Technical Reference #1673

1673.

Stimulation of EAAC1 in C6 Glioma Cells by Store-Operated Calcium Influx Andrew Murphy; Alice Vines; Gethin J. McBean, University College Dublin, Biochimica et Biophysica Acta, 1788(1673), (2009)

Abstract:
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 μM) produced a time-dependent increase in the Vmax for D-[3H] aspartate transport that reached a maximum at 15 min (143% of control; Pb0.001) 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; Pb0.01). 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 μM) or SKF 96365 (10 μM) 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

Keywords:
Glutamate; EAAC1; calcium; thapsigargin; C6 glioma

Materials & Methods:
SKF 96365 thapsigargin anti-rabbit secondary antibody and all RIPA buffer components unless indicated otherwise were purchased from Sigma-Aldrich Dorset UK; protease inhibitors were purchased from Roche Dublin Ireland. 2-APB was from Tocris Avon UK; PMA Bis-X H-89 KN-93 wortmannin and genistein were all supplied by Merck Biosciences U.K. Fura Red (AM) and Fluo-3 were obtained from Invitrogen/Molecular Probes Eugene OR U.S.A. and were dissolved in DMSO (3% giving a final concentration of DMSO in each assay of 0.06%) prior to use. Rabbit anti-rat EAAC1 primary antiserum was obtained from Alpha Diagnostic International TX USA. Sulfo-NHSbiotin and UltraLink Immobilized Monomeric Avidin beads were obtained from Pierce (Rockford IL USA). 35 mm glass bottom culture dishes were obtained from MatTek Corporation MA U.S.A.

Microscopic Technique
Confocal Microscopy

Cell Type(s)
C6