The Conformationally Sensitive Spatial Distance Between the TM3-4 Loop and Transmembrane Segment 7 in the Glutamate Transporter Revealed by Paired-Cysteine Mutagenesis.

Qi Qu,Shaogang Qu,Ji Wang,Guiping Li,Rongqing Chen

doi:10.3389/fcell.2021.737629

Abstract

Excitatory amino acid transporters can maintain extracellular glutamate concentrations lower than neurotoxic levels by transferring neurotransmitters from the synaptic cleft into surrounding glial cells and neurons. Previous work regarding the structural studies of GltPh, GltTK, excitatory amino acid transporter 1 (EAAT1), EAAT3 and alanine serine cysteine transporter 2 described the transport mechanism of the glutamate transporter in depth. However, much remains unknown about the role of the loop between transmembrane segment 3 and 4 during transport. To probe the function of this loop in the transport cycle, we engineered a pair of cysteine residues between the TM3-TM4 loop and TM7 in cysteine-less EAAT2. Here, we show that the oxidative cross-linking reagent CuPh inhibits transport activity of the paired mutant L149C/M414C, whereas DTT inhibits the effect of CuPh on transport activity of L149C/M414C. Additionally, we show that the effect of cross-linking in the mutant is due to the formation of the disulfide bond within the molecules of EAAT2. Further, L-glutamate or KCl protect, and D,L-threo-β-benzyloxy-aspartate (TBOA) increases, CuPh-induced inhibition in the L149C/M414 mutant, suggesting that the L149C and M414C cysteines are closer or farther away in the outward- or inward-facing conformations, respectively. Together, our findings provide evidence that the distance between TM3-TM4 loop and TM7 alter when substrates are transported.

Highlights

Glutamate is the predominant excitatory neurotransmitter in the brain (Curtis and Johnston, 1974; Fonnum, 1984) and is critical to numerous central nervous system (CNS) functions (Tang et al, 1999; Matsuzaki et al, 2004; Chantranupong and Sabatini, 2018; Boender et al, 2020; Stephenson-Jones et al, 2020)
To probe whether residues exist at EAAT2 TM3-4 and TM7 that are close enough to form disulfide bonds, we identified seven pairs of residues to study based on the crystal structural model of aspartate transporter from Pyrococcus horikoshii (GltPh).; corresponding double mutations were designed for each
The activities of the five other double mutants were less than 20% compared to the activity of CL-EAAT2, and we speculate that they may exhibit fewer transport activities in the presence of Cu(II)(1,10-phenanthroline)3 (CuPh), which is unfavorable under oxidative conditions

Summary

Introduction

Glutamate is the predominant excitatory neurotransmitter in the brain (Curtis and Johnston, 1974; Fonnum, 1984) and is critical to numerous central nervous system (CNS) functions (Tang et al, 1999; Matsuzaki et al, 2004; Chantranupong and Sabatini, 2018; Boender et al, 2020; Stephenson-Jones et al, 2020). Dysregulation of EAAT2 has been implicated in some neurodegenerative diseases including but not limited to amyotrophic lateral sclerosis (Takahashi et al, 2015; Jiang et al, 2019; Malik and Willnow, 2019), Alzheimer’s disease (Takahashi et al, 2015; Ugbode et al, 2017; Malik and Willnow, 2019; Sharma et al, 2019), and epilepsy (Takahashi et al, 2015; Guella et al, 2017; Malik and Willnow, 2019) For this reason, functional studies of EAAT2 are critical and may reveal potential genetically based therapeutic targets

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