Millisecond dynamics of an unlabeled amino acid transporter

Matin, Tina R.; Heath, George R.; Huysmans, Gerard H. M.; Boudker, Olga; Scheuring, Simon

doi:10.1038/s41467-020-18811-z

Cited by 32 publications

(31 citation statements)

References 61 publications

(119 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Such a state may be stabilized by the membrane-like environment provided by the nanodisc, which could support movements of the transport domain by surrounding it with a compliant lipid bilayer and providing an additional network of protein-lipid interaction 51 . Indeed, a recent study also observed an intermediate state between the OFS and the IFS of membrane-reconstituted Glt Ph 52 .…”

Section: Discussionmentioning

confidence: 84%

Glutamate transporters have a chloride channel with two hydrophobic gates

Chen

Pant

et al. 2021

Nature

View full text Add to dashboard Cite

Glutamate is the most abundant excitatory neurotransmitter in the central nervous system, therefore its precise control is vital for maintaining normal brain function and preventing excitotoxicity 1 . Removal of extracellular glutamate is achieved by plasma membrane-bound transporters, which couple glutamate transport to sodium, potassium and pH gradients using an elevator mechanism [2][3][4][5] . Glutamate transporters also conduct chloride ions via a channel-like process that is thermodynamically uncoupled from transport [6][7][8] . However, the molecular mechanisms that allow these dual-function transporters to carry out two seemingly contradictory roles are unknown. Here we report the cryo-electron microscopy structure of a glutamate transporter homologue in an open-channel state, revealing an aqueous cavity that is formed during the transport cycle. Using functional studies and molecular dynamics simulations, we show that this cavity is an aqueous-accessible chloride permeation pathway gated by two hydrophobic regions, and is conserved across mammalian and archaeal glutamate transporters. Our findings provide insight into the mechanism by which glutamate transporters support their dual function and add a crucial piece of information to aid mapping of the complete transport cycle shared by the SLC1A transporter family.

show abstract

Section: Discussionmentioning

confidence: 84%

Glutamate transporters have a chloride channel with two hydrophobic gates

Chen

Pant

et al. 2021

Nature

View full text Add to dashboard Cite

show abstract

“…Switching between the modes is rare, occurring on a time scale of hundreds of seconds and resulting in the so-called static disorder of the transport kinetics. These transport modes were attributed to sub-populations that show different elevator dynamics and intracellular substrate-release rates (Huysmans et al, 2021; Matin et al, 2020). Here, we observed a correlation, where nitrate changes the ratio of different substrate-binding states and increases the fraction of slow WT transporters ( Figure 1f ).…”

Section: Discussionmentioning

confidence: 99%

“…Recently, high-speed atomic force microscopy, single-molecule Förster resonance energy transfer (smFRET) total internal reflection fluorescence (TIRF) microscopy, and 19 F-NMR revealed a more complex picture of Glt Ph transport and dynamics (Akyuz, Altman, Blanchard, & Boudker, 2013; Akyuz et al, 2015; Ciftci et al, 2020; Erkens et al, 2013; Huang et al, 2020; Huysmans, Ciftci, Wang, Blanchard, & Boudker, 2021; Matin, Heath, Huysmans, Boudker, & Scheuring, 2020). These studies established the existence of additional conformational states in OFS and IFS, of which some translocate and transport at different rates.…”

Section: Main Textmentioning

confidence: 99%

Heterogeneous substrate binding in a glutamate transporter homologue

Reddy

Ciftci

Scopelliti

et al. 2021

Preprint

Self Cite

View full text Add to dashboard Cite

Integral membrane glutamate transporters couple the concentrative substrate transport to ion gradients. There is a wealth of structural and mechanistic information about this family, including kinetic models of transport. Recent studies have revealed transport rate heterogeneity in an archaeal glutamate transporter homologue GltPh, inconsistent with simple kinetic models. The structural and mechanistic determinants of this heterogeneity remain undefined. In a mutant form of GltPh, we demonstrate substrate binding heterogeneity in the outward-facing state, modulated by temperature and salts. We observe similar trends in wild-type GltPh that correlate with changes in the transport rate. Extensive cryo-EM analysis of the fully bound mutant GltPh provides multiple potential explanations of heterogeneous substrate binding. At equilibrium, we show subtle differences in tilts of protomers in the outward-facing state and configurations of the substrate-binding pocket. Within seconds of substrate binding, we observe perturbed helical packing of the extracellular half of the substrate-binding domain. Some or all of these may contribute to the heterogeneity observed in binding and transport.

show abstract

“…In 2004, researchers identified and described the crystal structure of Glt Ph , a prokaryotic homolog of EAATs that originated from archaeal ortholog Pyrococcus horikoshii ( Yernool et al, 2004 ). This homolog shares ∼37% sequence identity with human EAAT2 although is more conservative in the C-terminal (critical for facilitating the transport core to bind substrates and cations; Matin et al, 2020 ). Notably, Glt Ph is a homotrimer, and each subunit with an independent permeation pathway has eight transmembrane segments (TM1-8) and two re-entrant hairpin loops (1 and 2; HP1-2; Yernool et al, 2004 ).…”

Section: Introductionmentioning

confidence: 99%

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

Wang

et al. 2021

Front. Cell Dev. Biol.

View full text Add to dashboard Cite

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.

show abstract

Millisecond dynamics of an unlabeled amino acid transporter

Cited by 32 publications

References 61 publications

Glutamate transporters have a chloride channel with two hydrophobic gates

Glutamate transporters have a chloride channel with two hydrophobic gates

Heterogeneous substrate binding in a glutamate transporter homologue

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

Contact Info

Product

Resources

About