Abstract

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 IP₃R1. 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 IP₃R1 (L15 fibroblasts) or IP₃R3 (Lvec fibroblasts), even though the IP₃R3 does not contain Ca²⁺-dependent CaM-binding sites. Furthermore, we have found that CaM₁₂₃₄, 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.