Stimulation of EAAC1 in C6 glioma cells by store-operated calcium influx.
This study investigated how modulation of intracellular calcium alters the functional activity of the EAAC1 glutamate transporter in C6 glioma cells. Pre-incubation of C6 glioma cells with the endoplasmic reticulum Ca2+ ATP pump inhibitor, thapsigargin (10 microM) produced a time-dependent increase in the Vmax for D-[3H]aspartate transport that reached a maximum at 15 min (143% of control; P<0.001) that was accompanied by increased plasma membrane expression of EAAC1 and was blocked by inhibition of protein kinase C. Pre-incubation of C6 glioma cells with phorbol myristate-3-acetate (100 nM for 20 min) also caused a significant increase in the Vmax of sodium-dependent D-[3H]aspartate transport (190% of control; P<0.01). In contrast, in the absence of extracellular calcium, thapsigargin caused a significant inhibition in D-[3H]aspartate transport that was not mediated by protein kinase C. Blockade of store-operated calcium channels with 2-aminoethoxydiphenyl borate (50 microM) or SKF 96365 (10 microM) caused a net inhibition of D-[3H]aspartate uptake. Co-incubation of C6 glioma cells with both thapsigargin and 2-aminoethoxydiphenyl borate (but not SKF 96365) prevented the increase in D-[3H]aspartate transport that was observed in the presence of thapsigargin alone. Furthermore, 2-aminoethoxydiphenyl borate, but not SKF 96365, reduced the increase in intracellular calcium that occurred following pre-incubation of the cells with thapsigargin. It is concluded that, in C6 glioma cells, stimulation of EAAC1-mediated glutamate transport by thapsigargin is dependent on entry of calcium via the NSCC-1 subtype of store operated calcium channel and is mediated by protein kinase C. In contrast, in the absence of store operated calcium entry, thapsigargin inhibits transport.
Murphy
Andrew
A
UCD School of Biomolecular and Biomedical Science, Conway Institute for Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland.
Vines
Alice
A
McBean
Gethin J
GJ
D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism*; D045744:Cell Line, Tumor; D050580:Excitatory Amino Acid Transporter 3 / Q000378:metabolism*; D005110:Extracellular Space / Q000187:drug effects / Q000378:metabolism; D005910:Glioma / Q000378:metabolism*; D051379:Mice; D011493:Protein Kinase C / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D019284:Thapsigargin / Q000494:pharmacology
D050580:Excitatory Amino Acid Transporter 3; D047428:Protein Kinase Inhibitors; C493399:Slc1a1 protein, mouse; D001224:Aspartic Acid; D019284:Thapsigargin; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium