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

We examined the function of GFP-IP₃R3 (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-IP₃R3 fluorescence largely overlapped with the distribution of endoplasmic reticulum, whereas a portion of GFP-IP₃R3 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-IP₃R3. 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-IP₃R3-expressing IP₃R-KO cells, as well as in WT cells. In addition, BCR activation elicited Ca²⁺ entry in WT and GFP-IP₃R3-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-IP₃R3 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-IP₃R3 could compensate for native IP₃R in both IP₃-induced Ca²⁺ release and BCR-mediated Ca²⁺ entry.