Regulation of the presynaptic choline transporter

描述(申请人提供)：本研究的目标是利用基于结构和蛋白质组学的方法，阐明维持CHT在突触前胆碱能神经末梢的调节活性和定位的关键分子机制。CHT维持在突触小泡上，在必要的乙酰胆碱前体胆碱需求时期，被招募用于突触前膜的活动。这种独特的蛋白质运输的分子基础部分是基于一个基本的内吞作用基序。然而，在体内观察到的持续的CHT质膜活性不能完全用该基序的快速基础活性来解释。这项建议将利用CHT的功能和内吞分析来发现CHT细胞质序列中的其他调控基序。这些检测的基础是CHT对激酶连接通路的激活的敏感性，特别是PKC和PKA。结构分析将确定介导CHT调节内吞控制的序列基序。这些发现的最终结果将是检查基本的和受调控的内吞基序之间在调节CHT转运和细胞表面活性方面的相互作用，并验证培养的神经元制剂中运输的保守机制。基于蛋白质组学的方法将确定突触前末端CHT的蛋白结合伙伴，这些蛋白结合伙伴在质膜上调节CHT的活性及其囊泡定位。纹状体准备将与体外结合技术相结合，以捕获CHT的相关蛋白。捕获的关联的身份将通过LC/MS分析进行识别，并通过已建立的生化方法进行验证。发生在基础和调节性内吞作用模体上的蛋白质关联将通过体外结合准备的改变来探索。这一对CHT的分析将描述结构运输基序和蛋白质结合伙伴之间在调节CHT活动和突触前终末运输中的相互作用。对改进的胆碱能疗法有很大的需求，这项研究将通过表征这一新定义的对胆碱能神经元功能至关重要的分子靶点，为治疗干预提供新的靶点。公众：众所周知，体内胆碱能信号的中断会导致人类认知障碍，而胆碱能张力的增加会增强衰老和痴呆患者的注意力、学习和记忆能力。胆碱能信号的音调由突触前胆碱(CH)水平决定，受CHT(CH转运体)活性的限制。这项研究将确定调节胆碱能神经元中CHT活性的机制，并将为改善胆碱能治疗的公共卫生倡议做出贡献。

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.