Glutamate forward and reverse transport: From molecular mechanism to transporter‐mediated release after ischemia

Grewer, Christof; Gameiro, Armanda; Zhang, Zhou; Tao, Zhen; Braams, Simona; Rauen, Thomas

doi:10.1002/iub.98

Cited by 144 publications

(125 citation statements)

References 87 publications

(152 reference statements)

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“…Finally, the free energy profile suggests that the reverse transfer of aspartate from the IC to the EC side is not less favorable than the forward passage. The substrate translocation cycle has indeed been observed to be reversible (17). Fig.…”

Section: Resultsmentioning

confidence: 82%

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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Section: Resultsmentioning

confidence: 82%

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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“…Under these conditions, glycolysis may persist after oxygen has been depleted, but the reduction of oxidative metabolism of glucose leads to decreased ATP levels, while ADP and AMP levels increase (Hertz, 2008), causing a disruption of ionic homeostasis (Hansen, 1985), opening of anion channels (Kimelberg and Mongin, 1998), plasma membrane depolarization (Lipton, 1999), release of glutamate through astrocytic hemichannels (Ye et al, 2003) and downregulation of glutamate transporters (Harvey et al, 2011). The impairment of glutamate transporters, in addition to their operation in the reverse mode, leads to an accumulation of glutamate in the extracellular space (Grewer et al, 2008) and a consequent overactivation of postsynaptic glutamate receptors. Under these conditions, necrotic cell death occurs at the core region, while in the penumbra region the availability of ATP allows a delayed cell death by apoptosis (Broughton et al, 2009).…”

Section: Brain Ischemiamentioning

confidence: 99%

“…During stroke, membrane depolarization due to ATP breakdown leads to an increase in the release of glutamate, and the lack of energy blocks the reuptake of the excitatory amino acids at the synapse, leading to an extracellular accumulation of glutamate (Rossi et al, 2000;Grewer et al, 2008). The reversal of the glutamate transporters under these conditions (Rossi et al, 2000;Grewer et al, 2008) further contributes to the extracellular accumulation of glutamate, with a consequent toxic overactivation of postsynaptic glutamate receptors (excitotoxicity) (Olney, 1969;Simon et al, 1984;Choi et al, 1987;Ferreira et al, 1996Ferreira et al, , 1998Martel et al, 2012).…”

Section: Role Of Glutamatementioning

confidence: 99%

“…The reversal of the glutamate transporters under these conditions (Rossi et al, 2000;Grewer et al, 2008) further contributes to the extracellular accumulation of glutamate, with a consequent toxic overactivation of postsynaptic glutamate receptors (excitotoxicity) (Olney, 1969;Simon et al, 1984;Choi et al, 1987;Ferreira et al, 1996Ferreira et al, , 1998Martel et al, 2012). Upon oxygen and glucose deprivation (OGD), a well established in vitro model of global ischemia, glutamate is massively released by neurons, and the resulting increase in the intracellular Ca 2+ concentration ([Ca 2+ ] i ) (Goldberg and Choi, 1993) causes a delayed neuronal cell death (calcium overload hypothesis) (Manev et al, 1989).…”

Section: Role Of Glutamatementioning

confidence: 99%

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Role of the ubiquitin–proteasome system in brain ischemia: Friend or foe?

Caldeira

Salazar

Curcio

et al. 2014

Progress in Neurobiology

114

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A B S T R A C TThe ubiquitin-proteasome system (UPS) is a catalytic machinery that targets numerous cellular proteins for degradation, thus being essential to control a wide range of basic cellular processes and cell survival. Degradation of intracellular proteins via the UPS is a tightly regulated process initiated by tagging a target protein with a specific ubiquitin chain. Neurons are particularly vulnerable to any change in protein composition, and therefore the UPS is a key regulator of neuronal physiology. Alterations in UPS activity may induce pathological responses, ultimately leading to neuronal cell death. Brain ischemia triggers a complex series of biochemical and molecular mechanisms, such as an inflammatory response, an exacerbated production of misfolded and oxidized proteins, due to oxidative stress, and the breakdown of cellular integrity mainly mediated by excitotoxic glutamatergic signaling. Brain ischemia also damages protein degradation pathways which, together with the overproduction of damaged proteins and consequent upregulation of ubiquitin-conjugated proteins, contribute to the accumulation of ubiquitincontaining proteinaceous deposits. Despite recent advances, the factors leading to deposition of such aggregates after cerebral ischemic injury remain poorly understood. This review discusses the current knowledge on the role of the UPS in brain function and the molecular mechanisms contributing to UPS dysfunction in brain ischemia with consequent accumulation of ubiquitin-containing proteins. Chemical inhibitors of the proteasome and small molecule inhibitors of deubiquitinating enzymes, which promote the degradation of proteins by the proteasome, were both shown to provide neuroprotection in brain ischemia, and this apparent contradiction is also discussed in this review. ß

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“…Laser pulse photolysis experiments were performed as previously described. 32 4-Methoxy-7-nitroindolinyl (MNI)-caged glutamate (TOCRIS) was applied to the cells using a small quartz tube and then photolyzed with a light flash (355 nm Nd:YAG laser, Minilite series; Continuum, Santa Clara, CA) delivered through an optical fiber (350 μm diameter). The MNI-caged glutamate solutions were freshly prepared before starting each experiment.…”

Section: ■ Conclusionmentioning

confidence: 99%

Protonation State of a Conserved Acidic Amino Acid Involved in Na⁺ Binding to the Glutamate Transporter EAAC1

Mwaura

Tao

James

et al. 2012

ACS Chem. Neurosci.

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Substrate transport by glutamate transporters is coupled to the co-transport of 3 Na + ions and counter-transport of 1 K + ion. The highly conserved Asp454, which may be negatively charged, is of interest as its side chain may coordinate cations and/or contribute to charge compensation. Mutation to the nonionizable Asn resulted in a transporter that no longer catalyzed forward transport. However, Na + /glutamate exchange was still functional, as demonstrated by the presence of transient currents following rapid substrate application and voltage jumps. While the kinetics of Na + / glutamate exchange were slowed, the apparent valence (z) of the charge moved in EAAC1 D454N (0.71) was similar to that of EAAC1 WT (0.64). Valences calculated using the Poisson−Boltzmann equation were close to the experimental values for EAAC1 D454N (0.55), and with D454 protonated (0.45). In addition, pK a calculations performed for the bacterial homologue GltPh revealed a highly perturbed pK a (7.6 to >14) for D405 residue (analogous to D454), consistent with this site being protonated at physiological pH. In contrast to the D454N mutation, substitution to alanine resulted in a transporter that still bound glutamate, but could not translocate it. The results are consistent with molecular dynamics simulations, showing that the alanine but not the asparagine mutation resulted in defective Na + coordination. Our results raise the possibility that the protonated state of D454 supports transporter function.

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Glutamate forward and reverse transport: From molecular mechanism to transporter‐mediated release after ischemia

Cited by 144 publications

References 87 publications

Molecular simulations elucidate the substrate translocation pathway in a glutamate transporter

Molecular simulations elucidate the substrate translocation pathway in a glutamate transporter

Role of the ubiquitin–proteasome system in brain ischemia: Friend or foe?

Protonation State of a Conserved Acidic Amino Acid Involved in Na⁺ Binding to the Glutamate Transporter EAAC1

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Glutamate forward and reverse transport: From molecular mechanism to transporter‐mediated release after ischemia

Cited by 144 publications

References 87 publications

Molecular simulations elucidate the substrate translocation pathway in a glutamate transporter

Molecular simulations elucidate the substrate translocation pathway in a glutamate transporter

Role of the ubiquitin–proteasome system in brain ischemia: Friend or foe?

Protonation State of a Conserved Acidic Amino Acid Involved in Na+ Binding to the Glutamate Transporter EAAC1

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Protonation State of a Conserved Acidic Amino Acid Involved in Na⁺ Binding to the Glutamate Transporter EAAC1