Huang Shiwei, Ryan Renae M, Vandenberg Robert J
Transporter Biology Group, Discipline of Pharmacology, School of Medical Sciences, Bosch Institute, University of Sydney, Sydney, New South Wales 2006, Australia.
J Biol Chem. 2009 Feb 13;284(7):4510-5. doi: 10.1074/jbc.M808495200. Epub 2008 Dec 12.
Glutamate transport is coupled to the co-transport of 3Na(+) and 1H(+) and the countertransport of 1 K(+). However, the mechanism of how this process occurs is not well understood. The crystal structure of an archaeal homolog of the human glutamate transporters, Glt(Ph), has provided the framework to begin to understand the mechanism of transport. The glutamate transporter EAAT2 is different from other subtypes in two respects. First, Li(+) cannot support transport by EAAT2, whereas it can support transport by the other excitatory amino acid transporters, and second, EAAT2 is sensitive to a wider range of blockers than other subtypes. We have investigated the relationship between the cation driving transport and whether the glutamate analogues, l-anti-endo-3,4-methanopyrrolidine-dicarboxylic acid (MPDC) and (2S,4R)-4-methylglutamate (4MG), are substrates or blockers of transport. We have also investigated the molecular basis for these differences. EAAT2 has a Ser residue at position 441 with hairpin loop 2, whereas the corresponding residue in EAAT1 is a Gly residue. We demonstrate that if the transporter has a Ser residue at this position, then 4MG and MPDC are poor substrates in Na(+), and Li(+) cannot support transport of any substrate. Conversely, if the transporter has a Gly residue at this position, then in Na(+) 4MG and MPDC are substrates with efficacy comparable with glutamate, but in Li(+) 4MG and MPDC are poor substrates relative to glutamate. This Ser/Gly residue is located between the bound substrate and one of the cation binding sites, which provides an explanation for the coupling of substrate and cation binding.
谷氨酸转运与3个Na(+)和1个H(+)的共转运以及1个K(+)的反向转运相偶联。然而，这一过程的发生机制尚未完全明确。人谷氨酸转运体的古菌同源物Glt(Ph)的晶体结构为开始理解转运机制提供了框架。谷氨酸转运体EAAT2在两个方面与其他亚型不同。第一，Li(+)不能支持EAAT2的转运，而它可以支持其他兴奋性氨基酸转运体的转运；第二，EAAT2对比其他亚型更广泛的阻断剂敏感。我们研究了阳离子驱动转运与谷氨酸类似物l-反式-内型-3,4-甲吡咯烷二羧酸(MPDC)和(2S,4R)-4-甲基谷氨酸(4MG)是转运底物还是阻断剂之间的关系。我们还研究了这些差异的分子基础。EAAT2在441位有一个Ser残基与发夹环2相连，而EAAT1中的相应残基是一个Gly残基。我们证明，如果转运体在该位置有一个Ser残基，那么4MG和MPDC在Na(+)中是不良底物，并且Li(+)不能支持任何底物的转运。相反，如果转运体在该位置有一个Gly残基，那么在Na(+)中4MG和MPDC是与谷氨酸效能相当的底物，但在Li(+)中4MG和MPDC相对于谷氨酸是不良底物。这个Ser/Gly残基位于结合的底物和一个阳离子结合位点之间，这为底物与阳离子结合的偶联提供了解释。
