Binding and transport of D-aspartate by the glutamate transporter homolog GltTk

Mammalian glutamate transporters are crucial players in neuronal communication as they perform neurotransmitter reuptake from the synaptic cleft. Besides L-glutamate and L-aspartate, they also recognize D-aspartate, which might participate in mammalian neurotransmission and/or neuromodulation. Much of the mechanistic insight in glutamate transport comes from studies of the archeal homologs GltPh from Pyrococcus horikoshii and GltTk from Thermococcus kodakarensis. Here, we show that GltTk transports D-aspartate with identical Na+: substrate coupling stoichiometry as L-aspartate, and that the affinities (Kd and Km) for the two substrates are similar. We determined a crystal structure of GltTk with bound D-aspartate at 2.8 Å resolution. Comparison of the L- and D-aspartate bound GltTk structures revealed that D-aspartate is accommodated with only minor rearrangements in the structure of the binding site. The structure explains how the geometrically different molecules L- and D-aspartate are recognized and transported by the protein in the same way.

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Elevator mechanism of aspartate (glutamate) transport across the membrane

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314085088 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C1491-C1491

Author(s):

Albert Guskov ◽

Sonja Jensen ◽

Stephan Rempel ◽

Inga Hänelt ◽

Dirk Slotboom

Keyword(s):

Crystal Structure ◽

Binding Site ◽

Binding Sites ◽

Glutamate Transporter ◽

Sodium Ions ◽

Binding Residue ◽

Sodium Binding ◽

Structural Insight ◽

Global Similarity ◽

Conserved Residue

Archaeal homologues of human neuronal glutamate transporter catalyze the coupled uptake of aspartate and three sodium ions. After the delivery of the substrate and sodium ions in the cytoplasm the empty binding site must reorient to the outward-facing conformation to reset the transporter. Here we present a crystal structure of the substrate-free transporter GltTk from Thermococcus kodakarensis, resolved at 3 Å resolution [1]. Despite the global similarity to the previously resolved structures of aspartate transporter GltPh, there are tremendous rearrangements in the substrate-binding site. The key binding residue Arg401 moves in and partially occupies the substrate's position, while the rotation of another conserved residue Met314 completely destroys the geometry of the sodium-binding sites. This structure provides direct structural insight in the mechanism of the essential reorientation step in the translocation cycle for this type of transporters.

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Activation of glutamate transporters in rods inhibits presynaptic calcium currents

Visual Neuroscience ◽

10.1017/s0952523803205095 ◽

2003 ◽

Vol 20 (5) ◽

pp. 557-566 ◽

Cited By ~ 14

Author(s):

KATALIN RABL ◽

ERIC J. BRYSON ◽

WALLACE B. THORESON

Keyword(s):

Glutamate Receptor ◽

Metabotropic Glutamate Receptor ◽

Imaging Techniques ◽

Glutamate Transporter ◽

Glutamate Transporters ◽

Glutamate Transport ◽

Calcium Currents ◽

Feedback Signal ◽

Group I ◽

Presynaptic Calcium

We found that L-glutamate (L-Glu) inhibits L-type Ca2+currents (ICa) in rod photoreceptors. This inhibition was studied in isolated rods or rods in retinal slices from tiger salamander using perforated patch whole cell recordings and Cl−-imaging techniques. Application of L-Glu inhibitedICaby ∼20% at 0.1 mM and ∼35% at 1 mM. L-Glu also produced an inward current that reversed aroundECl. The metabotropic glutamate receptor (mGluR) agonists t-ADA (Group I), DCG-IV (Group II), and L-AP4 (Group III) had no effect onICa. However, the glutamate transport inhibitor, TBOA (0.1 mM), prevented L-Glu from inhibitingICa. D-aspartate (D-Asp), a glutamate transporter substrate, also inhibitedICawith significantly more inhibition at 1 mM than 0.1 mM. Using Cl−imaging, L-Glu (0.1–1 mM) and D-Asp (0.1–1 mM) were found to stimulate a Cl−efflux from terminals of isolated rods whereas the ionotropic glutamate receptor agonists NMDA, AMPA, and kainate and the mGluR agonist, 1S,3R-ACPD, did not. Glutamate-evoked Cl−effluxes were blocked by the glutamate transport inhibitors TBOA and DHKA. Cl−efflux inhibits Ca2+channel activity in rod terminals (Thoreson et al. (2000),Visual Neuroscience17, 197). Consistent with the possibility that glutamate-evoked Cl−efflux may play a role in the inhibition, reducing intraterminal Cl−prevented L-Glu from inhibitingICa. In summary, the results indicate that activation of glutamate transporters inhibitsICain rods possibly as a consequence of Cl−efflux. The neurotransmitter L-Glu released from rod terminals might thus provide a negative feedback signal to inhibit further L-Glu release.

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Investigation of the allosteric coupling mechanism in a glutamate transporter homolog via unnatural amino acid mutagenesis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1907852116 ◽

2019 ◽

Vol 116 (32) ◽

pp. 15939-15946 ◽

Cited By ~ 4

Author(s):

Erika A. Riederer ◽

Francis I. Valiyaveetil

Keyword(s):

Amino Acid ◽

Binding Site ◽

Glutamate Transporter ◽

Glutamate Transporters ◽

Unnatural Amino Acid ◽

Coupling Mechanism ◽

Molecular Sequence ◽

Unnatural Amino Acid Mutagenesis ◽

Sequence Of Events ◽

Amino Acid Mutagenesis

Glutamate transporters harness the ionic gradients across cell membranes for the concentrative uptake of glutamate. The sodium-coupled Asp symporter, GltPh is an archaeal homolog of glutamate transporters and has been extensively used to understand the transport mechanism. A critical aspect of the transport cycle in GltPh is the coupled binding of sodium and aspartate. Previous studies have suggested a major role for hairpin-2 (HP2), which functions as the extracellular gate for the aspartate binding site, in the coupled binding of sodium and aspartate to GltPh. In this study, we develop a fluorescence assay for monitoring HP2 movement by incorporating tryptophan and the unnatural amino acid, p-cyanophenylalanine into GltPh. We use the HP2 assays to show that HP2 opening with Na+ follows an induced-fit mechanism. We also determine how residues in the substrate binding site affect the opening and closing of HP2. Our data, combined with previous studies, provide the molecular sequence of events in the coupled binding of sodium and aspartate to GltPh.

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Estimating the glutamate transporter surface density in mouse hippocampal astrocytes

10.1101/2021.05.08.443234 ◽

2021 ◽

Author(s):

Anca Radulescu ◽

Cassandra Williams ◽

Gabrielle Todd ◽

Alex Lemus ◽

Haley Chesbro ◽

...

Keyword(s):

Surface Density ◽

Glutamate Transporter ◽

Reaction Diffusion ◽

Glutamate Transporters ◽

Synaptic Cleft ◽

Average Density ◽

Mouse Hippocampus ◽

Ampa And Nmda Receptors ◽

Hippocampal Astrocytes ◽

Spatial Specificity

Glutamate transporters preserve the spatial specificity of synaptic transmission by limiting glutamate diffusion away from the synaptic cleft, and prevent excitotoxicity by keeping the extracellular concentration of glutamate at low nanomolar levels. Glutamate transporters are abundantly expressed in astrocytes. Previous estimates in the rat hippocampus suggest that the surface density of glutamate transporters in astrocytic membranes is ~10,800 μm−2. Here, we estimate their surface density in astrocytic membranes of the mouse hippocampus, at different ages. By using realistic 3D Monte Carlo reaction-diffusion models, we show that varying the local glutamate transporter expression in astrocytes can alter profoundly the activation of extrasynaptic AMPA and NMDA receptors. Our findings show that the average density of astrocyte membranes and their surface density of glutamate transporters is higher in mice compared to rats, and increases with mouse age. There are stark differences in the density of expression of these molecules in different subcellular compartments, indicating that the extent to which astrocytes limit extrasynaptic glutamate diffusion depends not only on the level of astrocytic coverage, but also on the identity of the astrocyte compartment in contact with the synapse. Together, these findings provide information on the spatial distribution of glutamate transporters in the mouse hippocampus, which can be used in mathematical models of the spatiotemporal profile of extracellular glutamate after synaptic release.

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Characterization of vesicular glutamate transporter in pancreatic α- and β-cells and its regulation by glucose

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00333.2002 ◽

2003 ◽

Vol 284 (5) ◽

pp. G808-G814 ◽

Cited By ~ 39

Author(s):

Liqun Bai ◽

Xiaohong Zhang ◽

Fayez K. Ghishan

Keyword(s):

Glucose Concentration ◽

Glutamate Release ◽

Glutamate Transporter ◽

Glutamate Transporters ◽

Glutamate Transport ◽

Vesicular Glutamate Transporter ◽

Β Cells ◽

Rate Limiting Step ◽

Low Glucose ◽

Vesicular Glutamate Transporters

Glutamate has been suggested to play an important role in the release of insulin and glucagon from pancreatic cells via exocytosis. Vesicular glutamate transporter is a rate-limiting step for glutamate release and is involved in the glutamate-evoked exocytosis. Two vesicular glutamate transporters (VGLUT1 and -2) have recently been cloned from the brain. In this report, we first functionally characterized vesicular glutamate transporter in cultured pancreatic α- and β-cells, and then detected mRNA expression of VGLUT1 and -2 in these cells. We also investigated the effect of high or low level of glucose on vesicular glutamate transport in cultured pancreas cells. Our results suggest that both α- and β-cells contain functional vesicular glutamate transporter. The transport characteristics are similar to the cloned neuronal VGLUT1 and -2 in regard to ATP dependence, substrate specificity, kinetics, and chloride dependence. VGLUT2 mRNA is expressed in both α- and β-cells, whereas VGLUT1 is only expressed in β-cells. High (12.8 mM) and low (2.8 mM) concentrations of glucose increased vesicular glutamate transport in β- and α-cells, respectively. VGLUT2 mRNA was significantly increased in β- and α-cells by high and low glucose concentration, respectively. This increase in VGLUT2 mRNA was suppressed by actinomycin D. We conclude that both α- and β-cells possess functional vesicular glutamate transporters regulated by alteration in glucose concentration, partly via the transcriptional mechanism.

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Faculty Opinions recommendation of Crystal structure of the ADP-dependent glucokinase from Pyrococcus horikoshii at 2.0-A resolution: a large conformational change in ADP-dependent glucokinase.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1009782.135407 ◽

2002 ◽

Author(s):

Patricia C Weber

Keyword(s):

Crystal Structure ◽

Conformational Change ◽

Pyrococcus Horikoshii ◽

Large Conformational Change

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Faculty Opinions recommendation of Crystal structure of an asymmetric trimer of a bacterial glutamate transporter homolog.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.14082956.793521065 ◽

2016 ◽

Author(s):

Louis De Felice

Keyword(s):

Crystal Structure ◽

Glutamate Transporter

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New Mechanistic Insight on the PIM-1 Kinase Inhibitor AZD1208 Using Multidrug Resistant Human Erythroleukemia Cell Lines and Molecular Docking Simulations

Current Topics in Medicinal Chemistry ◽

10.2174/1568026619666190509121606 ◽

2019 ◽

Vol 19 (11) ◽

pp. 914-926 ◽

Cited By ~ 2

Author(s):

Maiara Bernardes Marques ◽

Michael González-Durruthy ◽

Bruna Félix da Silva Nornberg ◽

Bruno Rodrigues Oliveira ◽

Daniela Volcan Almeida ◽

...

Keyword(s):

Molecular Docking ◽

Binding Site ◽

Cell Lines ◽

Chemotherapeutic Agents ◽

Atp Binding ◽

Human Leukemia ◽

Atp Binding Site ◽

Mechanistic Insight ◽

Multiple Drugs

Background:PIM-1 is a kinase which has been related to the oncogenic processes like cell survival, proliferation, and multidrug resistance (MDR). This kinase is known for its ability to phosphorylate the main extrusion pump (ABCB1) related to the MDR phenotype.Objective:In the present work, we tested a new mechanistic insight on the AZD1208 (PIM-1 specific inhibitor) under interaction with chemotherapy agents such as Daunorubicin (DNR) and Vincristine (VCR).Materials and Methods:In order to verify a potential cytotoxic effect based on pharmacological synergism, two MDR cell lines were used: Lucena (resistant to VCR) and FEPS (resistant to DNR), both derived from the K562 non-MDR cell line, by MTT analyses. The activity of Pgp was ascertained by measuring accumulation and the directional flux of Rh123. Furthermore, we performed a molecular docking simulation to delve into the molecular mechanism of PIM-1 alone, and combined with chemotherapeutic agents (VCR and DNR).Results:Our in vitro results have shown that AZD1208 alone decreases cell viability of MDR cells. However, co-exposure of AZD1208 and DNR or VCR reverses this effect. When we analyzed the ABCB1 activity AZD1208 alone was not able to affect the pump extrusion. Differently, co-exposure of AZD1208 and DNR or VCR impaired ABCB1 activity, which could be explained by compensatory expression of abcb1 or other extrusion pumps not analyzed here. Docking analysis showed that AZD1208 is capable of performing hydrophobic interactions with PIM-1 ATP- binding-site residues with stronger interaction-based negative free energy (FEB, kcal/mol) than the ATP itself, mimicking an ATP-competitive inhibitory pattern of interaction. On the same way, VCR and DNR may theoretically interact at the same biophysical environment of AZD1208 and also compete with ATP by the PIM-1 active site. These evidences suggest that AZD1208 may induce pharmacodynamic interaction with VCR and DNR, weakening its cytotoxic potential in the ATP-binding site from PIM-1 observed in the in vitro experiments.Conclusion:Finally, the current results could have a pre-clinical relevance potential in the rational polypharmacology strategies to prevent multiple-drugs resistance in human leukemia cancer therapy.

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