TM4 of the glutamate transporter GLT-1 experiences substrate-induced motion during the transport cycle.
TM4 of the glutamate transporter GLT-1 experiences substrate-induced motion during the transport cycle.
复制标题
谷氨酸转运蛋白 GLT-1 的 TM4 在转运周期中经历底物诱导的运动
DOI：
10.1038/srep34522
复制
发表时间：
2016-10-04
影响因子：
4.6
通讯作者：
Qu S
中科院分区：
文献类型：
--
作者：
Rong X;Tan F;Wu X;Zhang X;Lu L;Zou X;Qu S
兴奋性氨基酸转运体2（EAAT 2），也称为胶质谷氨酸转运体1型（GLT-1），在维持细胞外谷氨酸浓度低于神经毒性水平中起重要作用。GLT-1的高度保守的TM 2跨膜结构域在转运周期中保持稳定的位置;然而，转运周期对TM 4拓扑结构的影响尚未完全确定。为了进一步揭示TM 4的功能，使用定点突变在TM 2和TM 4之间引入两个半胱氨酸对。在I93 C/V241 C和I97 C/V241 C突变体中观察到运输活性的显著降低，在应用氧化交联剂铜（II）（1，10-菲咯啉）3（CuPh）后，这表明构象转变对运输活性是必不可少的。此外，由CuPh交联的活性降低在具有谷氨酸或钾的外部介质中增强，这表明TM 2和TM 4在转运体的面向内的构象中假定更接近。我们的研究结果表明，GLT-1和潜在的其他谷氨酸转运蛋白的TM 4结构域，在底物易位过程中经历了一个复杂的构象转变，这涉及到增加的TM 2和TM 4结构域的接近向内面向的构象。
Excitatory amino acid transporter 2 (EAAT2), also known as glial glutamate transporter type 1 (GLT-1), plays an important role in maintaining the extracellular glutamate concentrations below neurotoxic levels. The highly conserved TM2 transmembrane domain of GLT-1 maintains a stable position during the transport cycle; however, the effect of the transport cycle on the topology of TM4 in not well established. To further reveal the function of TM4, two cysteine pairs between TM2 and TM4 were introduced using site-directed mutagenesis. A significant decrease of transport activity was observed in the I93C/V241C and I97C/V241C mutants upon application of the oxidative cross-linking reagent, copper (II) (1,10-phenanthroline)3(CuPh), which suggests that a conformational shift is essential for transporter activity. Furthermore, the decrease in activity by CuPh crosslinking was enhanced in external media with glutamate or potassium, which suggests that TM2 and TM4 assume closer proximity in the inward-facing conformation of the transporter. Our results suggest that the TM4 domain of GLT-1 and potentially other glutamate transporters, undergoes a complex conformational shift during substrate translocation, which involves an increase in the proximity of the TM2 and TM4 domains in the inward-facing conformation.
登录
查看更多内容
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：
64.8
作者：
KANAI, Y;HEDIGER, MA
通讯作者：
HEDIGER, MA
DOI：
10.1073/pnas.93.1.300
发表时间：
1996-01-09
影响因子：
11.1
作者：
PerezGarcia, MT;Chiamvimonvat, N;Tomaselli, GF
通讯作者：
Tomaselli, GF
影响因子：
4.8
作者：
Grunewald, M;Kanner, BI
通讯作者：
Kanner, BI
影响因子：
3.6
作者：
Hussainzada, Naissan;Khandewal, Akash;Swaan, Peter W.
通讯作者：
Swaan, Peter W.