Functional analysis of the green fluorescent protein-tagged inositol 1,4,5-trisphosphate receptor type 3 in Ca2+ release and entry in DT40 B lymphocytes

被引:18
作者
Morita, T
Tanimura, A [1 ]
Nezu, A
Kurosaki, T
Tojyo, Y
机构
[1] Hlth Sci Univ Hokkaido, Sch Dent, Dept Dent Pharmacol, Ishikari, Hokkaido 0610293, Japan
[2] Kansai Med Univ, Dept Mol Genet, Inst Liver Res, Moriguchi, Osaka 5708506, Japan
[3] RIKEN, Res Ctr Allergy & Immunol, Lab Lymphocyte Differentiat, Turumi Ku, Yokohama, Kanagawa 2300045, Japan
关键词
Ca2+ entry; Ca2+ release; DT; 40; cell; green fluorescent protein; imaging; inositol 1,4,5-trisphosphate receptor;
D O I
10.1042/BJ20031970
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
We examined the function of GFP-IP(3)R3 (green fluorescent protein-tagged inositol 1,4,5-trisphosphate receptor type 3) in Ca2+ release and entry using a mutant DT40 cell line (IP3R-KO) in which all three IP3R genes had been disrupted. GFP-IP(3)R3 fluorescence largely overlapped with the distribution of endoplasmic reticulum, whereas a portion of GFP-IP(3)R3 apparently co-localized with the plasma membrane. The application of IP3 to permeabilized WT (wild-type) DT40 cells induced Ca2+ release from internal stores. Although this did not occur in IP3R-KO cells it was restored by expression of GFP-IP(3)R3. In intact cells, application of anti-IgM, an activator of the BCR (B-cell receptor), or trypsin, a protease-activated receptor 2 agonist, did not cause any Ca2+ response in IP3R-KO cells, whereas these treatments induced oscillatory or transient Ca2+ responses in GFP-IP(3)R3-expressing IP3R-KO cells, as well as in WT cells. In addition, BCR activation elicited Ca2+ entry in WT and GFP-IP(3)R3-expressing IP3R-KO cells but not in IP3R-KO cells. This BCR-mediated Ca2+ entry was observed in the presence of La3+, which blocks capacitative Ca2+ entry. Thapsigargin depleted Ca2+ stores and led to Ca2+ entry in IP3R-KO cells irrespective of GFP-IP(3)R3 expression. In contrast with BCR stimulation, thapsigargin-induced Ca2+ entry was completely blocked by La3+, suggesting that the BCR-mediated Ca2+ entry pathway is distinct from the capacitative Ca2+ entry pathway. The present study demonstrates that GFP-IP(3)R3 could compensate for native IP3R in both IP3-induced Ca2+ release and BCR-mediated Ca2+ entry.
引用
收藏
页码:793 / 801
页数:9
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