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

本研究计划的总体目标是进一步了解二级活性钠偶联溶质转运体（如主要在胆碱能神经元中表达的高亲和力胆碱转运体CHT）的调节机制。胆碱能神经元存在于从无脊椎动物到人类的生物体中，它们利用神经化学递质乙酰胆碱（ACh）向各种目标器官和细胞传递信息。在哺乳动物中，它们在周围和中枢神经系统中传递关键信息，控制着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多种功能。乙酰胆碱是由胆碱乙酰转移酶合成的，胆碱被CHT吸收到胆碱能神经元中，但对CHT功能的调节和协调的动态事件知之甚少。这项拨款提案将在令人兴奋的新方向上研究CHT，以确定和表征调节CHT的关键事件。我们提出了一个模型，在神经元活动期间，与细胞蛋白相互作用的CHT蛋白回收池在胆碱能末端受到调节，以驱动乙酰胆碱合成；这些相互作用的蛋白质在功能上或物理上偶联，定位CHT在细胞中的位置，促进乙酰胆碱的有效合成。CHT在细胞内运输或与其他蛋白质形成功能复合物的机制，以及调节这些过程的生理信号，都不为人所知。本研究计划的目的是确定和定义与CHT蛋白的转运和亚细胞分布相关的急性调节[目标1]和与其他细胞蛋白的相互作用[目标2]所涉及的细胞分子机制。在亚细胞运输和加工过程中，一些转运蛋白会形成功能性的同型二聚体或低聚物，但这对于CHT来说是未知的。需要确定的关键点是二聚化在细胞中发生的位置，复合物的功能和寿命，刺激其形成的信号，以及在细胞通信中的作用。基于我们之前的研究，我们已经确定了潜在的CHT相互作用蛋白，可以调节其在亚细胞区室和细胞表面之间的运输，在细胞表面表达其功能性溶质运输活性。LC2和NECAP就是两个这样的候选蛋白。在我们的研究方法中，我们将我们在胆碱能神经元生物学方面的经验和专业知识结合起来，使用多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，当这些研究结合在一起时，这些研究中获得的许多信息也将与介导溶质转运到细胞中的一系列质膜蛋白的调节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.