Author:

Abstract:

Background: The anti-apoptotic B-cell lymphoma 2 (Bcl-2) protein not only counteracts apoptosis at the mitochondria by scaffolding pro-apoptotic Bcl-2-family members, but also acts at the endoplasmic reticulum, thereby controlling intracellular Ca2+ dynamics. Bcl-2 suppresses Ca2+ release by targeting the inositol 1,4,5-trisphosphate receptor (IP3R). Sequence analysis revealed that the Bcl-2-binding site on the IP3R displays strong homology with a conserved sequence present in all three ryanodine-receptor (RyR) isoforms. Subject: Here, we studied whether Bcl-2 could bind and regulate RyR channels, a major class of the family of tetrameric intracellular Ca2+-release channels and identified the molecular determinants responsible for RyR/Bcl-2-complex formation. Results: We found that ectopic expression of RyR isoforms in HEK293 cell models, resulting in an up-regulation of endogenous Bcl-2. Endogenous and ectopically expressed Bcl-2 co-immunoprecipitated with RyRs in lysates obtained from HEK293 cells and from native rat hippocampi. These data indicate the existence of endogenous RyR/Bcl-2 complexes. Purified RyR domains (from RyR1, RyR2 or RyR3 channels) containing the putative Bcl-2-binding site bound full-length Bcl-2 in GST-pull-down experiments and interacted with Bcl-2’s BH4 domain in surface-plasmon-resonance experiments, showing a direct interaction between Bcl-2 and the different RyR isoforms. Exogenous expression of full-length Bcl-2 or electroporation loading of Bcl-2’s BH4-domain dampened caffeine-induced RyR-mediated Ca2+ release in HEK293 cell models. To exclude that the observed inhibitory effects of Bcl-2 on Ca2+ signaling were not due to an inhibition of IP3R channels, a set of key experiments using xestospongin B, a selective IP3R inhibitor, or using Bcl-2K17D, a mutant version of Bcl-2 that is severely compromised in binding and inhibiting IP3Rs, were performed. Finally, introducing the BH4-domain peptide into GCAMP3-expressing primary hippocampal neurons via the patch pipette suppressed caffeine-induced RyR-mediated Ca2+ release. Conclusion: This study is the first to show that anti-apoptotic Bcl-2 proteins via their BH4 domain act as novel inhibitors of RyR-based intracellular Ca2+-release channels.