MOLECULAR STUDIES OF CNS GLUTAMATE TRANSPORTERS

描述：(改编自申请人摘要)中环 大多数神经系统细胞外和突触的浓度 经典的神经递质受到特定的、高水平的 介导快速重摄取的亲和力转运体 突触前终末及周围的神经胶质细胞。传送器为 兴奋性氨基酸神经递质的位置是有一个主要的 对突触信号和神经毒性作用的影响 由谷氨酸和天冬氨酸介导，但在许多情况下，它们往往被忽视 研究这些神经递质在中枢神经系统中的作用。这个 这项建议的目的是更好地定义生理功能 通过1)建立兴奋性氨基酸递质转运体 决定其容量的克隆转运蛋白的动力学特性 摄取和释放谷氨酸，2)鉴定结构和 运输所需的拓扑特征，3)确定其 人脑中的区域和细胞定位以及4)研究 信号转导机制起到调节再摄取过程的作用。 最初的实验将针对详细的表征 三个克隆的兴奋性基因的动力学、离子依赖性和生电性 氨基酸转运蛋白已经在卵母细胞和 转基因细胞。跨膜离子梯度的变化， 重新吸收的驱动力可以对方向产生巨大的影响 因此，在各种情况下，例如 在缺血期间发生，这些变化会导致净谷氨酸 通过传送器反向释放。智能网的发展 检测谷氨酸外流和离子的电生理方法 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应该有助于深入了解 运营商如何正常运作，以及它们可能如何为 神经元兴奋性毒性的机制。这些研究的其他目标 将解决运营商的区域和蜂窝本地化问题 以进一步评估它们对人类中枢神经系统的潜在贡献 神经退行性疾病。尽管该项目最初的重点是 在三个人类载体上，这些载体已经在申请人的 实验室，它将扩大到解决额外的作用 载体亚型和编码不同谷氨酸转运体的cDNA 他们被确认了身份。 认识其功能、定位和规范的重要性 不同的氨基酸转运蛋白亚型的差异被许多 临床和实验研究已涉及异常或 在退行性疾病中递质再积累不足，例如 肌萎缩侧索硬化症、亨廷顿病、缺血诱导的神经毒性，以及 阿尔茨海默氏症。

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.