Department of Biochemistry and Molecular Biology, Hebrew University Hadassah Medical School, Jerusalem 91120, Israel.
Department of Chemistry, Binghamton University, Binghamton, New York 13902.
J Biol Chem. 2011 Dec 2;286(48):41381-41390. doi: 10.1074/jbc.M111.291021. Epub 2011 Oct 7.
In the brain, transporters of the major excitatory neurotransmitter glutamate remove their substrate from the synaptic cleft to allow optimal glutamatergic neurotransmission. Their transport cycle consists of two sequential translocation steps, namely cotransport of glutamic acid with three Na(+) ions, followed by countertransport of K(+). Recent studies, based on several crystal structures of the archeal homologue Glt(Ph), indicate that glutamate translocation occurs by an elevator-like mechanism. The resolution of these structures was not sufficiently high to unambiguously identify the sites of Na(+) binding, but functional and computational studies suggest some candidate sites. In the Glt(Ph) structure, a conserved aspartate residue (Asp-390) is located adjacent to a conserved tyrosine residue, previously shown to be a molecular determinant of ion selectivity in the brain glutamate transporter GLT-1. In this study, we characterize mutants of Asp-440 of the neuronal transporter EAAC1, which is the counterpart of Asp-390 of Glt(Ph). Except for substitution by glutamate, this residue is functionally irreplaceable. Using biochemical and electrophysiological approaches, we conclude that although D440E is intrinsically capable of net flux, this mutant behaves as an exchanger under physiological conditions, due to increased and decreased apparent affinities for Na(+) and K(+), respectively. Our present and previous data are compatible with the idea that the conserved tyrosine and aspartate residues, located at the external end of the binding pocket, may serve as a transient or stable cation binding site in the glutamate transporters.
在大脑中，主要兴奋性神经递质谷氨酸的转运体将其底物从突触间隙中移除，以允许最佳的谷氨酸能神经传递。它们的转运循环由两个连续的转运步骤组成，即谷氨酸与三个 Na(+)离子的共转运，然后是 K(+)的反向转运。最近的研究基于几种古菌同源物 Glt(Ph)的晶体结构，表明谷氨酸转运是通过电梯样机制发生的。这些结构的分辨率不够高，无法明确识别 Na(+)结合的位点，但功能和计算研究提出了一些候选位点。在 Glt(Ph)结构中，一个保守的天冬氨酸残基（Asp-390）位于一个保守的酪氨酸残基旁边，该残基以前被证明是大脑谷氨酸转运体 GLT-1 离子选择性的分子决定因素。在这项研究中，我们对神经元转运体 EAAC1 的 Asp-440 突变体进行了表征，该残基是 Glt(Ph)中 Asp-390 的对应物。除了被谷氨酸取代外，该残基在功能上是不可替代的。使用生化和电生理方法，我们得出结论，尽管 D440E 本身具有净通量能力，但由于对 Na(+)和 K(+)的表观亲和力分别增加和降低，该突变体在生理条件下表现为交换体。我们目前和以前的数据与以下观点一致，即位于结合口袋外部末端的保守酪氨酸和天冬氨酸残基可能在谷氨酸转运体中作为瞬时或稳定的阳离子结合位点。
