Measurements of [Ca2+] using fura-2 in glioma C6 cells expressing calretinin with GFP as a marker of transfection: no Ca2+-buffering provided by calretinin

Glioma C6 cells were transfected with a plasmid containing the calretinin ( CR) and green fluorescent protein (GFP) coding regions to analyze the effec t of CR's presence on [Ca2+](i). Positive transfectants were identified by the detection of GFP and [Ca2+](i) was measured using fura-2 as a probe. We found that neither the basic [Ca2+](i) nor activated [Ca2+](i) achieved by exposure to ionomycin, ADP or thapsigargin were affected by CR's presence in transfected cells, despite the ability of CR to bind Ca2+ as part of fus ion protein. The level of expressed CR was estimated as at least 1 mu M. Th e presented results suggest that CR's function is unlikely to be an intrace llular Ca2+-buffer and support the hypothesis that CR might be involved in a specific Ca2+-dependent process. The results of this work also show that the S65T mutant of GFP is compatible with fura-2 measurements of intracellu lar [Ca2+]. We have demonstrated that the presence of GFP, as a transfectio n marker of glioma C6 cells, does not disturb fura-2 fluorescence, the basa l or activated [Ca2+](i) in these cells. (C) 1999 Published by Elsevier Sci ence B.V, All rights reserved.