MOLECULAR STUDIES OF CNS GLUTAMATE TRANSPORTERS

描述：（改编自申请人的摘要） 神经系统细胞外和突触浓度的大多数 经典的神经递质是由特定的，高的， 亲和转运蛋白，介导快速重摄取到 突触前末梢和周围的胶质细胞。 运输机为 兴奋性氨基酸神经递质被定位为具有主要的 对突触信号传导和神经毒性作用的影响 由谷氨酸和天冬氨酸介导，但它们往往被忽视，在许多 研究这些神经递质在CNS中的作用。 的 该提案的目的是更好地定义生理功能 兴奋性氨基酸递质转运蛋白的1）建立 克隆的转运蛋白的动力学特性决定了它们的能力 吸收和释放谷氨酸，2）确定结构和 运输所需的拓扑特征，3）确定其 区域和细胞定位在人脑和4）研究如何 信号转导机制起调节再摄取过程的作用。 最初的实验将针对的详细表征， 研究了三种克隆的兴奋性神经元的动力学、离子依赖性、产电性、 已经在卵母细胞中表达的氨基酸转运蛋白， 转染细胞 跨膜离子梯度的改变， 再摄取的驱动力可以对方向产生巨大的影响， 因此，在各种条件下，例如 这些变化会导致谷氨酸的净含量增加， 通过转运体逆转释放。 制定一项 用于检测谷氨酸流出和离子型谷氨酸的电生理测定 运输的要求，以及哺乳动物细胞表达系统 研究放射性标记底物的通量， 运营商如何正常运作，以及它们如何有助于 神经元兴奋性毒性的机制。 这些研究的其他目的 将解决运营商的区域和蜂窝定位问题 在人类中枢神经系统中，以进一步评估其对 神经退行性疾病 虽然该项目最初的重点是 在申请人的三个克隆人类载体上， 实验室，它将扩大，以解决额外的作用， 载体亚型和编码不同谷氨酸转运蛋白的cDNA， 它们被识别。 理解功能、定位和调控的重要性 不同氨基酸转运子亚型的差异被许多 临床和实验研究表明， 在退行性疾病中， ALS、亨廷顿病、缺血诱导的神经毒性和 老年痴呆症

DESCRIPTION: (adapted from Applicant's Abstract) Within the central nervous system extracellular and synaptic concentrations of most classical neurotransmitters are tightly regulated by specific, high affinity transporters that mediate the rapid reuptake into the presynaptic terminal and surrounding glial cells. Transporters for the excitatory amino acid neurotransmitters are positioned to have a major influence on both synaptic signalling and on the neurotoxic actions mediated by glutamate and aspartate, yet they are often ignored in many studies examining the role of these neurotransmitters in the CNS. The purpose of this proposal is to better define the physiological functions of excitatory amino acid transmitter transporters by 1) establishing the kinetic properties of cloned transporters that determine their capacity to take up and release glutamate, 2) identifying the structural and topological features required for transport, 3) determining their regional and cellular localization in human brain and 4) examining how signal transduction mechanisms act to modulate the reuptake process. Initial experiments will be directed at a detailed characterization of the kinetics, ion dependence, electrogenicity of three cloned excitatory amino acid transporters that have been expressed in oocytes and transfected cells. Alterations in transmembrane ion gradients, the driving forces for reuptake can have dramatic effects on the direction of transport, and thus, under a variety of conditions such as those which occur during ischemia, these changes can lead to net glutamate release through transporter reversal. The development of an electrophysiological assay for examining glutamate efflux and the ionic requirements of transport, as well as mammalian cell expression system to study the flux of radiolabeled substrates should provide insight into how the carriers function normally and how they may contribute to mechanisms of neuronal excitotoxicity. Additional goals of these studies will be to address the regional and cellular localization of carriers in the human CNS to further evaluate their potential contribution to neurodegenerative disease. Although the project is focused initially on three human carriers that have been cloned in the applicant's laboratory, it will be expanded to address the role of additional carrier subtypes and cDNAs encoding different glutamate transporters as they are identified. The importance of understanding the function, localization and regulation of different amino acid transporter subtypes is underscored by the many clinical and experimental studies which have implicated abnormal or inadequate transmitter reaccumulation in degenerative disorders such as ALS, Huntington's disease, ischemia-induced neurotoxicity, and Alzheimer's dementia.