Conformational Ensemble of Glutamate Transporters: Structure and Ionic Modulatio

描述（由申请人提供）：兴奋性氨基酸转运蛋白（EAAT）在神经传递后从突触间隙清除谷氨酸，并负责所有细胞类型对酸性和中性氨基酸的摄取。在预先存在的电化学梯度的驱动下，EAAT将底物摄取与钠离子和质子的共运输以及钾的反向运输相结合。以前的晶体学研究从Pyrococcus horikoshii，GltPh的细菌同源物已经揭示了在面向外的状态下与底物结合位点可访问的细胞外溶液和封闭时，结合到一个基板或暴露时，绑定到一个阻滞剂的转运。这些结构已经解释了底物和钠离子如何从细胞外溶液到达它们的结合位点，但没有回答这些溶质如何穿过膜并释放到细胞质中。据信，转运蛋白构象转变为面向内的状态，其中底物结合位点可接近细胞内溶液，是底物解离到细胞质中的先决条件。在本申请中，我们提出使用双半胱氨酸诱变和交联来描绘在转运蛋白转变成面向内的状态时发生的结构重排，并探测该过程的动力学。我们进一步提出，以确定的晶体结构的转运蛋白分子共价约束在面向内的状态，并可能在其他中间运输状态的半胱氨酸交联。最后，我们将采用定点诱变结合生物化学和结构分析，以探测在GltPh离子结合和渗透位点的位置，并以此类推，在EAAT的公共卫生相关性：谷氨酸转运蛋白是膜蛋白负责清除神经递质谷氨酸从突触后的神经传递轮。它们的功能障碍与许多疾病和病理状态相关，包括神经变性、癫痫、精神分裂症、创伤性脑损伤和中风。我们的研究重点是这些分子泵的机制和原子结构，这些分子泵对中枢神经系统的正常发育和功能至关重要。

DESCRIPTION (provided by applicant): The excitatory amino acid transporters (EAATs) clear glutamate from the synaptic cleft following rounds of neurotransmission and are responsible for the uptake of acidic and neutral amino acids by all cell types. Driven by pre-existing electrochemical gradients, EAATs couple substrate uptake to the co-transport of sodium ions and protons and to the counter-transport of potassium. Previous crystallographic studies on a bacterial homologue from Pyrococcus horikoshii, GltPh have revealed the transporter in the outward facing states with the substrate-binding site accessible to the extracellular solution and either occluded when bound to a substrate or exposed when bound to a blocker. These structures have explained how the substrate and sodium ions reach their binding sites from the extracellular solution but left unanswered how these solutes are translocated across the membrane and released into the cytoplasm. It is believed that a conformational transition of the transporter into an inward facing state, in which the substrate-binding site is accessible to the intracellular solution, is a prerequisite for the substrate dissociation into the cytoplasm. In the current application, we propose to use double cysteine mutagenesis and cross-linking to delineate the structural re- arrangements that occur upon transition of the transporter into the inward facing state and to probe the dynamics of the process. We further propose to determine the crystal structures of the transporter molecules covalently constrained in the inward facing state and possibly in other intermediate transport states by cysteine cross-linking. Finally, we will employ the site directed mutagenesis in conjuncture with biochemical and structural analyses to probe the location of the ion binding and permeation sites in GltPh and, by analogy, in EAATs PUBLIC HEALTH RELEVANCE: Glutamate transporters are membrane proteins responsible for the clearance of the neurotransmitter glutamate from the synapses following rounds of neurotransmission. Their malfunction is associated with numerous diseases and pathological states including neurodegeneration, epilepsy, schizophrenia, traumatic brain injury and stroke. We focus our study on the mechanism and atomic structure of these molecular pumps essential for the proper development and function of the central nervous system.