Conformational Ensemble of Glutamate Transporters: Structure and IonicModulation

描述(申请人提供)：谷氨酸转运体通过利用离子跨膜电化学梯度形式储存的能量，将神经递质从突触裂隙泵入神经胶质细胞和神经元的细胞质中，浓度梯度达到一百万倍。具体地说，它们将每个谷氨酸分子的摄取与三个钠离子和一个质子的结合以及一个钾离子的反结合结合在一起。它们的功能障碍与一系列神经疾病以及创伤性损伤和中风后的广泛脑损伤有关。谷氨酸转运体的完整转运循环包括底物和共生离子与转运体的外向构象结合，转运体异构化为内向构象，底物和离子释放到细胞质中，反向转运的钾离子结合，转运体返回外向状态。关于这个家族的结构信息来自对细菌同系物GltPh的结晶学研究，它将天冬氨酸的摄取与三个钠离子的共生结合起来，而不是与其他离子的运动结合起来。在我们之前的资助期间，我们主要关注GltPh的大规模构象转变，它是底物和耦合离子跨膜转移的基础。我们现在试图在结构和机制水平上研究与底物和膜两侧离子结合和释放相关的事件，这些事件是转运蛋白功能特异性的核心。我们进一步建议使用突变来重建在哺乳动物转运蛋白中观察到的质子和钾离子偶联的GltPh。最后，我们建议通过模仿该家族质子偶联成员离子结合位点的氨基酸组成，在GltPh中实现从使用钠梯度到使用质子驱动运输的特异性转换。通过将这些研究与骨质子偶联细菌谷氨酸转运体的结构研究相结合，我们旨在了解保守的蛋白质结构和结构机制是如何适应功能多样化的。我们的长期目标是实现对这些转运体催化循环的完整机制描述，并获得对其功能特性的合理控制

DESCRIPTION (provided by applicant): 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