Bcl-2 binds to and inhibits ryanodine receptors

Abstract

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 inhibits 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. We now report that, Bcl-2 co-immunoprecipitated with RyRs in ectopic expression systems and in native rat hippocampi, indicating the existence of endogenous RyR/Bcl-2 complexes. Purified RyR domains containing the putative Bcl-2-binding site bound full-length Bcl-2 in pull-down experiments and interacted with Bcl-2's BH4 domain in surface-plasmon-resonance experiments, suggesting a direct interaction. Exogenous expression of full-length Bcl-2 or electroporation loading of Bcl-2's BH4-domain dampened RyR-mediated Ca2+ release in HEK293 cell models. Finally, introducing the BH4-domain peptide into hippocampal neurons via a patch pipette decreased RyR-mediated Ca2+ release. In conclusion, this study identifies Bcl-2 as a novel inhibitor of RyR-based intracellular Ca2+-release channels.