The N-terminal Ca2+-independent calmodulin-binding site on the inositol 1,4,5-trisphosphate receptor is responsible for calmodulin inhibition, even though this inhibition requires Ca2+

Calmodulin (CaM) is a ubiquitous Ca2+-sensor protein that plays an important role in regulating a large number of Ca2+ channels, including the inositol 1,4,5-trisphosphate receptor (IP3R). CaM binds to the IP3R at Ca2+-dependent as well as at Ca2+-independent interaction sites. In this study, we have investigated the Ca2+-independent CaM-binding site for its role in the regulation of the Ca2+-dependent bell-shaped activation curve of the IP3R. Suramin, a polysulfonated napthylurea, displaced CaM in both the presence and the absence of Ca2+. Suramin competed with CaM for binding to different peptides representing the previously identified CaM-binding sites on IP(3)R1. By interacting with the N-terminal Ca2+-independent CaM-binding site, suramin mimicked the functional effect of CaM and induced an allosteric but competitive inhibition of IP3 binding. Therefore, suramin also potently inhibited IP3-induced Ca2+ release (IICR) from permeabilized cells predominantly expressing IP(3)R1 (L15 fibroblasts) or IP(3)R3 (Lvec fibroblasts), even though the IP(3)R3 does not contain Ca2+-dependent CaM-binding sites. Furthermore, we have found that CaM 1234, a CaM mutated in its four EF hands, inhibited IICR in a Ca2+-dependent way with the same potency as CaM. We conclude that CaM inhibits IICR via the N-terminal binding site. The inhibition requires Ca2+ but CaM itself is not the Ca2+ sensor for the inhibition of the IP3R.