Functional analysis of the green fluorescent protein-tagged inositol 1,4,5-trisphosphate receptor type 3 in Ca(2+) release and entry in DT40 B lymphocytes.
We examined the function of GFP-IP(3)R3 (green fluorescent protein-tagged inositol 1,4,5-trisphosphate receptor type 3) in Ca(2+) release and entry using a mutant DT40 cell line (IP(3)R-KO) in which all three IP(3)R 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 IP(3) to permeabilized WT (wild-type) DT40 cells induced Ca(2+) release from internal stores. Although this did not occur in IP(3)R-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 Ca(2+) response in IP(3)R-KO cells, whereas these treatments induced oscillatory or transient Ca(2+) responses in GFP-IP(3)R3-expressing IP(3)R-KO cells, as well as in WT cells. In addition, BCR activation elicited Ca(2+) entry in WT and GFP-IP(3)R3-expressing IP(3)R-KO cells but not in IP(3)R-KO cells. This BCR-mediated Ca(2+) entry was observed in the presence of La(3+), which blocks capacitative Ca(2+) entry. Thapsigargin depleted Ca(2+) stores and led to Ca(2+) entry in IP(3)R-KO cells irrespective of GFP-IP(3)R3 expression. In contrast with BCR stimulation, thapsigargin-induced Ca(2+) entry was completely blocked by La(3+), suggesting that the BCR-mediated Ca(2+) entry pathway is distinct from the capacitative Ca(2+) entry pathway. The present study demonstrates that GFP-IP(3)R3 could compensate for native IP(3)R in both IP(3)-induced Ca(2+) release and BCR-mediated Ca(2+) entry.
Morita
Takao
T
Department of Dental Pharmacology, School of Dentistry, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Hokkaido 061-0293, Japan.
Tanimura
Akihiko
A
Nezu
Akihiro
A
Kurosaki
Tomohiro
T
Tojyo
Yosuke
Y
D000818:Animals; D001402:B-Lymphocytes / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D002645:Chickens; D003600:Cytosol / Q000378:metabolism; D049452:Green Fluorescent Proteins; D015544:Inositol 1,4,5-Trisphosphate / Q000502:physiology*; D053496:Inositol 1,4,5-Trisphosphate Receptors; D018613:Microscopy, Confocal; D018160:Receptors, Cytoplasmic and Nuclear / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D013347:Subcellular Fractions / Q000378:metabolism
D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D015544:Inositol 1,4,5-Trisphosphate; D002118:Calcium