Cross-linking of IgE receptors (FCRI) by antigens in rat basophilic leukemia cells (RBL-2H3), results in the production of inositol trisphosphate (IP₃) and release of Ca²⁺ from intracellular stores, triggering exocytosis of secretory granules. FCRI activation also leads to changes in IP₃ receptor (IP₃R) distribution. At rest IP₃R distribution is relatively homogenous throughout the cell. Upon activation, IP₃Rs form discrete clusters in the cell.

In our experiments RBL-2H3 cells were primed with anti-DNP-IgE (1 µg/ml, 12 h) and activated with DNP-BSA (1 µg/ml, 1 h). Immunohistochemistry in combination with fluorescent constructs were used to investigate the redistribution of the IP₃Rs and endoplasmic reticulum (ER) in live and fixed cells.

Staining of IP₃R in fixed RBL-2H3 cells showed that 1 h after cell activation the number of IP₃R clusters increased significantly from 8.19 ± 1.29 clusters per 100 µm² [± S.E.M. n = 20 cells] to 14.13 ± 1.09 clusters per 100 µm² [± S.E.M. n = 9 cells] (Student’s t test, P < 0.05). Furthermore the size of the clusters increased significantly from 0.15 ± 0.01 µm² to 0.28 ± 0.03 µm² (Student’s t test, P < 0.001). We observed similar results in living RBL-2H3 cells expressing YFP-IP₃R.Fluorescence recovery after photobleaching (FRAP) of YFP-IP₃R showed that the IP₃R was mobile within the ER. The half life of recovery of the IP₃R is 39.04 ± 2.64 s [± S.E.M. n = 24 cells] and this decreased significantly following cell activation to 25.10 ± 2.44 s [± S.E.M. n = 15 cells] (Student’s t test, P < 0.001). The immobile fraction of IP₃R was not significantly altered.

To investigate if the ER itself was responsible for IP₃R movement we stained the ER using a BiP antibody. After cell activation the gross structure of the ER changed and, like the IP₃R, becomes concentrated in the peri-nuclear region wherue the microtubule organising centre is typically found. DsRed-ER expression in live cells demonstrated the ER was highly mobile. Expression of YFP-IP₃R and DsRed-ER showed that the IP₃R redistribution following activation was the consequence of ER redistribution. The IP₃R clusters appear and disappear dynamically at nodules in the ER over periods of less than 30 s. In conclusion, where RBL-2H3 cell activation induces the redistribution of the IP₃R, FRAP studies show an increase in IP₃R mobility which was likely to be due to increased movement and reorganization of the ER. This restructuring of the ER causes the transient formation of nodules at branch points in the ER and the IP₃R clusters in these regions. These clusters of IP₃R remain contiguous with the ER itself and increase in number and size in the 1 h following cell activation. We speculate the clustering of the IP₃R may have an effect on the calcium signal generated and functionally on the secretory response.

This study was sponsored by the MRC.