Dynamics of the Extracellular Gate and Ion-Substrate Coupling in the Glutamate Transporter

Huang, Zhijian; Tajkhorshid, Emad

doi:10.1529/biophysj.108.133421

Cited by 77 publications

(121 citation statements)

References 59 publications

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“…Ligand-induced movement of HP2 in GLT-1 is consistent with the proposal, based on the Glt Ph structure, that HP2 serves as an external gate of the transporter, which moves back and forth to allow substrate into the binding site from the extracellular side (11,23). A very recently published molecular dynamics simulation study has provided further support for this idea (37).…”

Section: Discussionsupporting

confidence: 86%

Substrates and Non-transportable Analogues Induce Structural Rearrangements at the Extracellular Entrance of the Glial Glutamate Transporter GLT-1/EAAT2

Kanner

2008

Journal of Biological Chemistry

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To explore rearrangements of the reentrant loop HP2 relative to transmembrane domains (TMs) 7 and 8 during transport by the glial glutamate transporter GLT-1/EAAT2, cysteine pairs were introduced at the extracellular ends of these structural elements. The pairs were introduced around 10 -15 Å "above" the residues, which make contact with substrate in the related archaeal homologue Glt Ph . Transport by the double mutants M449C/L466C (HP2/TM 8), L453C/I463C (HP2/TM 8), and I411C/I463C (TM 7/TM 8) was inhibited by copper(II)(1,10-phenanthroline) 3 (CuPh) and by Cd 2؉ . Inhibition was only observed when the two cysteines were present in the same construct, but not with the respective single cysteine mutants or when only one cysteine was paired with a mutation to another residue. Glutamate and potassium, both expected to increase the proportion of inward-facing transporters, significantly protected against the inhibition of transport activity of M449C/ L466C by CuPh. The non-transportable analogues kainate and D,L-threo-␤-benzyloxyaspartate are expected to stabilize an outward-facing conformation, but only the latter potentiated the effect of CuPh on M449C/L466C. However, both analogues increased the aqueous accessibility of the cysteines introduced at positions 449 and 466 to a membrane-impermeant sulfhydryl reagent. Inhibition of L453C/I463C by CuPh was protected not only by glutamate but also by the two analogues. In contrast, these ligands had very little effect on the inhibition of I411C/ I463C by CuPh. Our results are consistent with the proposal that HP2 serves as the extracellular gate of the transporter and indicate that glutamate and the two analogues induce distinct conformations of HP2.

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Section: Discussionsupporting

confidence: 86%

Substrates and Non-transportable Analogues Induce Structural Rearrangements at the Extracellular Entrance of the Glial Glutamate Transporter GLT-1/EAAT2

Kanner

2008

Journal of Biological Chemistry

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“…5B). The role of Na2 in sealing the binding site has also been reported in recent simulations of the glutamate transporter (18). Interestingly, a similar role has been proposed for a Na ϩ in a leucine transporter (19).…”

Section: Two Residues Distinguished By Their Persistent Mediating Rolsupporting

confidence: 68%

Molecular simulations elucidate the substrate translocation pathway in a glutamate transporter

Shrivastava²,

Amara

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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Glutamate transporters are membrane proteins found in neurons and glial cells, which play a critical role in regulating cell signaling by clearing glutamate released from synapses. Although extensive biochemical and structural studies have shed light onto different aspects of glutamate transport, the time-resolved molecular mechanism of substrate (glutamate or aspartate) translocation, that is, the sequence of events occurring at the atomic level after substrate binding and before its release intracellularly remain to be elucidated. We identify an energetically preferred permeation pathway of Ϸ23 Å between the helix HP1b on the hairpin HP1 and the transmembrane helices TM7 and TM8, using the high resolution structure of the transporter from Pyrococcus horikoshii (Glt Ph ) in steered molecular dynamics simulations. Detailed potential of mean force calculations along the putative pathway reveal 2 energy barriers encountered by the substrate (aspartate) before it reaches the exit. The first barrier is surmounted with the assistance of 2 conserved residues (S278 and N401) and a sodium ion (Na2); and the second, by the electrostatic interactions with D405 and another sodium ion (Na1). The observed critical interactions and mediating role of conserved residues in the core domain, the accompanying conformational changes (in both substrate and transporter) that relieve local strains, and the unique coupling of aspartate transport to Na ؉ dislocation provide insights into methods for modulating substrate transport.

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“…We used two separate homology models based on templates 2NWX, [termed ASCT2(2NWX)] and 2NWW [termed ASCT2(2NWW)] (Boudker et al, 2007). The former represents the substrate-bound conformation (closed re-entrant loop, RL2; termed closed-loop form), whereas the latter was proposed to be more representative of the substrate-free, apo state of the transporter [open RL2, termed open-loop form (Boudker et al, 2007;Huang and Tajkhorshid, 2008)]. L-Asparagine is a well characterized transported substrate, which was used first due to its high homology to the aspartate molecule bound to the Glt Ph template.…”

Section: In Silico Docking Studies Predict Open-loop or Closed-loop Pmentioning

confidence: 99%

Defining Substrate and Blocker Activity of Alanine-Serine-Cysteine Transporter 2 (ASCT2) Ligands with Novel Serine Analogs

Albers

Marsiglia²,

Thomas³

et al. 2011

Mol Pharmacol

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The neutral amino acid transporter alanine-serine-cysteine transporter 2 (ASCT2) belongs to the solute carrier 1 (SLC1) family of solute transporters and transports small, neutral amino acids across the membrane, including the physiologically important and ubiquitous amino acid glutamine. Our understanding of the involvement of ASCT2 in the physiological processes involving glutamine is hampered by a lack of understanding of its pharmacology and the absence of high-affinity inhibitors. In this study, we combined an in silico docking approach with experimental investigation of binding parameters to develop new ASCT2 inhibitors and substrates, a series of serine esters, and to determine structural parameters that govern their functional effects. The series of compounds was synthesized using standard methods and exhibited a range of properties, from inhibitors to partial substrates and full substrates. Our results suggest that amino acid derivatives with small side-chain volume and low side-chain hydrophobicity interact strongly with the closed-loop form of the binding site, in which re-entrant loop 2, the presumed extracellular gate for the substrate binding site, is closed off. However, these derivatives bind weakly to the open-loop form (external gate open to the extracellular side), acting as transported substrates. In contrast, inhibitors bind preferentially to the open-loop form. An aromatic residue in the side chain is required for high-affinity interaction. One of the compounds, the L-serine ester serine biphenyl-4-carboxylate reversibly inhibits ASCT2 function with an apparent affinity of 30 M.

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Dynamics of the Extracellular Gate and Ion-Substrate Coupling in the Glutamate Transporter

Cited by 77 publications

References 59 publications

Substrates and Non-transportable Analogues Induce Structural Rearrangements at the Extracellular Entrance of the Glial Glutamate Transporter GLT-1/EAAT2

Substrates and Non-transportable Analogues Induce Structural Rearrangements at the Extracellular Entrance of the Glial Glutamate Transporter GLT-1/EAAT2

Molecular simulations elucidate the substrate translocation pathway in a glutamate transporter

Defining Substrate and Blocker Activity of Alanine-Serine-Cysteine Transporter 2 (ASCT2) Ligands with Novel Serine Analogs

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