Modification of a Putative Third Sodium Site in the Glycine Transporter GlyT2 Influences the Chloride Dependence of Substrate Transport
Modification of a Putative Third Sodium Site in the Glycine Transporter GlyT2 Influences the Chloride Dependence of Substrate Transport
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DOI：
10.3389/fnmol.2018.00347
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发表时间：
2018-09-24
影响因子：
4.8
通讯作者：
Lopez-Corcuera, Beatriz
中科院分区：
文献类型：
--
作者：
Benito-Munoz, Cristina;Perona, Almudena;Lopez-Corcuera, Beatriz
甘氨酸介导的突触中神经递质的去除依赖于两种钠驱动的高亲和力质膜 GlyT，它们控制着神经递质的可用性。大多数胶质细胞 GlyT1 是甘氨酸突触水平的主要调节因子，而神经元 GlyT2 促进突触甘氨酸的循环并为突触前小泡的再填充提供神经递质。 GlyT 的钠：甘氨酸对称化学计量不同，显示 GlyT1 为 2:1，GlyT2 为 3:1 钠：甘氨酸偶联。钠在两个保守的 Na+ 位点上与 GlyT 结合：Na1 和 Na2。尽管 Glu650 参与了协调，但 GlyT2 Na3 位点的位置仍然未知。在这里，我们通过将 Na3 离子放置在两个不同的位置，对 GlyT2 模型进行了比较 MD 模拟，该模型与来自果蝇的结晶 DAT 同源。通过结合通过 GlyTs 突变体的生化和电生理学分析获得的计算机和实验数据，我们提供了证据表明 GlyT2 第三个钠离子由 Glu-250 和 Glu-650 保持，在涉及阳离子特异性敏感性的具有强大变构特性的区域内。用 GlyT1 中相应的甲硫氨酸取代 GlyT2 中的 Glu650，可将电荷通量比降低至 GlyT1 的水平，而不会产生传输解偶联。在该 GlyT2 突变体中，甘氨酸转运的氯依赖性几乎被消除，但同时用中性氨基酸取代 Glu250 和 Glu650 挽救了氯敏感性，表明 Glu250 的质子化/去质子化替代了氯功能。等效 GlyT1 突变的差异行为维持了推定的 Na3 位点和氯位点之间的 GlyT2 特异性变构耦合。
Neurotransmitter removal from glycine-mediated synapses relies on two sodium-driven high-affinity plasma membrane GlyTs that control neurotransmitter availability. Mostly glial GlyT1 is the main regulator of glycine synaptic levels, whereas neuronal GlyT2 promotes the recycling of synaptic glycine and supplies neurotransmitter for presynaptic vesicle refilling. The GlyTs differ in sodium: glycine symport stoichiometry, showing GlyT1 a 2: 1 and GlyT2 a 3: 1 sodium: glycine coupling. Sodium binds to the GlyTs at two conserved Na+ sites: Na1 and Na2. The location of GlyT2 Na3 site remains unknown, although Glu650 has been involved in the coordination. Here, we have used comparative MD simulations of a GlyT2 model constructed by homology to the crystalized DAT from Drosophila melanogaster by placing the Na3 ion at two different locations. By combination of in silico and experimental data obtained by biochemical and electrophysiological analysis of GlyTs mutants, we provide evidences suggesting the GlyT2 third sodium ion is held by Glu-250 and Glu-650, within a region with robust allosteric properties involved in cation-specific sensitivity. Substitution of Glu650 in GlyT2 by the corresponding methionine in GlyT1 reduced the charge-to-flux ratio to the level of GlyT1 without producing transport uncoupling. Chloride dependence of glycine transport was almost abolished in this GlyT2 mutant but simultaneous substitution of Glu250 and Glu650 by neutral amino acids rescued chloride sensitivity, suggesting that protonation/deprotonation of Glu250 substitutes chloride function. The differential behavior of equivalent GlyT1 mutations sustains a GlyT2-specific allosteric coupling between the putative Na3 site and the chloride site.