Conformational Ensemble of Glutamate Transporters: Structure and IonicModulation

项目摘要 谷氨酸转运蛋白将神经递质从突触间隙泵入神经胶质细胞的细胞质， 神经元对抗浓度梯度达到一百万倍，通过利用能量储存的形式， 离子的跨膜电化学梯度。具体地说，它们将每个分子的摄取 谷氨酸盐与三个钠离子和一个质子的同向转运和一个钾离子的反向转运。他们的 功能障碍与一系列神经系统疾病和广泛的脑损伤有关， 外伤和中风。谷氨酸转运蛋白的完整转运循环涉及谷氨酸转运蛋白的结合。 底物和共输离子的转运蛋白的面向外的构象，异构化的 转运蛋白转化为面向内的构象，底物和离子释放到细胞质中， 反向转运的钾离子的结合和转运蛋白返回到面向外的状态。 这个家族的结构信息来自于对细菌同源物的晶体学研究， GltPh，其将天冬氨酸摄取与三个钠离子的同向转运偶联，但不与其他钠离子的运动偶联。 离子。在我们之前的资助期间，我们主要专注于大规模构象 GltPh的转换，基础的跨膜易位的基板和耦合的离子。我们现在 寻求在结构和机制水平上研究与基质和离子相关的事件 在膜的两侧结合和释放，这是膜的功能特异性的核心。 运输机我们进一步提出采用诱变来重建与质子的GltPh偶联， 在哺乳动物转运蛋白中观察到的钾离子。最后，我们建议在GltPh中实现 从采用钠梯度到使用质子通过模拟氨基酸来驱动转运的特异性 该家族的质子偶联成员的离子结合位点的组成。通过结合这些研究 通过对细菌谷氨酸转运蛋白的结构研究， 了解保守的蛋白质结构和结构机制是如何适应的， 功能多样化。我们的长期目标是实现催化反应的完整机理描述。 这些转运蛋白的循环，并获得合理的控制其功能特性。

Project Summary Glutamate transporters pump the neurotransmitter from the synaptic cleft into the cytoplasm of glial cells and neurons against concentration gradients reaching a million-fold by harnessing the energy stored in the form of the trans-membrane electrochemical gradients of ions. Specifically, they couple uptake of each molecule of glutamate to the symport of three sodium ions and a proton and to the antiport of a potassium ion. Their dysfunction is associated with a range of neurological disorders and the extensive brain damage following traumatic injury and stroke. The complete transport cycle of glutamate transporters involves binding of the substrate and the symported ions to the outward facing conformation of the transporter, isomerization of the transporter into the inward facing conformation, the release of the substrate and ions into the cytoplasm, binding of the counter-transported potassium ion and the return of the transporter into the outward facing state. The structural information on this family comes from the crystallographic studies on a bacterial homologue, GltPh, which couples aspartate uptake to the symport of three sodium ions, but not to the movements of other ions. During our previous funded period, we have primarily focused on the large-scale conformational transitions of GltPh that underlie the trans-membrane translocation of the substrate and coupled ions. We now seek to investigate at the structural and mechanistic level the events associated with the substrate and ions binding and release on the two sides of the membrane that are at the heart of the functional specificity of the transporters. We further propose to employ mutagenesis to reconstitute in GltPh coupling to protons and potassium ions observed in the mammalian transporters. Finally, we propose to effectuate in GltPh a switch of specificity from employing sodium gradients to using protons to drive transport by mimicking the amino acid composition of the ion-binding sites of the proton-coupled members of the family. By combining these studies with the structural studies on the bone fide proton coupled bacterial glutamate transporters, we aim to understand how the conserved protein architecture and the structural mechanisms are adapted to allow functional diversification. Our long-term goal is to achieve a complete mechanistic description of the catalytic cycle of these transporters and to gain rational control over their functional properties.