It has recently been demonstrated that CaBP interacts with and activates InsP₃Rs, providing a novel mechanism for calcium release independent of InsP₃ production (Yang et al. 2002). Using a combination of calcium imaging, calcium flux studies and biochemical approaches, we have investigated the role of CaBPs in regulating InsP₃Rs.

Glutathione S-transferase (GST) pull-down experiments using GST-tagged NH₂-terminal regions of InsP₃Rs as bait and purified CaBP as prey in the presence of 200 µM calcium concentration revealed that CaBP interacted with a region of the type 1 InsP₃R encompassing amino acids 1-224. Peptide mapping of the site of interaction revealed that CaBPs interacted with a region encompassing amino acids proline 49 to asparagine 81, which corresponded to the calcium-independent calmodulin binding region. Furthermore, unlike the previously reported findings (Yang et al. 2002), we demonstrate that this interaction occurs completely independently of calcium. Imaging of fura-2-loaded COS-7 cells overexpressing GFP-tagged CaBPs revealed that CaBP could inhibit calcium release from internal stores in response to an InsP₃-generating agonist (ATP; 0.5 to 100 µM). Data are expressed as means ± S.E.M. and were analysed for significance using a Student’s t test. With 0.5 µM ATP, the integrated calcium response was significantly reduced from 12 618 ± 1879 nM s (n = 27 cells) in control cells to 4921 ± 1839 nM s (n = 4 cells) in CaBP-expressing cells (P < 0.05). With 1 µM ATP, the integrated calcium response was decreased from 24 187 ± 1861 nM s in control cells (n = 32 cells) to 9655 ± 1757 nM s (n = 13) in CaBP-expressing cells (P < 0.05). In addition to decreasing the magnitude of ATP-evoked calcium signals, CaBP expression reduced the proportion of responding cells. With 0.5 µM ATP, 73 % of control cells (n = 44) and 18 % of CaBP-expressing cells (n = 22) responded. The calcium release in response to a supramaximal ATP concentration (100 µM) was not significantly different between control and CaBP-expressing cells, indicating that the intracellular calcium stores were not compromised by CaBP expression. To directly test the effect of CaBPs on InsP₃-induced calcium release we performed ⁴⁵Ca²⁺ flux experiments using saponin-permeabilised A7r5 cells. In the presence of 100 nM cytosolic calcium, 1 µM InsP₃ released 30 % of the total calcium store. CaBP, however, did not release calcium from internal stores. In conclusion, CaBPs do not appear to act as endogenous activators for InsP₃Rs. Rather they inhibit InsP₃-mediated calcium release.