Dataset Information

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

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.

SUBMITTER: Rong X

PROVIDER: S-EPMC5048300 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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

Rong Xiuliang X Tan Feng F Wu Xiaojuan X Zhang Xiuping X Lu Lingli L Zou Xiaoming X Qu Shaogang S

Scientific reports 20161004

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 intro ...[more]

PMID: 27698371

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Project description:Historically, glutamate uptake in the CNS was mainly attributed to glial cells for three reasons: 1) none of the glutamate transporters were found to be located in presynaptic terminals of excitatory synapses; 2) the putative glial transporters, GLT-1 and GLAST are expressed at high levels in astrocytes; 3) studies of the constitutive GLT-1 knockout as well as pharmacological studies demonstrated that >90% of glutamate uptake into forebrain synaptosomes is mediated by the operation of GLT-1. Here we summarize the history leading up to the recognition of GLT-1a as a presynaptic glutamate transporter. A major issue now is understanding the physiological and pathophysiological significance of the expression of GLT-1 in presynaptic terminals. To elucidate the cell-type specific functions of GLT-1, a conditional knockout was generated with which to inactivate the GLT-1 gene in different cell types using Cre/lox technology. Astrocytic knockout led to an 80% reduction of GLT-1 expression, resulting in intractable seizures and early mortality as seen also in the constitutive knockout. Neuronal knockout was associated with no obvious phenotype. Surprisingly, synaptosomal uptake capacity (Vmax) was found to be significantly reduced, by 40%, in the neuronal knockout, indicating that the contribution of neuronal GLT-1 to synaptosomal uptake is disproportionate to its protein expression (5-10%). Conversely, the contribution of astrocytic GLT-1 to synaptosomal uptake was much lower than expected. In contrast, the loss of uptake into liposomes prepared from brain protein from astrocyte and neuronal knockouts was proportionate with the loss of GLT-1 protein, suggesting that a large portion of GLT-1 in astrocytic membranes in synaptosomal preparations is not functional, possibly because of a failure to reseal. These results suggest the need to reinterpret many previous studies using synaptosomal uptake to investigate glutamate transport itself as well as changes in glutamate homeostasis associated with normal functions, neurodegeneration, and response to drugs.

Dataset Information

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

Publications

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

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