Regulation of the presynaptic choline transporter

描述（由申请人提供）：本研究的目的是使用基于结构和蛋白质组学的方法阐明维持突触前胆碱能神经末梢CHT调节活性和定位的关键分子机制。CHT维持在突触囊泡上，并在需要必需的乙酰胆碱前体胆碱的时期在突触前膜上被募集用于活动。这种独特的蛋白质运输的分子基础部分基于基础内吞作用基序。然而，观察到持续的CHT质膜活性在体内不能完全解释的快速基础活动，这个主题。该建议将利用CHT的功能和内吞测定来揭示CHT胞质序列中的其他调控基序。这些试验的基础是CHT对激酶相关途径（特别是PKC和PKA）活化的敏感性。结构分析将鉴定介导CHT的调节性内吞控制的序列基序。这些发现的高潮将是检查的基础和调节内吞基序之间的相互作用，在调节CHT贩运和细胞表面活性，并验证一个保守的机制，在培养的神经元制剂贩运。基于蛋白质组学的方法将确定在突触前末端的CHT的蛋白质结合伙伴，其在质膜和囊泡定位的CHT活性的调节中具有作用。将纹状体制备物与体外结合技术相结合以捕获CHT的相关蛋白。将通过LC/MS分析识别捕获的关联的身份，并通过已建立的生物化学方法进行验证。蛋白质协会发生在基础和监管的内吞基序将探讨在体外结合制剂的改变。CHT的这种分析将描述结构运输基序和蛋白质结合伙伴之间的相互作用，在调节CHT活性和突触前末端的运输。有很大的需求，改善胆碱能疗法，这项研究将提供新的目标，通过表征这个新定义的分子靶点的胆碱能神经元功能的治疗干预。公众：已经确定，体内胆碱能信号传导的破坏诱导人的认知缺陷，并且胆碱能张力的增强增强衰老和痴呆中的注意力、学习和记忆。胆碱能信号的音调由突触前胆碱（Ch）水平决定，其受CHT（Ch转运体）活性限制。这项研究将确定胆碱能神经元中CHT活性的调节机制，并将有助于改善胆碱能治疗的公共卫生倡议。

DESCRIPTION (provided by applicant): The goal of this study is to elucidate the molecular mechanisms critical to maintain the regulated activity and localization of CHT at presynaptic cholinergic nerve terminals using structural and proteomics based approaches. CHT is maintained on synaptic vesicles and is recruited for activity at the presynaptic membrane in periods of demand for the essential acetylcholine precursor, choline. The molecular basis for this unique protein trafficking is partly based on a basal endocytosis motif. However, observed sustained CHT plasma membrane activity in vivo can not be entirely explained by the rapid basal activity of this motif. This proposal will utilize functional and endocytosis assays of CHT to uncover additional regulatory motifs in CHT cytoplasmic sequence. The basis for these assays is the sensitivity of CHT to the activation of kinase linked pathways, specifically PKC and PKA. Structural analysis will identify sequence motifs that mediate regulatory endocytic control of CHT. The culmination of these finding will be the examination of interactions between the basal and regulated endocytic motifs in modulating CHT trafficking and cell surface activity, and verification of a conserved mechanism of trafficking in cultured neuronal preparations. The proteomics based approach will determine protein binding partners of CHT in the presynaptic terminal that have a role in the regulation of CHT activity at the plasma membrane and its vesicular localization. Striatal preparations will be combined with in-vitro binding techniques to capture associated proteins of CHT. The identity of captured associations will be discerned by LC/MS analysis and verified by established biochemical methodologies. Protein associations that occur at the basal and regulatory endocytosis motifs will be explored by alterations in the in-vitro binding preparations. This analysis of CHT will describe the interaction between structural trafficking motifs and protein binding partners in the regulation of CHT activity and trafficking in the presynaptic terminal. There is great demand for improved cholinergic therapies and this research will provide new targets for therapeutic interventions by characterizing this newly defined molecular target critical for cholinergic neuronal function. Public: It is well established that disruption of cholinergic signaling in vivo induces cognitive deficits in man and augmentation of cholinergic tone enhances attention, learning and memory in aging and dementias. The tone of cholinergic signaling is determined by pre-synaptic choline (Ch) levels limited by CHT (Ch transporter) activity. This research will define mechanisms that regulate CHT activity in cholinergic neurons and will contribute to the public health initiative to improve cholinergic therapies.