Cysteine transport through excitatory amino acid transporter 3 (EAAT3).
Cysteine transport through excitatory amino acid transporter 3 (EAAT3).
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DOI：
10.1371/journal.pone.0109245
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Amara SG
中科院分区：
文献类型：
--
作者：
Watts SD;Torres-Salazar D;Divito CB;Amara SG
兴奋性氨基酸转运蛋白（EAAT）限制谷氨酸能信号传导并维持细胞外谷氨酸浓度低于神经毒性水平。在五种已知的EAAT亚型（EAAT 1-5）中，只有神经元亚型EAAT 3（EAAC 1）可以有效地转运不带电荷的氨基酸L-半胱氨酸。EAAT 3介导的半胱氨酸转运被认为是神经元获得半胱氨酸以合成谷胱甘肽的主要机制，谷胱甘肽是防止氧化应激和神经元毒性的关键分子。EAAT 3选择性转运半胱氨酸的分子机制尚未阐明。在这里，我们提出，通过EAAT 3的半胱氨酸的运输需要在结合位点形成巯基形式的半胱氨酸。使用非洲爪蟾卵母细胞和HEK 293细胞表达EAAT 2和EAAT 3，我们评估了不同的底物的运输动力学和测量转运相关电流电生理。我们的研究结果表明，L-硒代半胱氨酸，半胱氨酸类似物，形成一个带负电荷的硒离子在生理pH值，有效地运输EAAT 1-3，并具有更高的表观亲和力的运输相比，半胱氨酸。使用膜栓系的GFP变体来监测与运输活性相关的细胞内pH变化，我们观察到EAAT 3运输L-谷氨酸或L-硒代半胱氨酸降低细胞内pH，而运输半胱氨酸导致细胞质碱化。当半胱氨酸应用于表达EAAT 2的细胞时，未观察到pH变化，EAAT 2显示可忽略的半胱氨酸转运。在有利于通过EAAT 3释放细胞内底物的条件下，我们观察到标记的细胞内谷氨酸的释放，但没有检测到半胱氨酸的释放。我们的研究结果支持一个模型，即半胱氨酸运输通过EAAT 3是通过半胱氨酸去质子化促进，一旦内部，硫醇迅速重新质子化。此外，这些发现表明，半胱氨酸转运主要是单向的，反向转运并不有助于细胞内半胱氨酸池的耗尽。
Excitatory amino acid transporters (EAATs) limit glutamatergic signaling and maintain extracellular glutamate concentrations below neurotoxic levels. Of the five known EAAT isoforms (EAATs 1–5), only the neuronal isoform, EAAT3 (EAAC1), can efficiently transport the uncharged amino acid L-cysteine. EAAT3-mediated cysteine transport has been proposed to be a primary mechanism used by neurons to obtain cysteine for the synthesis of glutathione, a key molecule in preventing oxidative stress and neuronal toxicity. The molecular mechanisms underlying the selective transport of cysteine by EAAT3 have not been elucidated. Here we propose that the transport of cysteine through EAAT3 requires formation of the thiolate form of cysteine in the binding site. Using Xenopus oocytes and HEK293 cells expressing EAAT2 and EAAT3, we assessed the transport kinetics of different substrates and measured transporter-associated currents electrophysiologically. Our results show that L-selenocysteine, a cysteine analog that forms a negatively-charged selenolate ion at physiological pH, is efficiently transported by EAATs 1–3 and has a much higher apparent affinity for transport when compared to cysteine. Using a membrane tethered GFP variant to monitor intracellular pH changes associated with transport activity, we observed that transport of either L-glutamate or L-selenocysteine by EAAT3 decreased intracellular pH, whereas transport of cysteine resulted in cytoplasmic alkalinization. No change in pH was observed when cysteine was applied to cells expressing EAAT2, which displays negligible transport of cysteine. Under conditions that favor release of intracellular substrates through EAAT3 we observed release of labeled intracellular glutamate but did not detect cysteine release. Our results support a model whereby cysteine transport through EAAT3 is facilitated through cysteine de-protonation and that once inside, the thiolate is rapidly re-protonated. Moreover, these findings suggest that cysteine transport is predominantly unidirectional and that reverse transport does not contribute to depletion of intracellular cysteine pools.
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DOI：
10.1083/jcb.108.2.613
发表时间：
1989-02
期刊：
The Journal of cell biology
影响因子：
--
作者：
Skene JH;Virág I
通讯作者：
Virág I
影响因子：
4.6
作者：
Grewer, Christof;Gameiro, Armanda;Zhang, Zhou;Tao, Zhen;Braams, Simona;Rauen, Thomas
通讯作者：
Rauen, Thomas
影响因子：
56.9
作者：
Kryukov, GV;Castellano, S;Gladyshev, VN
通讯作者：
Gladyshev, VN
影响因子：
5.3
作者：
Levy, LM;Warr, O;Attwell, D
通讯作者：
Attwell, D
影响因子：
2.9
作者：
Elsliger, MA;Wachter, RM;Remington, SJ
通讯作者：
Remington, SJ