scholarly journals Rapid recycling of glutamate transporters on the astroglial surface

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Piotr Michaluk ◽  
Janosch Peter Heller ◽  
Dmitri A Rusakov
Keyword(s):  
Single Molecule ◽  
Metabotropic Glutamate Receptors ◽  
Glutamate Uptake ◽  
Glutamate Transporter ◽  
Lateral Diffusion ◽  
Synaptic Cleft ◽  
Surface Pattern ◽  
Membrane Turnover ◽  
C Terminus ◽  
Localisation Microscopy

Glutamate uptake by astroglial transporters confines excitatory transmission to the synaptic cleft. The efficiency of this mechanism depends on the transporter dynamics in the astrocyte membrane, which remains poorly understood. Here, we visualise the main glial glutamate transporter GLT1 by generating its pH-sensitive fluorescent analogue, GLT1-SEP. FRAP-based imaging shows that 70-75% of GLT1-SEP dwell on the surface of rat brain astroglia, recycling with a lifetime of ~22 s. Genetic deletion of the C-terminus accelerates GLT1-SEP membrane turnover while disrupting its surface pattern, as revealed by single-molecule localisation microscopy. Excitatory activity boosts surface mobility of GLT1-SEP, involving its C-terminus, metabotropic glutamate receptors, intracellular Ca2+ and calcineurin-phosphatase activity, but not the broad-range kinase activity. The results suggest that membrane turnover, rather than lateral diffusion, is the main 'redeployment' route for the immobile fraction (20-30%) of surface-expressed GLT1. This finding reveals an important mechanism helping to control extrasynaptic escape of glutamate.

scholarly journals Rapid recycling of glutamate transporters on the astroglial surface

2020 ◽  
Author(s):  
Piotr Michaluk ◽  
Janosch Heller ◽  
Dmitri A. Rusakov
Keyword(s):  
Metabotropic Glutamate Receptor ◽  
Glutamate Uptake ◽  
Glutamate Transporter ◽  
Lateral Diffusion ◽  
Synaptic Cleft ◽  
Receptor Activation ◽  
Surface Pattern ◽  
Membrane Turnover ◽  
C Terminus ◽  
Calcineurin Phosphatase

ABSTRACTGlutamate uptake by high-affinity astroglial transporters confines excitatory transmission to the synaptic cleft. The efficiency of this mechanism depends on the transporter dynamics in the astrocyte membrane, which remains poorly understood. Here, we visualise the main glial glutamate transporter GLT1 by generating its functional pH-sensitive fluorescent analogue, GLT1-SEP. Combining FRAP-based methods with molecular dissection shows that 70-75% of GLT1-SEP are expressed on the astroglial surface, recycling with a lifetime of only ~22 s. Genetic deletion of the C-terminus accelerates GLT1-SEP membrane turnover by ~60% while disrupting its molecule-resolution surface pattern as revealed by dSTORM. Excitatory activity boosts surface mobility of GLT1-SEP, involving its C-terminus, metabotropic glutamate receptor activation, intracellular Ca2+ signalling and calcineurin-phosphatase activity, but not the broad-range kinase activity. The results suggest that membrane turnover, rather than than lateral diffusion, is the main ‘redeployment’ route for the immobile fraction (20-30%) of surface-expressed GLT1. This reveals a novel mechanism by which the brain controls extrasynaptic glutamate escape, in health and disease.

scholarly journals K+ efflux through postsynaptic NMDA receptors suppresses local astrocytic glutamate uptake

2020 ◽  
Author(s):  
Olga Tyurikova ◽  
Pei-Yu Shih ◽  
Yulia Dembitskaya ◽  
Leonid P. Savtchenko ◽  
Thomas J. McHugh ◽  
...  
Keyword(s):  
Nmda Receptors ◽  
Decay Time ◽  
Glutamate Uptake ◽  
Glutamate Release ◽  
Glutamate Transporter ◽  
Synaptic Cleft ◽  
Conditional Knockout ◽  
Glutamate Signaling ◽  
Local Cell ◽  
Activity Dependent

AbstractGlutamatergic transmission in the hippocampus prompts K+ efflux through postsynaptic N-methyl-D-aspartate receptors (NMDARs). This K+ efflux depolarizes local presynaptic terminals, boosting glutamate release, but whether it also depolarizes local astrocytic processes, thus affecting glutamate uptake, remains unknown. Here, we find that the pharmacological blockade, or conditional knockout, of NMDARs suppresses the progressive use-dependent increase in the amplitude and decay time of the astrocytic glutamate transporter current (IGluT), whereas blocking the astrocytic inward-rectifying K+ channels prevents the decay time increase only. Glutamate spot-uncaging reveals that local astrocyte depolarization, rather than extracellular K+ rises on their own, reduces the amplitude and prolong the decay of IGluT. Biophysical simulations of a realistic 3D astrocyte confirm that local transient elevations of extracellular K+ can inhibit local glutamate uptake in fine astrocytic processes. We conclude that K+ efflux through postsynaptic NMDARs can transiently depolarize local cell membranes, which facilitates presynaptic release while reducing local glutamate uptake. Optical glutamate sensor imaging and a two-pathway test relate postsynaptic K+ efflux to enhanced extrasynaptic glutamate signaling. Thus, the frequency of synaptic discharges can control the way the network handles its synaptic signal exchange.Significance statementA long-standing controversy in cellular neuroscience is the question of what controls well-documented extrasynaptic actions of glutamate, given that in baseline conditions, the high-affinity astrocytic transporters form a non-saturable protection shield around the synaptic cleft. The use-dependent mechanism that enables glutamate to pass this transporter shield during sustained activity remains unknown. Earlier, we suggested that activity-dependent K+ efflux through postsynaptic NMDA receptors was partially responsible for activity-dependent facilitation of glutamate release. Here, we provide evidence that this K+ efflux also depolarizes perisynaptic astrocytic leaflets, which reduces local glutamate uptake, thus enabling extrasynaptic glutamate spillover. Our mechanistic insights into the use-dependent suppression of glutamate transport are relevant to various pathologies involving disruption of extracellular K+ homeostasis, such as epilepsy or migraine.

Voltage-Dependent Uptake Is a Major Determinant of Glutamate Concentration at the Cone Synapse: An Analytical Study

1998 ◽  
Vol 80 (4) ◽  
pp. 1951-1960 ◽  
Author(s):  
Botond Roska ◽  
Lubor Gaal ◽  
Frank S. Werblin
Keyword(s):  
Analytical Study ◽  
Glutamate Uptake ◽  
Glutamate Transporter ◽  
Light Response ◽  
Synaptic Cleft ◽  
Major Determinant ◽  
Glutamate Concentration ◽  
Voltage Dependent ◽  
Interdependent Relationships ◽  
Close Fit

Roska, Botond, Lubor Gaal, and Frank S. Werblin. Voltage-dependent uptake is a major determinant of glutamate concentration at the cone synapse: an analytical study. J. Neurophysiol. 80: 1951–1960, 1998. It was suggested that glutamate concentration at the synaptic terminal of the cones was controlled primarily by a voltage-dependent glutamate transporter and that diffusion played a less important role. The conclusion was based on the observation that the rate of glutamate concentration during the hyperpolarizing light response was dramatically slowed when the transporter was blocked with dihydrokainate although diffusion remained intact. To test the validity of this notion we constructed a model in which the balance among uptake, diffusion, and release determined the flow of glutamate into and out of the synaptic cleft. The control of glutamate concentration was assumed here to be determined by two relationships; 1) glutamate concentration is the integral over the synaptic volume of the rates of release, uptake, and diffusion, and 2) membrane potential is the integral over the membrane capacitance of the dark, leak, and transporter-gated chloride current. These relationships are interdependent because glutamate uptake via the transporter is voltage dependent and because the transporter-gated current is concentration dependent. The voltage and concentration dependence of release and uptake, as well as the light-elicited, transporter-gated, and leak currents were measured in other studies. All of these measurements were incorporated into our predictive model of glutamate uptake. Our results show a good quantitative fit between the predicted and the measured magnitudes and rates of change of glutamate concentration, derived from the two interdependent relationships. This close fit supports the validity of these two relationships as descriptors of the mechanisms underlying the control of glutamate concentration, it verifies the accuracy of the experimental data from which the functions used in these relationships were derived, and it lends further support to the notion that glutamate concentration is controlled primarily by uptake at the transporter.

Astrocytic group I mGluR-dependent potentiation of astrocytic glutamate and potassium uptake

2013 ◽  
Vol 109 (9) ◽  
pp. 2404-2414 ◽  
Author(s):  
Prakash Devaraju ◽  
Min-Yu Sun ◽  
Timothy L. Myers ◽  
Kelli Lauderdale ◽  
Todd A. Fiacco
Keyword(s):  
Charge Transfer ◽  
Metabotropic Glutamate Receptors ◽  
Glutamate Uptake ◽  
Hippocampal Slices ◽  
Glutamate Transporter ◽  
Metabotropic Glutamate ◽  
Group I ◽  
Group I Mglurs ◽  
G Protein Coupled

One of the most important functions of astrocytes is removal of glutamate released during synaptic transmission. Surprisingly, the mechanisms by which astrocyte glutamate uptake is acutely modulated remain to be clarified. Astrocytes express metabotropic glutamate receptors (mGluRs) and other G protein-coupled receptors (GPCRs), which are activated during neuronal activity. Here, we test the hypothesis that astrocytic group I mGluRs acutely regulate glutamate uptake by astrocytes in situ. This hypothesis was tested in acute mouse hippocampal slices. Activation of astrocytic mGluRs, using a tetanic high-frequency stimulus (HFS) applied to Schaffer collaterals, led to potentiation of the amplitude of the synaptically evoked glutamate transporter currents (STCs) and associated charge transfer without changes in kinetics. Similar potentiation of STCs was not observed in the presence of group I mGluR antagonists or the PKC inhibitor, PKC 19–36, suggesting that HFS-induced potentiation of astrocyte glutamate uptake is astrocytic group I mGluR and PKC dependent. Pharmacological stimulation of a transgenic GPCR (MrgA1R), expressed exclusively in astrocytes, also potentiated STC amplitude and charge transfer, albeit quicker and shorter lasting compared with HFS-induced potentiation. The amplitude of the slow, inward astrocytic current due to potassium (K+) influx was also enhanced following activation of the endogenous mGluRs or the astrocyte-specific MrgA1 Gq GPCRs. Taken together, these findings suggest that astrocytic group I mGluR activation has a synergistic, modulatory effect on the uptake of glutamate and K+.

Glutamate Uptake Controls Expression of a Slow Postsynaptic Current Mediated by mGluRs in Cerebellar Purkinje Cells

2002 ◽  
Vol 87 (4) ◽  
pp. 1974-1980 ◽  
Author(s):  
W. Reichelt ◽  
T. Knöpfel
Keyword(s):  
Glutamate Receptors ◽  
Metabotropic Glutamate Receptors ◽  
Metabotropic Glutamate Receptor ◽  
Glutamate Uptake ◽  
Glutamate Transporter ◽  
Excitatory Postsynaptic Current ◽  
Metabotropic Glutamate ◽  
Glutamatergic Synaptic Transmission ◽  
Cerebellar Purkinje Cells ◽  
Cell Synapse

At the cerebellar parallel fiber-Purkinje cell synapse, isolated presynaptic activity induces fast excitatory postsynaptic currents via ionotropic glutamate receptors while repetitive, high-frequency, presynaptic activity can also induce a slow excitatory postsynaptic current that is mediated by metabotropic glutamate receptors (mGluR1-EPSC). Here we investigated the involvement of glutamate uptake in the expression of the mGluR1-EPSC. Inhibitors of glutamate uptake led to a large increase of the mGluR1-EPSC. d-aspartate (0.4 mM) andl(−)-threo-3-hydroxyaspartate (0.4 mM) increased the mGluR1-EPSC ∼4.5 and ∼9-fold, respectively, while dihydrokainic acid (1 mM), had no significant effect on the mGluR1-EPSC.d-aspartate (0.4 mM) shifted the concentration-response curve of the depression of the mGluR1-EPSC by the low-affinity mGluR1 antagonist ( S)-a-Methyl-4-carboxyphenylglycine [( S)-MCPG] to higher concentrations and decreased the stimulus intensity and the number of necessary stimuli to evoke an mGluR1-EPSC. Depression of the mGluR1-EPSC by rapid pressure application of ( S)-MCPG at varying time intervals after tetanic stimulation of the parallel fibers indicated that the glutamate concentration in the peri- and extrasynaptic space decayed with time constants of 36 and 316 ms under control conditions and with inhibition of glutamate uptake, respectively. These results show that expression of the slow mGluR-mediated excitatory postsynaptic current is controlled by glutamate transporter activity. Thus in contrast to fast glutamatergic synaptic transmission, metabotropic glutamate receptor-mediated transmission is critically dependent on the activity and capacity of glutamate uptake.

scholarly journals GLT-1 overexpression attenuates bladder nociception and local/cross-organ sensitization of bladder nociception

2011 ◽  
Vol 300 (6) ◽  
pp. F1353-F1359 ◽  
Author(s):  
M. Yang ◽  
K. Roman ◽  
D.-F. Chen ◽  
Z.-G. Wang ◽  
Y. Lin ◽  
...  
Keyword(s):  
Glutamate Uptake ◽  
Glutamate Transporter ◽  
Vehicle Group ◽  
Trinitrobenzene Sulfonic Acid ◽  
Nociceptive Response ◽  
Bladder Distension ◽  
Beneficial Effects ◽  
Visceromotor Response ◽  
Colonic Distension ◽  
Uptake Activity

Glutamatergic pathways mediate transmission of pain. Strategies to reduce glutamatergic neurotransmission may have beneficial effects to mitigate nociception. Recent work revealed that overexpression of the astrocytic glutamate transporter (GLT-1) by transgenic or pharmacologic approaches produced a diminished visceral nociceptive response to colonic distension. The purpose of this study was to determine the effect of GLT-1 overexpression on the visceromotor response to bladder distension. Increased glutamate uptake activity produced by 1-wk ceftriaxone (CTX) treatment attenuated 60–64% the visceromotor response to graded bladder distension compared with vehicle-treated mice. One-hour pretreatment with selective GLT-1 antagonist dihydrokainate reversed the blunted visceromotor response to bladder distension produced by 1-wk CTX, suggesting that GLT-1 overexpression mediated the analgesic effect of CTX. Moreover, sensitization of the visceromotor response to bladder distension produced by local bladder irritation (acrolein) was also attenuated by 1-wk CTX treatment. A model of cross-organ sensitization of bladder visceromotor response to distension was next studied to determine whether increased expression of GLT-1 can mitigate colon to bladder sensitization. Intracolonic trinitrobenzene sulfonic acid (TNBS) administered 1 h before eliciting the visceromotor response to graded bladder distension produced a 75–138% increase in visceromotor response compared with animals receiving intracolonic vehicle. In marked contrast, animals treated with 1-wk CTX + intracolonic TNBS showed no enhanced visceromotor response compared with the 1-wk vehicle + intracolonic vehicle group. The study suggests that GLT-1 overexpression attenuates the visceromotor response to bladder distension and both local irritant-induced and cross-organ-sensitized visceromotor response to bladder distension.

scholarly journals Ischemic Preconditioning Reveals that GLT1/EAAT2 Glutamate Transporter is a Novel PPARγ Target Gene Involved in Neuroprotection

2007 ◽  
Vol 27 (7) ◽  
pp. 1327-1338 ◽  
Author(s):  
Cristina Romera ◽  
Olivia Hurtado ◽  
Judith Mallolas ◽  
Marta P Pereira ◽  
Jesús R Morales ◽  
...  
Keyword(s):  
Nervous System ◽  
Ischemic Preconditioning ◽  
Transcriptional Activity ◽  
Target Gene ◽  
Glutamate Uptake ◽  
Glutamate Release ◽  
Glutamate Transporter ◽  
Glutamate Transport ◽  
Extracellular Glutamate ◽  
Pparγ Agonist

Excessive levels of extracellular glutamate in the nervous system are excitotoxic and lead to neuronal death. Glutamate transport, mainly by glutamate transporter GLT1/EAAT2, is the only mechanism for maintaining extracellular glutamate concentrations below excitotoxic levels in the central nervous system. We recently showed that neuroprotection after experimental ischemic preconditioning (IPC) involves, at least partly, the upregulation of the GLT1/EAAT2 glutamate transporter in astrocytes, but the mechanisms were unknown. Thus, we decided to explore whether activation of the nuclear receptor peroxisome proliferator-activated receptor (PPAR)γ, known for its antidiabetic and antiinflammatory properties, is involved in glutamate transport. First, we found that the PPARγ antagonist T0070907 inhibits both IPC-induced tolerance and reduction of glutamate release after lethal oxygen-glucose deprivation (OGD) (70.1% ± 3.4% versus 97.7% ± 5.2% of OGD-induced lactate dehydrogenase (LDH) release and 61.8% ± 5.9% versus 85.9% ± 7.9% of OGD-induced glutamate release in IPC and IPC + T0070907 1 μmol/L, respectively, n = 6 to 12, P < 0.05), as well as IPC-induced astrocytic GLT-1 overexpression. IPC also caused an increase in nuclear PPARγ transcriptional activity in neurons and astrocytes (122.1% ± 8.1% and 158.6% ± 22.6% of control PPARγ transcriptional activity, n = 6, P < 0.05). Second, the PPARγ agonist rosiglitazone increased both GLT-1/EAAT2 mRNA and protein expression and [3H]glutamate uptake, and reduced OGD-induced cell death and glutamate release (76.3% ± 7.9% and 65.5% ± 15.1% of OGD-induced LDH and glutamate release in rosiglitazone 1 μmol/l, respectively, n = 6 to 12, P < 0.05). Finally, we have identified six putative PPAR response elements (PPREs) in the GLT1/EAAT2 promoter and, consistently, rosiglitazone increased fourfold GLT1/EAAT2 promoter activity. All these data show that the GLT1/EAAT2 glutamate transporter is a target gene of PPARγ leading to neuroprotection by increasing glutamate uptake.

Effect of Acute Exposure to Ammonia on Glutamate Transport in Glial Cells Isolated From the Salamander Retina

2001 ◽  
Vol 86 (2) ◽  
pp. 836-844 ◽  
Author(s):  
Dominic Mort ◽  
Païkan Marcaggi ◽  
James Grant ◽  
David Attwell
Keyword(s):  
Glial Cells ◽  
Glutamate Uptake ◽  
Ph Dependence ◽  
Glutamate Transporter ◽  
Ammonia Concentration ◽  
Ammonia Level ◽  
Glutamate Transport ◽  
Acute Exposure ◽  
Ph Change ◽  
Glutamine Synthesis

A rise of brain ammonia level, as occurs in liver failure, initially increases glutamate accumulation in neurons and glial cells. We investigated the effect of acute exposure to ammonia on glutamate transporter currents in whole cell clamped glial cells from the salamander retina. Ammonia potentiated the current evoked by a saturating concentration ofl-glutamate, and decreased the apparent affinity of the transporter for glutamate. The potentiation had a Michaelis-Menten dependence on ammonia concentration, with a K m of 1.4 mM and a maximum potentiation of 31%. Ammonia also potentiated the transporter current produced by d-aspartate. Potentiation of the glutamate transport current was seen even with glutamine synthetase inhibited, so ammonia does not act by speeding glutamine synthesis, contrary to a suggestion in the literature. The potentiation was unchanged in the absence of Cl− ions, showing that it is not an effect on the anion current gated by the glutamate transporter. Ammonium ions were unable to substitute for Na+in driving glutamate transport. Although they can partially substitute for K+ at the cation counter-transport site of the transporter, their occupancy of these sites would produce a potentiation of <1%. Ammonium, and the weak bases methylamine and trimethylamine, increased the intracellular pH by similar amounts, and intracellular alkalinization is known to increase glutamate uptake. Methylamine and trimethylamine potentiated the uptake current by the amount expected from the known pH dependence of uptake, but ammonia gave a potentiation that was larger than could be explained by the pH change, and some potentiation of uptake by ammonia was still seen when the internal pH was 8.8, at which pH further alkalinization does not increase uptake. These data suggest that ammonia speeds glutamate uptake both by increasing cytoplasmic pH and by a separate effect on the glutamate transporter. Approximately two-thirds of the speeding is due to the pH change.

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