Proximity of transmembrane segments 5 and 8 of the glutamate transporter GLT-1 inferred from paired cysteine mutagenesis.
Proximity of transmembrane segments 5 and 8 of the glutamate transporter GLT-1 inferred from paired cysteine mutagenesis.
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DOI：
10.1371/journal.pone.0021288
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Qu S
中科院分区：
文献类型：
--
作者：
Zhang X;Qu S
GLT-1是一种神经胶质谷氨酸转运蛋白，其维持兴奋性神经递质的低突触浓度，从而实现有效的突触传递。基于细菌同源物GltPh的晶体结构，已经提出连接跨膜结构域（TM）7和8的折返环HP 2移动以打开和关闭从细胞外介质进入结合口袋。然而，TM 5和TM 8在转运过程中的构象变化尚不清楚。我们使用配对半胱氨酸诱变与铜（II）（1，10-菲咯啉）3（CuPh）的治疗相结合，以验证位于GLT-1的这些结构元件的残基的预测接近度。为了评估跨膜结构域（TM）5在通过神经胶质谷氨酸转运蛋白GLT-1/EAAT 2转运期间相对于TM 8的接近性，在这些结构元件的细胞外末端引入半胱氨酸对。在双突变体I295 C/I463 C和G297 C/I463 C中观察到铜（II）（1，10-菲咯啉）3对转运的完全抑制，但在相应的单突变体中未观察到。谷氨酸和钾，都预计将增加面向内的转运蛋白的比例，显着保护对I295 C/I463 C和G297 C/I463 C的转运活性的抑制CuPh。双突变体I295 C/I463 C和G297 C/I463 C的转运也受到Cd ~（2+）的抑制。我们的研究结果表明，TM 5（Ile-295，Gly-297）是在接近TM 8（Ile-463）在哺乳动物转运蛋白，这些结构域之间的空间关系在运输周期中被改变。
GLT-1 is a glial glutamate transporter which maintains low synaptic concentrations of the excitatory neurotransmitter enabling efficient synaptic transmission. Based on the crystal structure of the bacterial homologue GltPh, it has been proposed that the reentrant loop HP2, which connects transmembrane domains (TM) 7 and 8, moves to open and close access to the binding pocket from the extracellular medium. However the conformation change between TM5 and TM8 during the transport cycle is not clear yet. We used paired cysteine mutagenesis in conjunction with treatments with Copper(II)(1,10-Phenanthroline)3 (CuPh), to verify the predicted proximity of residues located at these structural elements of GLT-1. To assess the proximity of transmembrane domain (TM) 5 relative to TM8 during transport by the glial glutamate transporter GLT-1/EAAT2, cysteine pairs were introduced at the extracellular ends of these structural elements. A complete inhibition of transport by Copper(II)(1,10-Phenanthroline)3 is observed in the double mutants I295C/I463C and G297C/I463C, but not in the corresponding single mutants. Glutamate and potassium, both expected to increase the proportion of inward-facing transporters, significantly protected against the inhibition of transport activity of I295C/I463C and G297C/I463C by CuPh. Transport by the double mutants I295C/I463C and G297C/I463C also was inhibited by Cd2+. Our results suggest that TM5 (Ile-295, Gly-297) is in close proximity to TM8 (Ile-463) in the mammalian transporter, and that the spatial relationship between these domains is altered during the transport cycle.
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影响因子：
5.3
作者：
Levy, LM;Warr, O;Attwell, D
通讯作者：
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影响因子：
5.6
作者：
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5.3
作者：
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影响因子：
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作者：
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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