Regulation of function of the solute carrier sodium-coupled choline transporter CHT

这项研究的总体目标是更深入地了解次级活性钠偶联溶质转运蛋白的调节机制，例如主要在胆碱能神经元中表达的高亲和力胆碱转运蛋白CHT。胆碱能神经元存在于从无脊椎动物到人类的有机体中，它们使用神经化学递质乙酰胆碱(ACh)与各种靶器官和细胞交流信息。在哺乳动物中，它们在外周和中枢神经系统中传递关键信息，控制从神经肌肉收缩和激素分泌到睡眠、注意力和高级认知功能的各种功能。胆碱乙酰转移酶由胆碱乙酰转移酶合成，胆碱被CHT吸收进入胆碱能神经元，但对CHT功能的调节和协调的动态事件知之甚少。这项拨款提案将对CHT的研究引向令人振奋的新方向，以确定和描述调控CHT的关键事件。我们提出了一个模型，其中与细胞蛋白相互作用的CHT蛋白的循环池在神经元活动期间被调节在胆碱能终末以驱动ACh的合成；这些相互作用的蛋白在功能上或物理上偶联到CHT在细胞中的位置，并促进高效的ACh合成。CHT在细胞内运输或与其他蛋白质形成功能复合体的机制，以及调节这些过程的生理信号，都是未知的。这项研究计划的目的是确定和确定与CHT蛋白的运输和亚细胞分布[目标1]以及与其他细胞蛋白的相互作用[目标2]相关的急性调节CHT蛋白的细胞-分子机制。与其亚细胞运输和加工有关，一些转运体形成功能性同源二聚体或寡聚体，但对于CHT来说这是未知的。需要确定的关键点包括细胞内发生二聚化的位置、复合体的功能和寿命、刺激它们形成的信号以及在细胞通讯中的作用。基于我们以前的研究，我们已经确定了潜在的CHT相互作用蛋白，这些蛋白可能调节CHT在亚细胞室和细胞表面之间的运输，在那里它表达了其功能溶质运输活性。Lc2和NECAP就是两个这样的候选蛋白。在我们进行这些研究的过程中，我们结合了我们在胆碱能神经元生物学方面的经验和专业知识，使用各种细胞和分子生物学方法以及先进的活细胞成像技术来解决这些长期存在的问题。重要的是，当综合起来，这些研究中获得的许多信息也将与调节一系列介导溶质进入细胞的质膜蛋白具有更广泛的相关性。

The overall objective of this research program is to develop greater understanding of mechanisms involved in regulation of secondary-active sodium-coupled solute transporters such as the high-affinity choline transporter CHT that is expressed predominantly in cholinergic neurons. Cholinergic neurons are found in organisms from invertebrates to humans where they use the neurochemical transmitter acetylcholine (ACh) to communicate information to a variety of target organs and cells. In mammals, they transmit critical information in both peripheral and central nervous systems, controlling diverse functions from neuromuscular contraction and hormone secretion to sleep, attention and higher-order cognitive functions. ACh is synthesized by choline acetyltransferase from choline that is taken up into cholinergic neurons by CHT, but little is known about the dynamic events that regulate and coordinate CHT function. This grant proposal takes studies on CHT in exciting new directions to identify and characterize key events regulating CHT. We propose a model where a recycling pool of CHT proteins interacting with cellular proteins are regulated in cholinergic terminals during neuron activity to drive ACh synthesis; these interacting proteins are functionally or physically coupled to position CHT in the cell and facilitate efficient ACh synthesis. Neither the mechanisms by which CHT is trafficked within the cell or forms functional complexes with other proteins, nor the physiological signals that regulate these processes, are known. The aims of this research program are to identify and define cellular-molecular mechanisms involved in acute regulation of CHT proteins related to their trafficking and subcellular distribution [Aim #1] and interaction with other cellular proteins [Aim #2]. In relation to their subcellular trafficking and processing, some transporters form functional homo-dimers or oligomers, but this is unknown for CHT. Key points to be identified are where dimerization occurs in the cell, the function and longevity of complexes, signals that stimulate their formation, and the role in cellular communication. Based on our previous studies, we have identified potential CHT interacting proteins that may regulate its trafficking between subcellular compartments and the cell surface where it expresses its functional solute transport activity. LC2 and NECAP are two such candidate proteins. In our approach to these investigations, we being together our experience in cholinergic neuron biology and expertise using a diverse range of cell and molecular biology methods and advanced imaging technology in live cells to solve these long-standing questions. Importantly, when taken together, much of the information gained in these studies will also have more general relevance to the regulation of a range of plasma membrane proteins that mediate solute transport into cells.