Modulation of Human Glutamate Transporter Activity by Phorbol Ester

Ganel, Raquelli; Crosson, Craig E.

doi:10.1046/j.1471-4159.1998.70030993.x

Cited by 50 publications

(21 citation statements)

References 35 publications

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“…In the present study, the primary astrocyte cultures were prepared from cerebral cortex of rats where GLT-1 represents the predominant glial transporter (O'Shea et al, 2006;Marco and Michael, 2004). It has been reported that activation of PKC caused internalization of GLT-1 and decreased in cellsurface expression of GLT-1 (Kalandadze et al, 2002); PKC activation also inhibited the traffic process of GLT-1 whereby PKC phosphorylation reduced the function of GLT-1 (Ganel and Crosson, 1998). The present study showed that treatment with diazoxide and iptakalim inhibited PKC phosphorylation, thus it is reasonable to presume that inhibiting PKC phosphorylation by KCOs facilitates glutamate uptake.…”

Section: Kcos Suppressed Pkc Activity In Mpp + -Treated Astrocyte Culsupporting

confidence: 61%

KATP Channel Openers Facilitate Glutamate Uptake by GluTs in Rat Primary Cultured Astrocytes

Sun

Zeng

Zhou

et al. 2007

Neuropsychopharmacol

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Increasing evidence, including from our laboratory, has revealed that opening of ATP sensitive potassium channels (K ATP channels) plays the neuronal protective roles both in vivo and in vitro. Thus K ATP channel openers (KCOs) have been proposed as potential neuroprotectants. Our previous studies demonstrated that K ATP channels could regulate glutamate uptake activity in PC12 cells as well as in synaptosomes of rats. Since glutamate transporters (GluTs) of astrocytes play crucial roles in glutamate uptake and K ATP channels are also expressed in astrocytes, the present study showed whether and how K ATP channels regulated the function of GluTs in primary cultured astrocytes. The results showed that nonselective KCO pinacidil, selective mitochondrial KCO diazoxide, novel, and blood-brain barrier permeable KCO iptakalim could enhance glutamate uptake, except for the sarcolemmal KCO P1075. Moreover pinacidil, diazoxide, and iptakalim reversed the inhibition of glutamate uptake induced by 1-methyl-4-phenylpyridinium (MPP + ). These potentiated effects were completely abolished by mitochondrial K ATP blocker 5-hydroxydecanoate. Furthermore, either diazoxide or iptakalim could inhibit MPP + -induced elevation of reactive oxygen species (ROS) and phosphorylation of protein kinases C (PKC). These findings are the first to demonstrate that activation of K ATP channel, especially mitochondrial K ATP channel, improves the function of GluTs in astrocytes due to reducing ROS production and downregulating PKC phosphorylation. Therefore, the present study not only reveals a novel pharmacological profile of KCOs as regulators of GluTs, but also provides a new strategy for neuroprotection.

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Section: Kcos Suppressed Pkc Activity In Mpp + -Treated Astrocyte Culsupporting

confidence: 61%

KATP Channel Openers Facilitate Glutamate Uptake by GluTs in Rat Primary Cultured Astrocytes

Sun

Zeng

Zhou

et al. 2007

Neuropsychopharmacol

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“…29 -31). The effects of PKC activators on the activity of GLT-1 are varied and may depend on cellular environment (12)(13)(14)(15). Therefore, we initially examined the effect of shortterm activation of PKC on the cell surface expression of GLT-1 using primary co-cultures of neurons and astrocytes derived from embryonic rat cortex.…”

Section: Effects Of a Pkc Activator On Cell Surface Expression Andmentioning

confidence: 99%

“…For example, activation of PKC causes an increase in activity when GLT-1 is expressed in HeLa cells using vaccinia virus (12) but has no effect on the activity of GLT-1 stably transfected into HeLa cells or two other peripheral cell lines (13). In a cell line that endogenously expresses the human variant of GLT-1 (Y-79 human retinoblastoma), activation of PKC causes a decrease in activity by increasing the K m value for transport (14). In a preliminary study, activation of PKC caused a decrease in GLT-1 activity in stably transfected Madin-Darby canine kidney cells (15).…”

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confidence: 99%

Protein Kinase C Activation Decreases Cell Surface Expression of the GLT-1 Subtype of Glutamate Transporter

Kalandadze

Robinson

2002

Journal of Biological Chemistry

135

136

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Na؉ -dependent glutamate transporters are required for the clearance of extracellular glutamate and influence both physiological and pathological effects of this excitatory amino acid. In the present study, the effects of a protein kinase C (PKC) activator on the cell surface expression and activity of the GLT-1 subtype of glutamate transporter were examined in two model systems, primary co-cultures of neurons and astrocytes that endogenously express GLT-1 and C6 glioma cells transfected with GLT-1. In both systems, activation of PKC with phorbol ester caused a decrease in GLT-1 cell surface expression. This effect is opposite to the one observed for the EAAC1 subtype of glutamate transporter (Davis, K. E., Straff, D. J., Weinstein, E. A., Bannerman, P. G., Correale, D. M., Rothstein, J. D., and Robinson, M. B. (1998) J. Neurosci. 18, 2475-2485). Several recombinant chimeric proteins between GLT-1 and EAAC1 transporter subtypes were generated to identify domains required for the subtype-specific redistribution of GLT-1. We identified a carboxyl-terminal domain consisting of 43 amino acids (amino acids 475-517) that is required for PKC-induced GLT-1 redistribution. Mutation of a non-conserved serine residue at position 486 partially attenuated but did not completely abolish the PKC-dependent redistribution of GLT-1. Although we observed a phorbol ester-dependent incorporation of 32 P into immunoprecipitable GLT-1, mutation of serine 486 did not reduce this signal. We also found that chimeras containing the first 446 amino acids of GLT-1 were not functional unless amino acids 475-517 of GLT-1 were also present. These non-functional transporters were not as efficiently expressed on the cell surface and migrated to a smaller molecular weight, suggesting that a subtypespecific interaction is required for the formation of functional transporters. These studies demonstrate a novel effect of PKC on GLT-1 activity and define a unique carboxyl-terminal domain as an important determinant in cellular localization and regulation of GLT-1.Glutamate is the predominant excitatory neurotransmitter in the central nervous system (CNS) 1 (1, 2) and is removed from the synaptic cleft by sodium-dependent glutamate transport activity. This activity is mediated by a family of five subtypes of transporters that share up to 60% sequence identity (for reviews, see Refs. 3-5). Expression of these transporters is generally restricted to particular cell types and brain regions. Two subtypes, GLT-1 and GLAST, are astroglial and two others, EAAC1 and EAAT4, are neuronal. Expression of the fifth transporter, EAAT5, is restricted to the retina (for reviews, see Refs. 4 and 5). Both the neuronal and glial glutamate transporters control the amplitude and/or duration of synaptic responses (6, 7) and prevent an extracellular accumulation of this potential excitotoxin (8 -10). For several different reasons, it is thought that the glial transporter, GLT-1, may represent the predominant route for the clearance of extracellular glutamate in forebrain (f...

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“…Although Casado and colleagues originally suggested that activation of PKC increases activity in GLT-1-transfected HeLA cells (Casado et al, 1993), we were unable to replicate this result (Tan et al, 1999). In primary cultures derived from rat brain and Y-79 human retinoblastoma cells that endogenously express GLT-1, activation of PKC rapidly (within min) decreases GLT-1-mediated transport activity and reduces the amount of GLT-1 that is observed at the plasma membrane (Ganel andCrosson, 1998 Kalandadze et al, 2002;Zhou and Sutherland, 2004;Guillet et al, 2005). Similar effects have been observed in C6 glioma cells or Madin-Darby canine kidney cells transfected with GLT-1 (Carrick and Dunlop, 1999;Kalandadze et al, 2002;Zhou and Sutherland, 2004).…”

Section: Introductionmentioning

confidence: 99%

Internalization and degradation of the glutamate transporter GLT-1 in response to phorbol ester

Susarla

Robinson

2008

Neurochemistry International

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Activation of protein kinase C (PKC) decreases the activity and cell surface expression of the predominant forebrain glutamate transporter, GLT-1. In the present study, C6 glioma were used as a model system to define the mechanisms that contribute to this decrease in cell surface expression and to determine the fate of internalized transporter. As was previously observed, phorbol 12-myristate 13-acetate (PMA) caused a decrease in biotinylated GLT-1. This effect was blocked by sucrose or by co-expression with a dominant-negative variant of dynamin 1, and it was attenuated by co-expression with a dominant-negative variant of the clathrin heavy chain. Depletion of cholesterol with methyl-β-cyclodextrin, co-expression with a dominant-negative caveolin-1 mutant (Cav1/S80E), co-expression with dominant-negative variants of Eps15 (epidermal-growth-factor receptor pathway substrate clone 15), or co-expression with dominant-negative Arf6 (T27N) had no effect on the PMA-induced loss of biotinylated GLT-1. Long-term treatment with PMA caused a time-dependent loss of biotinylated GLT-1 and decreased the levels of GLT-1 protein. Inhibitors of lysosomal degradation (chloroquine or ammonium chloride) or co-expression with a dominantnegative variant of a small GTPase implicated in trafficking to lysosomes (Rab7) prevented the PMAinduced decrease in protein and caused an intracellular accumulation of GLT-1. These results suggest that the PKC-induced redistribution of GLT-1 is dependent upon clathrin-mediated endocytosis. These studies identify a novel mechanism by which the levels of GLT-1 could be rapidly downregulated via lysosomal degradation. The possibility that this mechanism may contribute to the loss of GLT-1 observed after acute insults to the CNS is discussed.

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Modulation of Human Glutamate Transporter Activity by Phorbol Ester

Cited by 50 publications

References 35 publications

KATP Channel Openers Facilitate Glutamate Uptake by GluTs in Rat Primary Cultured Astrocytes

KATP Channel Openers Facilitate Glutamate Uptake by GluTs in Rat Primary Cultured Astrocytes

Protein Kinase C Activation Decreases Cell Surface Expression of the GLT-1 Subtype of Glutamate Transporter

Internalization and degradation of the glutamate transporter GLT-1 in response to phorbol ester

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