Conformational Ensemble of Glutamate Transporters: Structure and IonicModulation

描述（由申请人提供）：谷氨酸转运蛋白通过利用以离子的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神经胶质细胞和神经元的细胞质中，浓度梯度达到百万倍。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同向转运以及一个钾离子的反向转运偶联。他们的功能障碍与一系列神经系统疾病和创伤性损伤和中风后的广泛脑损伤有关。谷氨酸转运蛋白的完整转运循环涉及底物和共转运离子与转运蛋白的面向外的构象结合，转运蛋白异构化为面向内的构象，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。关于这个家族的结构信息来自于对细菌同系物GltPh的晶体学研究，GltPh将天冬氨酸摄取与三个钠离子的同向转运偶联，但不与其他离子的运动偶联。在我们以前的资助期间，我们主要集中在大规模的构象转变的GltPh的基础跨膜易位的基板和耦合离子。我们现在寻求调查在结构和机制水平的事件与基板和离子的结合和释放的两侧的膜，在心脏的功能特异性的转运。我们进一步建议采用诱变重组GltPh耦合到质子和钾离子中观察到的哺乳动物转运。最后，我们建议在GltPh中实现从采用钠梯度到使用质子来驱动运输的特异性的切换，通过模仿该家族的质子偶联成员的离子结合位点的氨基酸组成。通过结合这些研究与结构研究的真正的质子耦合细菌谷氨酸转运蛋白，我们的目标是了解保守的蛋白质结构和结构机制是如何适应，使功能多样化。我们的长期目标是实现这些转运蛋白的催化循环的完整的机理描述，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