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

被引:15
作者
Billing-Marczak, K
Przybyszewska, M
Kuznicki, J
机构
[1] M Nencki Inst Expt Biol, Dept Mol & Cellular Neurobiol, PL-02093 Warsaw, Poland
[2] Ctr Canc, Dept Cell Biol, PL-02781 Warsaw, Poland
来源
BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH | 1999年 / 1449卷 / 02期
关键词
calretinin; Ca2+] imaging; green fluorescent protein; transfection;
D O I
10.1016/S0167-4889(99)00010-5
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Glioma C6 cells were transfected with a plasmid containing the calretinin (CR) and green fluorescent protein (GFP) coding regions to analyze the effect 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 fusion protein. The level of expressed CR was estimated as at least 1 mu M. The presented results suggest that CR's function is unlikely to be an intracellular 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 intracellular [Ca2+]. We have demonstrated that the presence of GFP, as a transfection marker of glioma C6 cells, does not disturb fura-2 fluorescence, the basal or activated [Ca2+](i) in these cells. (C) 1999 Published by Elsevier Science B.V, All rights reserved.
引用
收藏
页码:169 / 177
页数:9
相关论文
共 35 条
[1]   Ataxia and altered dendritic calcium signaling in mice carrying a targeted null mutation of the calbindin D28k gene [J].
Airaksinen, MS ;
Eilers, J ;
Garaschuk, O ;
Thoenen, H ;
Konnerth, A ;
Meyer, M .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1997, 94 (04) :1488-1493
[2]   CHANGES IN CA2+ CONCENTRATION IN PHORBOL ESTER AND THAPSIGARGIN TREATED GLIOMA C6 CELLS - THE ROLE OF PROTEIN-KINASE-C IN REGULATION OF CA2+ ENTRY [J].
BARANSKA, J ;
CHABAN, V ;
CZARNY, M ;
SABALA, P .
CELL CALCIUM, 1995, 17 (03) :207-215
[3]  
Braunewell KH, 1997, J NEUROCHEM, V68, P2129
[4]   A SUBSET OF CALRETININ-POSITIVE NEURONS ARE ABNORMAL IN ALZHEIMERS-DISEASE [J].
BRION, JP ;
RESIBOIS, A .
ACTA NEUROPATHOLOGICA, 1994, 88 (01) :33-43
[5]   IDENTIFICATION AND ULTRASTRUCTURAL-LOCALIZATION OF A CALRETININ-LIKE CALCIUM-BINDING PROTEIN (PROTEIN-10) IN THE GUINEA-PIG AND RAT INNER-EAR [J].
DECHESNE, CJ ;
WINSKY, L ;
KIM, HN ;
GOPING, G ;
VU, TD ;
WENTHOLD, RJ ;
JACOBOWITZ, DM .
BRAIN RESEARCH, 1991, 560 (1-2) :139-148
[6]  
FELLAY BS, 1996, 4 EUR S CALC BIND PR, pP49
[7]   CALRETININ-IMMUNOREACTIVE NEURONS IN THE NORMAL HUMAN TEMPORAL CORTEX AND IN ALZHEIMERS-DISEASE [J].
FONSECA, M ;
SORIANO, E .
BRAIN RESEARCH, 1995, 691 (1-2) :83-91
[8]   EARLY DEGENERATION OF CALRETININ-CONTAINING NEURONS IN THE RAT HIPPOCAMPUS AFTER ISCHEMIA [J].
FREUND, TF ;
MAGLOCZKY, Z .
NEUROSCIENCE, 1993, 56 (03) :581-596
[9]   EXPRESSION OF THE CALCIUM-BINDING PROTEIN CALRETININ IN WIDR CELLS AND ITS CORRELATION TO THEIR CELL-CYCLE [J].
GOTZOS, V ;
SCHWALLER, B ;
HETZEL, N ;
BUSTOSCASTILLO, M ;
CELIO, MR .
EXPERIMENTAL CELL RESEARCH, 1992, 202 (02) :292-302
[10]  
Gotzos V, 1996, ANTICANCER RES, V16, P3491