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 subtype-specific 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.
Highlights
Na؉-dependent glutamate transporters are required for the clearance of extracellular glutamate and influence both physiological and pathological effects of this excitatory amino acid
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
When GLT-1 was introduced into C6 glioma cells, a central nervous system (CNS)-derived cell line that endogenously expresses the neuronal transporter, EAAC1, a PKC activator caused a decrease in the cell surface expression of GLT-1 and a decrease in GLT-1-mediated transport activity
Summary
Materials—DMEM, L-glutamine, and penicillin/streptomycin were obtained from Invitrogen (Gaithersburg, MD). Cells were grown in DMEM supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 units/ml penicillin, 100 g/ml streptomycin and maintained at 37 °C in 5% CO2 incubator. Cells were grown in a monolayer on 12-well plates and rinsed twice with 1 ml of warmed (37 °C) sodium- or choline-containing buffer prior to incubation with radioisotope (0.5 M L-[3H]Glu) in sodium- or cholinecontaining buffer for 5 min. Plates were rinsed with ice-cold phosphate-buffered saline containing 0.1 mM CaCl2 and 0.1 mM MgCl2 and were incubated in this same solution supplemented with 1 mg/ml sulfosuccinimidobiotin for 20 min at 4 °C. Following removal of the primary antibody, membranes were washed and incubated with horseradish peroxidase-linked donkey anti-rabbit IgG (1:5,000), and proteins were visualized by enhanced chemiluminescence. For proteins labeled with 32P, the dried gels were rehydrated in 50 mM ammonium bicarbonate for 30 min, and the proteins were transferred to a polyvinylidene fluoride membrane
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