Mitrovic A D, Amara S G, Johnston G A, Vandenberg R J
Department of Pharmacology, University of Sydney, New South Wales 2006, Australia.
J Biol Chem. 1998 Jun 12;273(24):14698-706. doi: 10.1074/jbc.273.24.14698.
Glutamate transporters serve the important function of mediating removal of glutamate released at excitatory synapses and maintaining extracellular concentrations below excitotoxic levels. Excitatory amino acid transporter subtypes EAAT1 and EAAT2 have a high degree of sequence homology and similar predicted topology and yet display a number of functional differences. Several recombinant chimeric transporters were generated to identify domains that contribute to functional differences between EAAT1 and EAAT2. Wild-type transporters and chimeric transporters were expressed in Xenopus laevis oocytes, and electrogenic transport was studied under voltage clamp conditions. The differential sensitivity of EAAT1 and EAAT2 to transport blockers, kainate, threo-3-methylglutamate, and (2S, 4R)-4-methylglutamate as well as L-serine-O-sulfate transport and chloride permeability were employed to characterize chimeric transporters. One particular region, transmembrane domains 9 and 10, plays an important role in defining these functional differences. The intracellular carboxyl-terminal region may also play a minor role in conferring an effect on chloride permeability. This study provides important insight into the identification of functional domains that determine differences among glutamate transporter subtypes.
谷氨酸转运体发挥着重要作用，介导兴奋性突触释放的谷氨酸的清除，并将细胞外浓度维持在兴奋性毒性水平以下。兴奋性氨基酸转运体亚型EAAT1和EAAT2具有高度的序列同源性和相似的预测拓扑结构，但仍表现出许多功能差异。生成了几种重组嵌合转运体，以确定导致EAAT1和EAAT2功能差异的结构域。野生型转运体和嵌合转运体在非洲爪蟾卵母细胞中表达，并在电压钳制条件下研究电转运。利用EAAT1和EAAT2对转运阻滞剂、海人酸、苏-3-甲基谷氨酸和(2S,4R)-4-甲基谷氨酸的不同敏感性以及L-丝氨酸-O-硫酸盐转运和氯通透性来表征嵌合转运体。一个特定区域，即跨膜结构域9和10，在定义这些功能差异中起重要作用。细胞内羧基末端区域在赋予氯通透性影响方面可能也起次要作用。这项研究为确定谷氨酸转运体亚型之间差异的功能结构域的鉴定提供了重要见解。
