转运蛋白的C末端结构域相互作用，改变转运蛋白的离子渗透性，从而增加多巴胺流出并减少底物摄取，而不影响多巴胺转运蛋白表面水平。该项目将通过以下具体目标来解决这一假设：1）确定α-突触核蛋白是否调节多巴胺转运蛋白的生物物理特性，及其功能，例如多巴胺转运蛋白介导的全细胞电流、底物的正向和反向转运，以及这些功能是否是通过转运体表面水平的改变来介导的； 2) 确定a-突触核蛋白对多巴胺转运蛋白功能的调节是否是通过与多巴胺转运蛋白的物理相互作用，通过与多巴胺转运蛋白C末端结构域上的钙钙调蛋白激酶IIα（CaMKIIa）共享的结合结构域； 3) 确定α-突触核蛋白过度表达对人多能细胞中多巴胺转运蛋白功能的影响，该细胞分化为源自帕金森病患者和正常受试者的成纤维细胞的中脑多巴胺能神经元。我们将使用同步全细胞膜片钳电生理学，通​​过多巴胺转运蛋白释放的多巴胺进行电流定量，并测量底物摄取，以研究小鼠中脑多巴胺能神经元原代培养物中多巴胺转运蛋白的α-突触核蛋白调节。使用荧光共振能量转移、生化和免疫沉淀策略，我们将确定 a-突触核蛋白过表达对多巴胺转运蛋白与 CaMKIIa 关联的调节作用，以及这些条件下转运蛋白的磷酸化状态。此外，这些创新方法将用于确定α-突触核蛋白调节多巴胺转运蛋白功能对从正常人类受试者和特发性帕金森病患者和具有α-突触核蛋白三倍体的帕金森病个体获得的人中脑多巴胺能神经元的影响。 由于α-突触核蛋白和多巴胺转运蛋白都与帕金森病等神经退行性疾病有关，因此我们的研究结果将为我们了解这些疾病状态的机制知识提供重要的见解，并可用于开发疾病建模和靶向药物的新策略发现。