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

Abstract

Bcl-2 protein has emerged as a critical regulator of intracellular Ca 2+ dynamics by directly targeting and inhibiting the IP3 receptor (IP3R), a major intracellular Ca 2+ -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. Consistent with this, 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.

Ca2+ Fluxes and Cancer

“…In this context, inositol 1,4,5-trisphosphate receptor type 3 (ITPR3, best known as IP 3 R3) and ITPR2 (best known as IP 3 R2) may play a predominant role as compared to ITPR1 (best known as IP 3 R1), at least potentially reflecting their elevated capacity to transmit Ca 2+ signals to mitochondria (Bartok et al, 2019;Mendes et al, 2005;Sun et al, 2019). However, whether BCL2 and other antiapoptotic Bcl-2 proteins limit agonist-induced Ca 2+ release through IP 3 Rs by inhibiting them (Ivanova et al, 2019;Rong et al, 2009), promoting some degree of activation at baseline by increasing the sensitivity to IP 3 (Eckenrode et al, 2010;White et al, 2005) or supporting a cytoprotective Ca 2+ leak from the ER via other mechanisms (Bassik et al, 2004;Palmer et al, 2004;Pinton et al, 2000Pinton et al, , 2001, remains to be clarified. These apparently contrasting observations may at least in part relate to the ability of both pro-and antiapoptotic Bcl-2 family members to regulate mitochondrial VDAC opening (Chong et al, 2020;Shimizu et al, 1999;Tajeddine et al, 2008), and the highly divergent expression of these regulators of apoptosis in cells from different tissues or tumor types, ultimately resulting in different priming of the apoptotic system at mitochondria (Potter and Letai, 2016).…”

Partners in Crime: Towards New Ways of Targeting Calcium Channels

“…Here, we will focus on the protein Bcl-2 who exerts its anti-apoptotic role in two ways [109]: (i) by binding and inhibiting pro-apoptotic proteins of the Bcl-2 family such as Bax and Bak at the mitochondria level; (ii) by binding and modulating IP3R at the ER level [110]. Bcl-2 has been shown to directly interact with IP3R, and multiple binding sites have been described [111][112][113][114]. Via these interactions, Bcl-2 directly modulates calcium release through the IP3R [115], thus promoting pro-survival calcium oscillations [116,117] and inhibiting pro-apoptotic calcium release [112].…”

Regulation of Cell Death by Mitochondrial Transport Systems of Calcium and Bcl-2 Proteins

“…They work as regulators of cell cycle progression, or autophagy, as well as the mediators of Ca 2+ concentration, unfolded protein response and metabolism of the lipids and carbohydrates [ 169 , 185 , 190 , 191 ]. Importantly, Bcl-2 proteins are crucial regulators of Ca 2+ homeostasis and dynamics due to their ability to directly target and regulate the intracellular Ca 2+ -transport systems, in particular IP3 receptors and mitochondrial Ca 2+ transport systems [ 192 , 193 , 194 ].…”