The accessibility in the external part of the TM5 of the glutamate transporter EAAT1 is conformationally sensitive during the transport cycle.
The accessibility in the external part of the TM5 of the glutamate transporter EAAT1 is conformationally sensitive during the transport cycle.
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DOI：
10.1371/journal.pone.0030961
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发表时间：
2012
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
兴奋性氨基酸转运蛋白1 （Excitatory amino acid transporter 1, EAAT1）是谷氨酸转运蛋白，是谷氨酸突触作用终止的关键因素。它的作用是保持细胞外谷氨酸浓度低于神经毒性水平。然而，EAAT1的功能意义和残基在跨膜结构域（TM） 5中的可及性变化尚不清楚。我们采用半胱氨酸诱变的方法，用膜不渗透巯基试剂MTSET[（2-三甲基铵）甲乙硫磺酸]处理，研究了TM5的可及性变化。从EAAT1的无半胱氨酸版本的291到300位引入半胱氨酸突变体。我们检测了MTSET处理前后突变体的活性和动力学参数，进一步分析了底物和阻滞剂对MTSET抑制半胱氨酸突变体的影响。在突变体L296C、I297C和G299C中观察到MTSET对转运的抑制作用，而K300C在MTSET作用后活性更高。经MTSET处理后，L296C和G299C的Vmax较低，K300C的Vmax较高。L296C、G299C、K300C单半胱氨酸突变体表现出构象敏感的反应模式。谷氨酸和TBOA可增强L296C对MTSET的敏感性，而G299C对MTSET的敏感性仅由TBOA增强。这些事实表明，在运输周期中，TM5外部部分位置的可达性是构象敏感的。我们的研究结果表明，TM5的一些残基在运输周期中参与了运输途径。
Excitatory amino acid transporter 1 (EAAT1) is a glutamate transporter which is a key element in the termination of the synaptic actions of glutamate. It serves to keep the extracellular glutamate concentration below neurotoxic level. However the functional significance and the change of accessibility of residues in transmembrane domain (TM) 5 of the EAAT1 are not clear yet. We used cysteine mutagenesis with treatments with membrane-impermeable sulfhydryl reagent MTSET [(2-trimethylammonium) methanethiosulfonate] to investigate the change of accessibility of TM5. Cysteine mutants were introduced from position 291 to 300 of the cysteine-less version of EAAT1. We checked the activity and kinetic parameters of the mutants before and after treatments with MTSET, furthermore we analyzed the effect of the substrate and blocker on the inhibition of the cysteine mutants by MTSET. Inhibition of transport by MTSET was observed in the mutants L296C, I297C and G299C, while the activity of K300C got higher after exposure to MTSET. Vmax of L296C and G299C got lower while that of K300C got higher after treated by MTSET. The L296C, G299C, K300C single cysteine mutants showed a conformationally sensitive reactivity pattern. The sensitivity of L296C to MTSET was potentiated by glutamate and TBOA,but the sensitivity of G299C to MTSET was potentiated only by TBOA. All these facts suggest that the accessibility of some positions of the external part of the TM5 is conformationally sensitive during the transport cycle. Our results indicate that some residues of TM5 take part in the transport pathway during the transport cycle.
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