Biphasic regulation of InsP ₃ receptor gating by dual Ca ²⁺ release channel BH3-like domains mediates Bcl-x _L control of cell viability

Abstract

Antiapoptotic Bcl-2 family members interact with inositol trisphosphate receptor (InsP 3 R) Ca 2+ release channels in the endoplasmic reticulum to modulate Ca 2+ signals that affect cell viability. However, the molecular details and consequences of their interactions are unclear. Here, we found that Bcl-x L activates single InsP 3 R channels with a biphasic concentration dependence. The Bcl-x L Bcl-2 homology 3 (BH3) domain-binding pocket mediates both high-affinity channel activation and low-affinity inhibition. Bcl-x L activates channel gating by binding to two BH3 domain-like helices in the channel carboxyl terminus, whereas inhibition requires binding to one of them and to a previously identified Bcl-2 interaction site in the channelcoupling domain. Disruption of these interactions diminishes cell viability and sensitizes cells to apoptotic stimuli. Our results identify BH3-like domains in an ion channel and they provide a unifying model of the effects of antiapoptotic Bcl-2 proteins on the InsP 3 R that play critical roles in Ca 2+ signaling and cell viability.T he inositol trisphosphate receptors (InsP 3 R) are a family of intracellular cation channels that release Ca 2+ from the endoplasmic reticulum (ER) in response to a variety of extracellular stimuli (1). Three InsP 3 R isoforms are ubiquitously expressed and regulate diverse cell processes, including cell viability (1). Activation of the channels by InsP 3 elicits changes in cytoplasmic Ca 2+ concentration ([Ca 2+ ] i ) that provide versatile signals to regulate molecular processes with high spatial and temporal fidelity (1). Regions of close proximity to mitochondria enable localized Ca 2+ release events to be transduced to mitochondria (2, 3). Ca 2+ released from the ER during cell stimulation modulates activities of effector molecules and is taken up by mitochondria to stimulate oxidative phosphorylation and enhance ATP production (4-6) to match energetic supply with enhanced demand. In addition, cells in vivo are constantly exposed to low levels of circulating hormones, transmitters, and growth factors that bind to plasma membrane receptors to provide a background level of cytoplasmic InsP 3 (7) that generates low-level stochastic InsP 3 R-mediated localized or propagating [Ca 2+ ] i signals (8-10). Such signals also play an important role in maintenance of cellular bioenergetics (8). Nevertheless, under conditions of cell stress the close proximity of mitochondria to Ca 2+ release sites may result in mitochondrial Ca 2+ overload and initiate Ca 2+ -dependent forms of cell death, including necrosis and apoptosis (11-13). It has been suggested that high levels of ER Ca 2+ (14-16) and enhanced activity of the InsP 3 R (17-19) promote cell death by providing a higher quantity of released Ca 2+ to mitochondria (3,20,21).Protein interactions modulate the magnitude and quality of InsP 3 R-mediated [Ca 2+ ] i signals that regulate apoptosis and cell viability. Notable in this regard is the Bcl-2 protein family. Proapoptotic Bcl-2-related proteins Bax ...

The Stability and Expression Level of Bok Are Governed by Binding to Inositol 1,4,5-Trisphosphate Receptors

“…Proteolysis within the ARM3 domain has recently been suggested to provide a novel mode of IP 3 R regulation (49) and it is intriguing that such proteolysis is inhibited by Bok, most likely because of steric hindrance (14). In view of the ability of Bcl-2 to regulate IP 3 R channel activity (17)(18)(19)(20), and the recently described findings that Bcl-x L activates channel gating by interacting with BH3 domain-like helices in the IP 3 R C-terminal tail (50), it is somewhat surprising that initial experiments with control and Bok Ϫ/Ϫ mouse embryonic fibroblasts did not reveal a major effect of Bok on the Ca 2ϩ -mobilizing function of IP 3 Rs (14). However, a caveat with this study is that the Bok Ϫ/Ϫ cells have adapted in terms of IP 3 R1-3 expression (14) and thus, whether Bok binding to the ARM3 domain directly regulates the Ca 2ϩ mobilizing function of IP 3 Rs remains to be determined.…”

Inositol 1,4,5‐trisphosphate receptors and neurodegenerative disorders

“…The BH3 domain of Bcl-x L binds two BH3 domain-like helices, H1 and H4, in the C terminus of the IP 3 R (Fig. 2B) leading to IP 3 R gating activation [45]. Helix H1 represents residues 2571-2606 and helix H4 represents residues 2690-2732 in the IP 3 R1 sequence [45].…”

“…2B) leading to IP 3 R gating activation [45]. Helix H1 represents residues 2571-2606 and helix H4 represents residues 2690-2732 in the IP 3 R1 sequence [45]. According to cryo-EM data, the proposed H1 BH3 motif of IP 3 R1 represents most of the TM6 a-helix together with the first a-helix from the LNK domain while the proposed H4 BH3 motif represents the a-helix in the CTD [23].…”

“…According to cryo-EM data, the proposed H1 BH3 motif of IP 3 R1 represents most of the TM6 a-helix together with the first a-helix from the LNK domain while the proposed H4 BH3 motif represents the a-helix in the CTD [23]. Sequence alignment of BH3 domains from Bcl-2-related proteins has revealed conserved BH3-like sequences in both H1 and H4 [45]. Gating inhibition is observed when Bcl-x L binds to one BH3 domain-like helix and to a previously identified Bcl-2 interaction site in the coupling domain of the IP 3 R. Disruption of these interactions provokes apoptosis and reduces the viability of cells [45].…”

From dysfunctional endoplasmic reticulum-mitochondria coupling to neurodegeneration

“…Excessive IP 3 Rmediated Ca 2+ release leads to mitochondrial Ca 2+ overload, opening of the mitochondrial permeability transition pore (mPTP), cytochrome c release, caspase activation and apoptosis (Vervliet et al, 2016). Members of the anti-apoptotic protein family Bcl-2 were shown to localize at ERmitochondria contacts and modulate IP 3 R-mediated mitochondrial Ca 2+ transients (Chen et al, 2004;Meunier and Hayashi, 2010;Vervliet et al, 2016;Yang et al, 2016). Regulators of caspase-8 activation were also found at the ER-mitochondria interface (Iwasawa et al, 2011;Marini et al, 2015).…”