Conformational Ensemble of Glutamate Transporters: Structure and IonicModulation

描述（由申请人提供）：谷氨酸转运体通过利用以离子跨膜电化学梯度形式储存的能量，将神经递质从突触间隙泵入胶质细胞和神经元的细胞质，以对抗浓度梯度达到一百万倍。具体来说，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同向转运以及一个钾离子的反转运结合起来。它们的功能障碍与一系列神经系统疾病以及外伤性损伤和中风后的大面积脑损伤有关。谷氨酸转运体的完整转运周期包括底物和转运离子结合到转运体的外向构象，转运体异构成内向构象，底物和离子释放到细胞质中，与反转运的钾离子结合，转运体返回到外向状态。这个家族的结构信息来自对细菌同源物GltPh的晶体学研究，它将天冬氨酸的摄取与三个钠离子的同体结合，而不是与其他离子的运动结合。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，这是底物和偶联离子跨膜易位的基础。我们现在试图在结构和机制水平上研究与底物和离子在膜两侧的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。我们进一步建议采用诱变来重建在哺乳动物转运体中观察到的GltPh与质子和钾离子的偶联。最后，我们建议在GltPh中通过模拟质子偶联家族成员的离子结合位点的氨基酸组成，实现从使用钠梯度到使用质子驱动转运的特异性转换。通过将这些研究与骨键质子偶联细菌谷氨酸转运体的结构研究相结合，我们旨在了解保守蛋白的结构和结构机制如何适应功能多样化。我们的长期目标是实现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