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

Abstract

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...

EAAT2 regulation and splicing: relevance to psychiatric and neurological disorders

“…92 In primary neuron-astrocyte co-cultures, activation of PKC with phorbol esters led to a decrease in GLT1 protein at the cell surface without altering total GLT1 protein content. 93 This decrease in membrane expression was accompanied by a decrease in transport activity mediated by GLT1. Similarly, decreased translocation of PKC to the membrane has been demonstrated in rat brain slices and cell culture models following administration of lithium.…”

“…92 More recent studies have shown that PKC-a interacts with GLT1 to regulate its activity and possibly trafficking of GLT1 to the membrane. 93,94 In addition, brain derived neurotrophic factor (BDNF), a molecule that has been consistently implicated in mood disorders, causes upregulation of GLT1 expression in differentiated astrocytes that is accompanied by increased glutamate uptake. 95 This effect appears to be specific to BDNF and GLT1, as other neurotrophic factors did not influence GLT1 expression and BDNF had no effect on expression of the other glial glutamate transporter, GLAST.…”

Hallucinogen-Induced UP States in the Brain Slice of Rat Prefrontal Cortex: Role of Glutamate Spillover and NR2B-NMDA Receptors

“…Since glutamate uptake by transporters is known to be a major limiting factor on spillover under normal conditions in brain slice (Diamond, 2001;Huang and Bergles, 2004), their blockade is known to increase extracellular glutamate (Melendez et al, 2005), and G q -coupled mechanisms can decrease surface expression of glutamate transporters (Kalandadze et al, 2002), we tested whether blocking glutamate uptake with a relatively low concentration of TBOA could enhance the occurrence of late EPSCs similarly to psychedelic hallucinogens. Figure 5 shows that TBOA (30 mM, 3-5 min) only minimally enhanced late EPSCs.…”

Regulation of Neuronal Glutathione Synthesis

“…Phorbol 12-myristate 13-acetate (PMA) is a PKC activator and increases the cell surface expression and activity of EAAC1 (78), while decreasing those of GLT-1 (86). The carboxyl-terminal domain of EAAC1 is an intracellular tail and plays a critical role in trafficking to the membrane surface.…”