Conradt M, Stoffel W
Institute of Biochemistry, Medical Faculty, University of Cologne, Koeln, Germany.
J Biol Chem. 1995 Oct 20;270(42):25207-12. doi: 10.1074/jbc.270.42.25207.
The reuptake of excitatory amino acids, such as glutamate, terminates excitatory signals and prevents the persistence of excitotoxic levels of glutamate in the synaptic cleft. The L-glutamate/L-aspartate transporter (GLAST-1) is the first member of the recently discovered glutamate transporter family, which includes GLT-1 and EAAC1. The neutral amino acid carrier ASCT1 is structurally closely related to this new family of membrane proteins. Transmembrane transport of neutral amino acids is expected to differ in its binding site from that of the acidic excitatory amino acids glutamate and aspartate. Three positively charged amino acid residues, Arg-122, Arg-280, Arg-479, and one polar Tyr-405 are conserved in all glutamate transporters. They are replaced by apolar amino acid residues in the ASCT1 sequence. We exchanged these residues in the GLAST-1-specific cDNA by site-directed mutagenesis. cRNAs of these mutants were expressed in the Xenopus oocyte system. The functional characterization of the mutants R122I and R280V and the double mutant R122I, R280V revealed that the mutations have no influence on the intrinsic properties and kinetics of glutamate transport but alter the Km-values for L-aspartate and the competitive inhibitor D,L-threo-3-hydroxy aspartate. Substitutions of Tyr-405 by Phe (Y405F) and Arg-479 (R479T) by Thr completely inactivate the glutamate transporter. Immunoprecipitations of [35S]methionine-labeled transporter molecules indicate similar expression levels of wild-type and mutant transporters. Immunostaining of oocyte sections clearly proves the correct targeting to and integration of the mutant GLAST-1 proteins in the plasma membrane. Our results suggest the pivotal function of the hydroxy group of the highly conserved Tyr-405 and the positively charged Arg-479 in the binding of the negatively charged acidic neurotransmitter glutamate.
兴奋性氨基酸如谷氨酸的再摄取可终止兴奋性信号，并防止突触间隙中谷氨酸的兴奋性毒性水平持续存在。L-谷氨酸/L-天冬氨酸转运体（GLAST-1）是最近发现的谷氨酸转运体家族的首个成员，该家族还包括GLT-1和EAAC1。中性氨基酸载体ASCT1在结构上与这个新的膜蛋白家族密切相关。中性氨基酸的跨膜转运预计在其结合位点上与酸性兴奋性氨基酸谷氨酸和天冬氨酸不同。在所有谷氨酸转运体中，三个带正电荷的氨基酸残基，即精氨酸-122、精氨酸-280、精氨酸-479以及一个极性酪氨酸-405是保守的。在ASCT1序列中，它们被非极性氨基酸残基取代。我们通过定点诱变在GLAST-1特异性cDNA中替换了这些残基。这些突变体的cRNA在非洲爪蟾卵母细胞系统中表达。突变体R122I和R280V以及双突变体R122I、R280V的功能特性表明，这些突变对谷氨酸转运的内在特性和动力学没有影响，但改变了L-天冬氨酸和竞争性抑制剂D,L-苏式-3-羟基天冬氨酸的Km值。用苯丙氨酸取代酪氨酸-405（Y405F）以及用苏氨酸取代精氨酸-479（R479T）会使谷氨酸转运体完全失活。对[35S]甲硫氨酸标记的转运体分子进行免疫沉淀表明，野生型和突变型转运体的表达水平相似。对卵母细胞切片进行免疫染色清楚地证明了突变型GLAST-1蛋白正确靶向并整合到质膜中。我们的结果表明，高度保守的酪氨酸-405的羟基和带正电荷的精氨酸-479在带负电荷的酸性神经递质谷氨酸的结合中起关键作用。
