Dynamics and Energetics of Secondary-Active Glutamate Transport

次级活性谷氨酸转运的动力学和能量学

• 批准号: 1515028
• 负责人: Christof Grewer
• 金额: $ 53.1万
• 依托单位: SUNY at Binghamton
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-15 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1515028&HistoricalAwards=false
• 关键词: Dynamics; Energetics; Secondary; Active; Glutamate

The plasma membrane separates the interior of the cell from the extracellular space and provides a barrier to cell entry/exit for many molecules (for example nutrient or signaling molecules) that are essential for cell survival. Glutamate is one such molecule, which is not only an amino acid needed for the synthesis of proteins, but also the most important neurotransmitter in the mammalian brain, responsible for the majority of excitatory neurotransmission. Glutamate is transported across the plasma membrane through active glutamate transporters, which play essential roles in controlling the levels of glutamate in the mammalian brain and other tissue. This project explores the fundamental principles by which these transporters work. Understanding these principles is important because it will contribute to our knowledge not only of the role of glutamate and nutrient movement between cellular compartments in the mammalian brain, as well as other organs of the body, but also glutamate homeostasis, which is important for cellular function in general. Furthermore, the developed methods for analyzing transport will be applicable to potential future studies of other neurotransmitter and/or nutrient transporters and membrane proteins. On the educational level, the project provides an invaluable opportunity for undergraduate students to become involved in cutting-edge biophysical research, providing training in basic physical, quantitative approaches to be applied to a significant biological problem. The investigator is continuing previous efforts of undergraduate involvement (including members of under-represented groups), which has led to many publications with undergraduate students as co-authors, as well as students successfully moving on to careers in research. The investigator is also very active in local and regional activities that promote the sciences at all levels, including demonstrations at schools, science fair involvement, and assuming leadership roles in the local American Chemical Society (ACS) section and the Science Olympiad. Such activities are necessary to ensure a vibrant local science community and to foster excitement of the upcoming generations of young scientists for science and research. Active glutamate transport is a multi-step process that requires multiple ion/substrate binding steps, as well as conformational changes. Despite recent progress towards understanding the transport process through functional studies, as well as the identification of structural models of a bacterial homologue of mammalian glutamate transporters in several states, basic questions about the mechanism, specificity, and dynamics of interaction of the transporters with cations, in particular K+ and Li+, as well as the energetic contributions to energy barriers that control the transport rate and specific structural changes in the transport cycle remain unresolved. Answers to these questions are obtained by applying a combination of experimental and computational methods, allowing the investigation of the dynamics of glutamate transport in real time, with microsecond time resolution, and to test predictions from all-atom and simplified molecular models. This work has the potential to reveal new aspects of glutamate movement across the cellular membrane, in particular with respect to the cooperation of polar and non-polar forces, which are fine tuned to adjust the energy barriers associated with ion/substrate binding, as well as conformational changes. The novel insight may uncover more general concepts that are employed by membrane transport proteins, in order to provide a permeation pathway for polar molecules across the hydrophobic membrane.

质膜将细胞的内部与细胞外空间分开，并为许多对于细胞存活所必需的许多分子（例如营养或信号分子）提供了细胞进入/出口的障碍。谷氨酸是一种这样的分子，它不仅是蛋白质合成所需的氨基酸，而且是哺乳动物脑中最重要的神经递质，也是导致大多数兴奋性神经传递的原因。谷氨酸通过活性谷氨酸转运蛋白在质膜上运输，该转运蛋白在控制哺乳动物脑和其他组织中谷氨酸水平方面起着重要作用。该项目探讨了这些运输者使用的基本原理。理解这些原理很重要，因为它不仅会促进我们的知识，不仅对哺乳动物大脑中的细胞隔室以及人体其他器官之间的细胞隔室之间的作用以及谷氨酸稳态，这对于一般的细胞功能很重要。此外，开发的用于分析运输的方法将适用于其他神经递质和/或营养转运蛋白和膜蛋白的潜在研究。在教育层面上，该项目为本科生提供了一个宝贵的机会，可以参与尖端的生物物理研究，从而提供了基本的物理，定量方法的培训，以应用于重大的生物学问题。调查人员正在继续在本科参与（包括代表性不足的团体的成员）的先前努力，这导致了许多出版物作为本科生作为共同作者的出版物，以及学生成功地从事研究职业。研究人员在促进各个层面的科学的地方和区域活动中也非常积极，包括学校的示威，科学公平参与，并在当地的美国化学学会（ACS）部分和科学奥林匹克运动会中担任领导角色。这样的活动对于确保充满活力的当地科学界并促进即将到来的年轻科学家进行科学和研究的兴奋是必要的。主动谷氨酸转运是一个多步过程，需要多个离子/底物结合步骤以及构象变化。尽管最近取得了进展，但通过功能研究来理解运输过程，以及在几种状态下哺乳动物谷氨酸转运蛋白的细菌同源物的结构模型的鉴定，有关转运蛋白与阳离子相互作用的机制，特异性和动态的基本问题，尤其是对能源造成能量的能量促进的运输速率和转运率的促进速率和能量的变化速率。这些问题的答案是通过应用实验和计算方法的组合来获得的，从而可以实时研究谷氨酸传输的动力学，并通过微秒时间分辨率进行研究，并测试全原子和简化分子模型的预测。这项工作有可能揭示整个细胞膜的谷氨酸运动的新方面，尤其是在极性和非极性力的合作方面，这些方面经过微调以调节与离子/底物结合相关的能屏障以及构象变化。新颖的见解可能会发现膜转运蛋白采用的更多一般概念，以便为跨疏水膜的极性分子提供渗透途径。