TM4 of the glutamate transporter GLT-1 experiences substrate-induced motion during the transport cycle.

Xiuliang Rong,Shaogang Qu,Xiaojuan Wu,Xiaoming Zou,Lingli Lu,Xiuping Zhang,Feng Tan

doi:10.1038/srep34522

Abstract

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.

Highlights

Glutamate is the most important excitatory neurotransmitter in the central nervous system
Our results showed that the protein expression of all the mutants were significant reduced comparied with CL-glutamate transporter type 1 (GLT-1) (Fig. 2B,C), and the transport function of I93C, I93C/V241C and I97C/V241C were impaired (Fig. 2D)
As the main transporters for clearing glutamate in the synaptic cleft, excitatory amino acid transporters (EAATs) have been reported to be involved in the pathogenesis of various neurodegenerative diseases

Summary

Introduction

Glutamate is the most important excitatory neurotransmitter in the central nervous system. The members of the glutamate transporter family, which include the EAATs, as well as prokaryotic transporters and two Na+-dependent neutral amino acid transporters, are about 25 to 30% identical[7]. This homology results in a similar membrane topology and transport mechanism for all family members[8,9,10,11,12,13,14,15,16,17,18,19,20]. The experimentally determined membrane topology of the glutamate transporter EAAT2 ( known as glial glutamate transporter type 1, GLT-1) is supported by the crystal structure of a homolog. Our data suggest that there is a complex relative motion between TM2 and TM4 and that a conformational shift of TM4 may occur during substrate transport cycle

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Conclusion