Bcl-2 and IP₃ compete for the ligand-binding domain of IP₃Rs modulating Ca²⁺ signaling output

Bcl-2 proteins have emerged as critical regulators of intracellular Ca²⁺ dynamics by directly targeting and inhibiting the IP₃ receptor (IP₃R), a major intracellular Ca²⁺-release channel. Here, we demonstrate that such inhibition occurs under conditions of basal, but not high IP₃R activity, since overexpressed and purified Bcl-2 (or its BH4 domain) can inhibit IP₃R function provoked by low concentration of agonist or IP₃, while fails to attenuate against high concentration of agonist or IP₃. Surprisingly, Bcl-2 remained capable of inhibiting IP₃R1 channels lacking the residues encompassing the previously identified Bcl-2-binding site (a.a. 1380–1408) located in the ARM2 domain, part of the modulatory region. Using a plethora of computational, biochemical and biophysical methods, we demonstrate that Bcl-2 and more particularly its BH4 domain bind to the ligand-binding domain (LBD) of IP₃R1. In line with this finding, the interaction between the LBD and Bcl-2 (or its BH4 domain) was sensitive to IP₃ and adenophostin A, ligands of the IP₃R. Vice versa, the BH4 domain of Bcl-2 counteracted the binding of IP₃ to the LBD. Collectively, our work reveals a novel mechanism by which Bcl-2 influences IP₃R activity at the level of the LBD. This allows for exquisite modulation of Bcl-2’s inhibitory properties on IP₃Rs that is tunable to the level of IP₃ signaling in cells.